# The Astounding World of Glycans from Giant Viruses

Immacolata Speciale, Anna Notaro, Chantal Abergel, Rosa Lanzetta, Todd L Lowary, Antonio Molinaro, Michela Tonetti, James L van Etten, Cristina de Castro

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Immacolata Speciale, Anna Notaro, Chantal Abergel, Rosa Lanzetta, Todd L Lowary, et al. The Astounding World of Glycans from Giant Viruses. Chemical Reviews, 2022, 10.1021/acs.chemrev.2c00118. [https://doi.org/10.1021/acs.chemrev.2c00118](https://doi.org/10.1021/acs.chemrev.2c00118)

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# The Astounding World of Glycans from Giant Viruses

Immacolata Speciale,○ Anna Notaro,○ Chantal Abergel, Rosa Lanzetta, Todd L. Lowary, Antonio Molinaro, Michela Tonetti, James L. Van Etten, and Cristina De Castro*

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Cite This: https://doi.org/10.1021/acs.chemrev.2c00118

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**ABSTRACT:** Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed.

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## CONTENTS

1. Introduction B

2. Nucleocytoplasmic Large DNA Viruses B

2.1. Family Marseilleviridae C

2.2. Proposed *Medusaviridae* Family E

2.3. Family *Mimiviridae* E

2.3.1. Proposed Subfamily *Klosneuvirinae* E

2.3.2. Proposed Subfamily *Megavirinae* F

2.4. Proposed *Molliviridae* Family F

2.5. Proposed Family *Pandoraviridae* G

2.6. Family Phycodnaviridae G

2.6.1. Genus *Chlorovirus* G

2.6.2. Genus *Coccolithovirus* H

2.6.3. Genus *Phaeovirus* H

2.6.4. Genus *Prasinovirus* H

2.6.5. Genus *Prymnesiovirus* H

2.6.6. Genus *Raphidovirus* I

2.7. Proposed *Pithocedratviridae* Family I

3. Biosynthetic Machinery and the Glycans Produced by Giant Viruses I

3.1. Biosynthesis of Sugar Nucleotides J

3.1.1. Chloroviruses J

3.1.2. Megavirinae M

3.2. Glycans Structures U

3.2.1. Chloroviruses U

3.2.2. APMV Glycans Structures X

3.3. Glycosyltransferases Z

3.3.1. Chloroviruses Remodel the Host Cell Surface Z

3.3.2. Chloroviruses Encode the GTs that Synthesize Glycans Attached to Proteins AB

3.3.3. *Megavirinae* GTs: a Focus on APMV GTs AF

3.4. Glycosyl Hydrolases (GHs) and Lyases (PL) AG

3.4.1. Chlorovirus Chitinase and Chitosanase AG

3.4.2. PBCV-1 β-Glucanase AH

3.4.3. CVN1 Alginate Lyase AH

3.4.4. CVK2 Alginate Lyase AI

3.4.5. PBCV-1 A561L, a putative alginate lyase AI

4. Chemical Synthesis of Glycans Inspired from Giant Viruses AJ

4.1. Viosamine AJ

4.2. Chlorovirus N-Glycans AJ

5. Conclusions and Future Perspectives AN

Author Information AO

Corresponding Author AO

Authors AO

Author Contributions AO

**Special Issue:** Glycosciences

**Received:** February 14, 2022
<table><tr><td>Notes</td><td>AO</td></tr><tr><td>Biographies</td><td>AO</td></tr><tr><td>Acknowledgments</td><td>AP</td></tr><tr><td>Abbreviations</td><td>AP</td></tr><tr><td>References</td><td>AQ</td></tr></table>

# 1. INTRODUCTION

The general perception of viruses is that they are small entities and dangerous human pathogens. This view is understandable because, over the years, viruses have caused major disease burdens such as AIDS and the Ebola outbreak in 2014 or the ongoing pandemic caused by SARS-CoV-2. However, this view is biased because it only focuses on a restricted number of viruses and it neglects the fact that viruses target all cellular organisms. Indeed, viruses are the most abundant biological entities on the planet, with surface ocean water reported to contain ~10⁷ virus particles per milliliter.¹,² This value translates into ~10³¹ virus particles in the world's oceans, and this estimate does not include RNA viruses or viruses present in soils and other environments. Most of these viruses infect bacteria, with lesser numbers infecting microalgae and other organisms. Thus, viruses should be seen as integral components of any ecosystem where they contribute to the maintenance of the balance between species and resources, thus playing a positive role in the living community.³

Viruses are a heterogeneous ensemble of biological entities, all sharing the common trait that their replication requires a suitable host even though they vary in morphology, size, nature, and complexity of their genomic content. Depending on the virus, the genetic material can be either DNA or RNA, which exists either as double stranded (ds) or single stranded (ss) molecules while their physical size can vary by orders of magnitude. Viruses can be as small as 17 nm, e.g., porcine circovirus, which has a ssDNA genome of ~1.8 kilobases (kb) that encodes for three proteins,⁴ or they can reach 1500 nm in size like Pithovirus sibericum,⁵ or they can be as genetically complex as Pandoravirus salinus, the virus with the largest genome found to date, with a dsDNA genome of ~2500 kb, which encodes ~1500 predicted proteins.⁶ Finally, viral particles (virions) can have different morphologies: e.g., porcine circovirus particles have an icosahedral geometry while Pandoravirus salinus, visible under a light microscope as are bacteria, has amphora-shaped virions that resemble the morphology of some bacteria. In contrast, many plant viruses are rod shaped.⁷

When discussing species that produce carbohydrate-manipulating enzymes, viruses are seldom considered as they are often thought to lack these proteins. Again, this view is flawed from the knowledge acquired from viruses that are human pathogens, such as rhabdoviruses, herpesviruses, poxviruses, coronaviruses, and paramyxoviruses, all endowed with structural proteins that are glycosylated. These human pathogenic viruses use host-encoded glycosyltransferases (GTs) and glycosyl hydrolases (GH) located in the endoplasmic reticulum (ER) and Golgi apparatus of the host to add and remove N-linked sugar residues from virus glycoproteins either cotranslationally or shortly after translation of the protein. Thus, the glycoproteins produced by these pathogenic viruses are host-specific, and the structures of their glycans echo those of the host because they are synthesized by the same biosynthetic machinery as the host glycoproteins.⁸⁻¹⁰

However, certain viruses, the so-called large or giant viruses, differ from this general view because many of them are reported to either encode, or are predicted to encode, enzymes involved in making extracellular polysaccharides, sugar nucleotides, and glycans attached to their major capsid proteins, i.e., glycoproteins. In addition, many of these viruses also encode enzymes involved in polysaccharide degradation, such as GHs and polysaccharide lyases (PL).

The evidence that some viruses have carbohydrate biosynthetic enzymes began with the discovery that the green microalga *Chlorella variabilis* NC64A acquired the ability to synthesize hyaluronic acid when infected with Paramecium bursaria chlorella virus type 1 (PBCV-1),¹¹ a large virus discussed later in this review. Since this discovery in the late 1990s, reports of large and giant viruses that encode putative carbohydrate biosynthetic enzymes and produce their own glycan structures have steadily increased, with most of the reports occurring in the last 10 years. As a result, the concept of viral autonomous glycosylation has only recently entered into the literature.

The purpose of this review is to take readers on a journey into the world of large and giant viruses as seen through the lenses of carbohydrate structure, synthetic chemistry, and biochemistry. In doing so, this review will first describe the characteristics of these large and giant viruses that infect eukaryotic organisms, with a focus on those that have either known or putative genes encoding carbohydrate active enzymes. These include GTs, GHs, sugar methyltransferases, and those involved in the production of activated sugar nucleotide precursors.¹²⁻¹⁴ Then, the structure of the glycans, reported so far for only a few of these viruses, will be discussed along with how these findings have inspired synthetic organic chemistry. Finally, we will discuss how these findings open new avenues of research in many areas of chemistry and biochemistry.

# 2. NUCLEOCTOPLASMIC LARGE DNA VIRUSES

To introduce the reader to viruses that encode carbohydrate manipulating enzymes, we need to mention some changes that are occurring in the classification of viruses. There is an effort by the International Committee on the Taxonomy of Viruses (ICTV)¹⁵ to classify viruses into taxonomic schemes like those used for living organisms. The schemes include Kingdoms, Phyla, Classes, Orders, Families, Genera, and Species. Currently, there are 17 Phyla, and all the large dsDNA viruses considered in this review either belong to the Phylum *Nucleocytoviricota* and Class *Megaviricetes* or are predicted to eventually be classified in this phylum and class, such as pithoviruses, pandoraviruses, and molliviruses.¹⁶,¹⁷ For the families (or the subfamilies or the genera) whose names are proposed but not yet approved by the ICTV committee, the term "proposed" will precede their name when discussed in this review.

Collectively, large and giant viruses are often referred to as nucleocytoplasmic large DNA viruses (NCLDVs), and this term will be used in this review when referring to the group. The NCLDV group is rapidly expanding, and currently it includes several families of viruses, which differ in morphology (Figure 1), replication cycles in the host, and number of encoded genes. NCLDVs are found in diverse habitats and infect various organisms, mostly protists and microalgae. However, some NCLDVs, such as poxviruses and asfarviruses, impact animal health and food security¹⁸ but will not be
<figure><img src="image_8.png" /><figcaption>**Figure 1.** Electron microscopy images of selected giant viruses. SEM or TEM images of different giant viruses taken to display the morphology or other morphological features; their classification is reported in Table 1 along with other features. (a) PBCV-1, a member of the *Phycodnaviridae* family (genus *Chlorovirus*) and is an icosahedral virion (see also section 3.2.1). (b) Tupanvirus soda lake (clade D of *Mimivirus* genus): tridimensional image detailing the morphology of the virus, unique in having a long tubular tail (extending along the lower part of the image) attached to the capsid (top part), and fully covered with fibrils. Adapted with permission from ref 76. Copyright 2018 Springer Nature. (c) Different view of Tupanvirus soda lake virion: here the tail is defined from the two boundaries that extend down to the lower left corner of the image, while the capsid is at the up-right corner; both capsid and tail are covered with a dense layer of fibrils. Adapted with permission from ref 76. Copyright 2018 Springer Nature. (d) Pandoravirus dulcis (proposed *Pandoraviridae* family), an amphora shaped virus with an ostiole-like depression located at the left-end of the particle. (e) APMV (clade A of *Mimivirus* genus): the particle has an icosahedral shape covered with a dense layer of long fibrils. Adapted with permission from ref 77. Copyright 2008 Public Library of Science. (f) CroV and its virophage Mavirus (lower left corner). Reproduced with permission from ref 83. Copyright 2019 Public Library of Science. (g) Moumouvirus australiensis (clade B in *Mimivirus* genus): its icosahedral virion differs from the other in the same clade with a sparse number of fibrils attached to the capsid. (h) Moumouvirus maliensis (clade B in *Mimivirus* genus): image detailing the nature of the fibrils, shorter than those of APMV, and denser than those of Moumouvirus australiensis (g). (i) *Pithovirus sibericum*, an amphora shaped virus with a cork-like element at the left side of its longitudinal edge. (j) APMV image (cryo-EM) showing an area of lesser density (lighter gray shading) between five fibered faces, the stargate on mature particle. Adapted with permission from ref 84. Copyright 2009 Public Library of Science. (k) Mollivirus sibericum, a virus characterized by a round-shaped morphology. (l) Cedratvirus mature particle with the two cork-like elements visible at the opposite longitudinal edges. Adapted with permission from ref 85. Copyright 2009 Multidisciplinary Digital Publishing Institute. All of the images are from the authors of this review except where specified.</figcaption></figure>

considered in this review because they lack genes encoding
GTs. The dsDNA genomes of some NCLDVs are large, going
from about 300 kbp to over 2.5 Mbp (Table 1), and their
genetic complexity can be larger than many small cellular
organisms, like the bacterium *Mycoplasma genitalium* and the
archaean *Nanoarcheum equitans* or the parasitic eukaryotic
*Encephalitozoon* species.

Interestingly, NCLDV genomes encode a repertoire of
putative uncharacterized proteins because their amino acid
sequences lack any resemblance to annotated proteins, and for
this reason are termed ORFans. Regarding the restricted pool
of proteins with some homologies with known proteins, many
have cell-metabolism like properties, such as components of
the translational system, proteases, and other elements of the
protein degradation machinery. Moreover, a certain set of
these viruses encode putative proteins able to manipulate
sugars at different levels: from the biosynthesis of nucleotide
activated precursors, to those used to build glycans, namely

GTs, and the hydrolases involved in the degradation of these
molecules.

The next sections will briefly describe the properties of
representative members of each of the NCLDV families
discussed in this review (Table 1). Selection will be driven by
the presence of putative GT encoding genes in their genome,
as these genes more than others indicate the potential of these
viruses to synthesize either glycans independently, or with a
limited usage, of the host machinery.

## 2.1. Family Marseilleviridae

Since the discovery of the first member, Marseillevirus
marseillevirus, in 2009,¹⁹ the family has rapidly expanded
with new isolates coming from all kinds of environments. They
all infect *Acanthamoeba* and belong to at least five phylogeneti-
cally related clades, each labeled with a letter (Table 1).²⁰ This
family has some affinity with *Iridoviridae*, even though a
majority of their genes are ORFans. The icosahedral virions
have a diameter of ~250 nm and enclose AT-rich 350–390 kb
dsDNA circular genomes encoding 45–550 proteins.²¹⁻²³
<table><caption>Table 1. Principal Features of the Large and Giant Virus Isolates That Encode (Putative) Glycosyltransferases<sup>a</sup></caption><thead><tr><th rowspan="2">family</th><th rowspan="2">subfamily</th><th rowspan="2">genus</th><th rowspan="2">clade</th><th rowspan="2">species or<br>prototype</th><th colspan="3">virion</th><th rowspan="2">natural host or lab<br>replication host</th></tr><tr><th>type</th><th>size (nm)</th><th>dsDNA<br>(Mb)</th></tr></thead><tbody><tr><td rowspan="5">Marseilleviridae</td><td rowspan="5"></td><td rowspan="5">Marseillevirus</td><td>A</td><td>Marseillevirus<br>marseillevirus</td><td>icosahedral</td><td>250</td><td>0.35–0.38</td><td>Acanthamoeba</td></tr><tr><td>B</td><td>Lausannevirus</td><td></td><td></td><td>0.39</td><td></td></tr><tr><td>C</td><td>Insectomime<br>virus</td><td></td><td></td><td>0.36</td><td></td></tr><tr><td>D</td><td>Brazilian virus</td><td></td><td></td><td>0.36</td><td></td></tr><tr><td>E</td><td>Golden<br>marseillevirus</td><td></td><td></td><td>0.38</td><td></td></tr><tr><td>*Medusaviridae</td><td></td><td></td><td></td><td>Medusavirus</td><td>icosahedral</td><td>260</td><td>0.38</td><td>Acanthamoeba</td></tr><tr><td rowspan="3">Mimiviridae</td><td rowspan="3">*Klosneu-<br>virinae<sup>b</sup></td><td rowspan="3"></td><td></td><td>Bodo saltans virus</td><td>icosahedral with<br>fibrils</td><td>300</td><td>1.39</td><td>Bodo saltans</td></tr><tr><td></td><td>Yasminevirus</td><td></td><td>330</td><td>2.1</td><td>Vermamoeba</td></tr><tr><td></td><td>Fadolivirus</td><td></td><td>300</td><td>1.5</td><td></td></tr><tr><td rowspan="5">*Megavirinae</td><td rowspan="5"></td><td rowspan="5">Cafeteria-virus</td><td></td><td>CroV</td><td>icosahedral</td><td>300</td><td>0.69</td><td>Cafeteria<br>roenbergensis</td></tr><tr><td rowspan="4">Mimivirus</td><td>A</td><td>APMV</td><td>icosahedral with<br>fibrils</td><td>500 + 250</td><td>1.18</td><td>Acanthamoeba</td></tr><tr><td>B</td><td>Moumouvirus</td><td></td><td>420 + 200</td><td>1.02</td><td></td></tr><tr><td>C</td><td>Megavirus<br>chilensis</td><td></td><td>520 + 150</td><td>1.26</td><td></td></tr><tr><td>D</td><td>Tupanvirus deep<br>ocean</td><td>icosahedral with<br>fibrils and tail</td><td>450 + 150 + 550</td><td>1.44–1.51</td><td>Acanthamoeba,<br>Vermamoeba</td></tr><tr><td></td><td>E</td><td>Cotonvirus<br>japonicus</td><td>Icosahedral with<br>fibrils</td><td>400 + 200</td><td>1.48</td><td>Acanthamoeba</td></tr><tr><td>*Molliviridae</td><td></td><td></td><td></td><td>Mollivirus<br>sibericum</td><td>spherical</td><td>650</td><td>0.65</td><td>Acanthamoeba</td></tr><tr><td rowspan="2">*Pandoraviridae</td><td rowspan="2"></td><td rowspan="2"></td><td>A</td><td>Pandoravirus<br>salinus</td><td>amphora</td><td>1000 × 500</td><td>2.5</td><td>Acanthamoeba</td></tr><tr><td>B</td><td>Pandoravirus<br>neocaledonia</td><td></td><td></td><td>2.0</td><td></td></tr><tr><td rowspan="6">Phycodnaviridae</td><td rowspan="6"></td><td>Chlorovirus</td><td></td><td>PBCV-1</td><td>icosahedral</td><td>190</td><td>0.33</td><td>inland water green<br>microalgae</td></tr><tr><td>Coccolithovirus</td><td></td><td>Emiliania huxleyi<br>virus</td><td></td><td>100–220</td><td>0.41</td><td>Emiliania huxleyi</td></tr><tr><td>Phaeovirus</td><td></td><td>Ectocarpus<br>siliculosus virus<br>1</td><td></td><td>120–150</td><td>0.15–0.35</td><td>marine brown<br>algae</td></tr><tr><td>Prasinovirus</td><td></td><td>Micromonas<br>pusilla virus<br>SP1</td><td></td><td>100–130</td><td>0.18–0.22</td><td>marine green<br>microalgae</td></tr><tr><td>Prymnesiovirus</td><td></td><td>CpV PW1</td><td></td><td>150–250</td><td>0.37–1.42</td><td>marine microalgae</td></tr><tr><td>Raphidovirus</td><td></td><td>Heterosigma<br>akashiwo virus</td><td></td><td>100–220</td><td>0.30</td><td>marine microalgae</td></tr><tr><td rowspan="2">*Pithocedratviridae</td><td rowspan="2"></td><td>*Cedratvirus</td><td></td><td>Cedratvirus A11</td><td>amphora</td><td>1500 × 500</td><td>0.46–0.59</td><td>Acanthamoeba</td></tr><tr><td>*Pithovirus</td><td></td><td>Pithovirus<br>sibericum</td><td></td><td></td><td>0.61</td><td></td></tr></tbody></table>

The prototype Marseillevirus marseillevirus, codes for six GTs with only two characterized by biochemical studies: L230, hydroxylates the lysine residues of a collagen-like viral protein, and transfers a Glc unit to it,²⁴ and R707,²⁵ discussed latter in this review (section 3.3.3).

The *Marseilleviridae* family members appear to be prone to horizontal gene transfer and genome rearrangements, localized to one side of the genome.²⁶ Surprisingly, they encode histone doublets²⁷ that form unstable nucleosomes to compact and package the viral genome in capsids.²⁸,²⁹ They enter the host

<footnote><sup>a</sup>The classification follows the criteria used by the ICTV; for the viruses whose position is proposed but not yet defined, the name of the family or of the subfamily is starred. Families and/or subfamilies can be further divided into clades (denoted with a letter) or into genera (denoted with a name). For each family/subfamily, the prototype is indicated based on the ICTV classification in 2020. After this date, type species were abolished by the committee. <sup>b</sup>For this proposed subfamily, there is no prototype representative, accordingly the three isolates have been reported.</footnote>
by phagocytosis and lose their icosahedral appearance to
become spherical after disappearance of the vacuole mem-
brane. At this stage, the viral nucleosomes probably unfold to
allow early transcription. Marseilleviruses encode an RNA
polymerase that is not packaged in the virion and therefore
must rely on the host for early transcription. During early
transcription, the cell host nucleus starts changing appearance
and becomes leaky through an unknown mechanism triggered
by the viral infection.²⁰ The nuclear proteins are recruited to
the early viral factory (VF, a transitory organelle developed by
the virus in which replication and viral assembly takes place),
located in the cytoplasm, including the host RNA polymerase
that carries out early transcription. After a few hours post
infection (PI), the host nucleus morphology is restored and the
viral-encoded RNA polymerase performs intermediate and late
transcription in the VF.³⁰ A predicted conserved promoter
motif is not sufficient to explain the temporal classes of
transcription, suggesting transcription factors drive the
process.³⁰,³¹ The VF expands and icosahedral particles
assemble inside this host cytoplasmic region. Mature particles
gather in large vesicles³² and cell lysis leads to the release of
both individual virions and virus-filled vacuoles. The cryo-EM
structure of the capsid was obtained for two members of the
family at various resolutions revealing an icosahedral shape of
the virion and a complex organization of the capsids³³⁻³⁵ with
a major and many minor capsid proteins like the chlorovirus
PBCV-1 (Figure 1a, section 2.6.1)³⁶ and Cafeteria roenber-
gensis virus (CroV, a member of the Megavirinae subfamily,
section 2.3.2, Figure 1f).³⁷

## 2.2. Proposed *Medusaviridae* Family

Currently, there are just two isolates in this family,³⁸ both
named *Medusavirus*, which infect *Acanthamoeba*.³⁹,⁴⁰ A low-
resolution structure of the virion by cryo-EM reported an
icosahedral shape with the capsid covered by spherical-headed
14 nm spikes, each extending from the capsomer, the building
block of the capsid is in turn made of three copies of the major
capsid protein (MCP).⁴⁰ The icosahedral virions have a
diameter of 260 nm and enclose a 381 kb dsDNA GC-rich
genome, encoding more than 400 putative proteins, two-thirds
of which are ORFans. Virus DNA replication takes place in the
host nucleus while virions assemble in the VF in the cytoplasm;
information regarding the entry and egress of the virions has
not been reported. Genes used to manipulate the DNA
replication machinery, such as DNA topoisomerase II and
RNA polymerase, are lacking. However, a large repertoire of
histones are encoded by the viruses, with the majority of them
detected by in silico analyses and they are involved in the
packaging of the DNA into the capsids, similarly to that
reported for *Marseilleviridae*.²⁹ Regarding the ability of
medusaviruses to manipulate sugars, the two isolates encode
one putative GT each, and the isolate with the largest genome
also has a putative NDP-sugar dehydrogenase gene.

## 2.3. Family *Mimiviridae*

The first representative of the *Mimiviridae* family, Acantha-
moeba polyphaga Mimivirus (APMV), was isolated from a
cooling tower in Bradford (UK) in 1992. It was originally
mistaken as an intracellular bacterium infecting an *Acantha-
moeba* spp. because of its large size and positive Gram staining.
However, infected cells imaged by electron microscopy later
revealed a typical viral icosahedral morphology (Figure 1e,j),
and its viral origin was confirmed by genome sequencing.<sup>41,42</sup>
The genome is a 1.2 Mb dsDNA, with a GC content of 28%,

encoding more than 1000 proteins, including several
components of the protein translation machinery. The
APMV virion has a 500 nm diameter, and it is covered by a
dense layer of glycosylated fibers, 120–140 nm long (Figure
1e,j).

All members of this family have dsDNA AT-rich linear
genomes, and they have one internal lipidic membrane
underneath the capsid shell and one delineating the
nucleoprotein core. They all replicate in large VFs in the
host cell cytoplasm, where the infectious cycle starts.<sup>43-47</sup> All
of these viruses encode a RNA polymerase and transcription
maturation machinery that includes a mRNA capping enzyme,
a RNase, a poly A polymerase, and methyltransferases that are
packaged in the virions. As a result of this complete
transcription and replication machinery, the VF can be the
target of viral infection by so-called virophages, which are true
viruses of viruses (Figure 1f).<sup>48-54</sup> As of today, members of the
*Mimiviridae* family, along with those in the genus *Prymnesio-
virus* (section 2.6.5) are the only known viruses allowing the
replication of virophages. Finally, these viruses encode
complete DNA repair machinery,<sup>55,56</sup> an asparagine synthase,<sup>57</sup>
as well as numerous glyco-related genes.<sup>24,25,58-65</sup>

It has been proposed to divide the family into two
subfamilies, the *Klosneuvirinae* and the phylogenetically related
*amoeba-infecting Megavirinae* (Table 1).<sup>66</sup>

**2.3.1. Proposed Subfamily Klosneuvirinae.** All mem-
bers of this proposed subfamily were initially identified from
metagenomic data from bottom sediments of reservoirs at a
wastewater treatment plant in Klosterneuburg (Austria).<sup>67</sup>
Subsequently, three different viruses have been isolated,
enabling the characterization of their morphology, along with
their replication style.

Bodo saltans virus (BsV) was the first isolated member of
this subfamily.⁶⁸ It infects the marine kinetoplastid *Bodo
saltans*, a group of flagellated protists. The icosahedral capsids
are 300 nm in diameter and contain an AT-rich linear genome
of ~1.4 Mb, encoding ~1225 predicted proteins. Forty percent
of the genes correspond to ORFans and, like other members of
this subfamily, BsV encodes a complete DNA replication and
repair system, along with several genes able to manipulate
sugars at different levels: nine GTs, one GDP-Man 4,6-
dehydratase, a Man 6-phosphoisomerase, and a 4,6 dehy-
dratase 5-epimerase. The icosahedral capsid is made of two
proteinaceous layers surrounded by 40 nm long fibrils. An
apparent stargate-like structure, similar to that of APMV
(Figure 1j) and other mimiviruses (section 2.3.2.2), is present
at one vertex of the capsid. This structural motif is relevant
during the initial stage of viral infection because it mediates the
capsid opening and the creation of a channel for the delivery of
the viral genome into the host cytoplasm.⁶⁹ Finally, there are
two membranes, one lining the external protein shell and one
internal to the nucleoid compartment containing the genome.
In contrast to other members of the proposed *Megavirinae*
subfamily, BsV lacks tRNA genes, and there are only two genes
encoding aminoacyl tRNA synthetases (aaRS). The infectious
cycle is comparable to other members of the *Megavirinae*
except that the host nuclear genome is degraded by the end of
the infection cycle. Lipid vesicles are recruited for virion
assembly, which takes place at one side of the VF, and mature
virions are released by budding in vesicles from the host
membrane and ultimately after cell lysis.⁶⁸

Regarding the two other members of the *Klosneuvirinae*,
both have recently been isolated by using *Vermamoeba*
*vermiformis* as the host in laboratory conditions, thus, their natural host is yet to be defined.⁷⁰,⁷¹ In temporal order, Yasminevirus was the second member to be discovered. It has an icosahedral shape of about 330 nm and the capsid is covered with a thin layer of fibrils. Its dsDNA is ~2.1 Mb, with a GC content of 40.2%, and it codes for 1541 putative proteins including a nearly complete translational system, consisting of 70 tRNAs that are predicted to recognize 20 amino acids, along with 20 aaRSs and several translation initiation, termination, and elongation factors.⁷⁰ The repertoire of carbohydrate active enzymes includes 10 putative GTs, along with one glycan debranching enzyme, a glycosyl hydrolase, an UDP-Glc 4-epimerase, and enzymes active on (1→4)-α-glucans, along with a glucosamine-fructose 6-phosphate amidotransferase (GFAT).

The last member of this proposed subfamily was isolated in 2021 from an Algerian sewage sample. Fadolivirus has an icosahedral shape, with a diameter of about 300 nm covered with short fibrils. Its genome is ~1.5 Mb and encodes 1452 predicted proteins, including 66 tRNAs, 23 aaRSs, and a wide range of transcription factors, that collectively surpass the numbers found in *Klosneuvirinae* and other giant viruses.⁷¹ Regarding the number of predicted glycogenes, analysis of the annotated genome returns 30 GTs, three genes involved in Kdo (3-deoxy-D-manno-oct-2-ulosonic acid, see section 3.1.2.4) synthesis, and six other enzymes able to manipulate sugar nucleotides at different levels: dTDP-D-Glc 4,6-dehydratase, UDP-Glc 6-dehydrogenase, NDP-sugar epimerase, UDP-GlcNAc 2-epimerase WecB-like protein, NDP-sugar epimerase, NAD-dependent epimerase/dehydratase, along with a chitin synthase and a chitinase.

### 2.3.2. Proposed Subfamily *Megavirinae*. 2.3.2.1. *Genus Cafeteriavirus*
Related viruses belonging to the *Megavirinae* subfamily infecting unicellular eukaryotes other than amoeba have been described. Of note, the Cafeteria roenbergensis virus (CroV, Table 1, Figure 1f), the representative of the *Cafeteriavirus* genus, is a marine virus infecting the widespread heterotrophic flagellate *Cafeteria roenbergensis*.⁵⁹ Its icosahedral 300 nm in diameter capsid is devoid of fibrils, it has an internal lipid-containing membrane,³⁷ and it contains a 618 kb dsDNA AT-rich genome, with about a third of the genes in common with its closest relative APMV, including an isoleucyl-tRNA synthetase.

Analysis of the CroV genome identified a 38 kb cluster encoding several glycogenes. It is predicted to encode the complete pathway for Kdo biosynthesis (section 3.1.2.4), along with five putative GTs, one UDP-Glc 6-dehydrogenase, and additional sugar modifying enzymes, such as sugar methyl-transferases.⁷²

Currently, the CroV mode of infection is not completely understood, but a nucleoid structure was reported in the cytoplasm as well as extracellular empty capsids, supporting an external opening of the capsids followed by fusion of the internal membrane with the cell plasma membrane, allowing the transfer of the nucleoid to the cytoplasm. Virions contain about 150 proteins, including several DNA repair enzymes and an ion channel protein that could protect the genome from damages.⁷² The transcription machinery packaged in the virion initiates the early formation of the VF using the same stringent early promoter as APMV.⁵⁹ Neo-synthesized virions assemble during the late stage of infection and are released by cell lysis.

CroV was isolated in association with the virophage Mavirus (Figure 1f).⁴⁹ Mavirus uses the late VF for gene expression and

replication, interfering with the CroV infectious cycle. Mavirus is also able to enter the host cell and integrate its genome into the host genome, like lysogenic or temperate viruses. Mavirus remains silent until the cell becomes infected by CroV, which leads to VF formation and Mavirus replication. As a result, Mavirus virion production has a protecting effect on the algal cell population against CroV replication.⁷³

#### 2.3.2.2. *Genus Mimivirus*
Since the discovery of mimivirus APMV in 2003 (Figure 1e,j),⁴¹,⁴² several dozen members of the *Mimivirus* genus have been isolated worldwide that belong to five clades (Table 1).⁴⁶,⁷⁴⁻⁷⁶ They have large complex AT-rich genomes up to 1.5 Mb (Table 1), and they all have genes involved in translation initiation, elongation, and termination and can encode up to 20 aaRS.⁴²,⁵⁹,⁶⁴,⁶⁷,⁶⁸ They have icosahedral capsids of about 500 nm, decorated by a layer of glycosylated fibrils of various lengths. In *tupanviruses* (Figure 1b,c, clade D), there is a long tail linked to the capsid also with long fibrils.⁴²,⁶⁵,⁶⁸,⁷⁶

The virions carry an RNA polymerase and transcript maturation machinery into the host, and they have exclusively cytoplasmic replication cycles.⁴⁵,⁵⁹,⁶⁸,⁷⁷ They infect their amoeba host by triggering phagocytosis after adhesion to the cell membrane by the glycosylated fibrils. Once in the vacuole, a specific structure, the stargate (Figure 1j), located at one vertex of the icosahedron capsid opens, pulling the internal membrane outside the capsid to fuse with the vacuole membrane.⁶⁹ A compartment, coined nucleoid, is transferred into the host cytoplasm,⁴⁶,⁷²,⁷⁸ where early transcription begins using the virally encoded transcription machinery, which remains first confined in the nucleoid.⁴⁴ The VF in which nucleic acids accumulate due to active transcription and replication increases in size and neosynthesized virions start budding at its periphery, recycling host cell membranes derived from the ER,⁴⁴,⁷⁹,⁸⁰ or Golgi compartments, as recently described for Cotonvirus (clade E).⁸¹ The last step in virion maturation, after genome loading into the nucleoid, is the addition to the capsid of the fibril layer, which comprises many proteins and two different polysaccharides (section 3.2.2).⁸⁰,⁸² Hundreds of neosynthesized virions are released after cell lysis.

*Megavirinae* members can be infected by two kinds of virophages, Sputnik⁴⁸,⁵¹ and Zamilon.⁵¹ They can also be found associated with transpovirons, which are 7 kb linear episomes.⁵¹,⁸⁶

With regard to glyco-related genes, all of the members of this genus encode for several GTs and other enzymes involved in sugar metabolism, as discussed in section 3.1.2.5.⁸⁷

## 2.4. Proposed *Molliviridae* Family

The first member of the proposed *Molliviridae* family, Mollivirus sibericum (Figure 1k), was isolated from the same 30 000 year old Siberian permafrost sample as Pithovirus sibericum (Figure 1i).⁸⁸ Currently, there are only two members in this family, Mollivirus sibericum and Mollivirus kamchatka, both with some affinity with the *Pandoraviridae* family (Figure 1d), including the ability to create genes *de novo*.⁸⁹ The capsid, haloed by fibrils, appears almost spherical with a closed ostiole-like depression, a thick tegument made of at least two layers and an internal lipidic membrane. The capsid encloses linear dsDNA, GC-rich genomes up to 650 kb, encoding ~520 putative proteins. Two-thirds of the genes are ORFans and 16% have their closest homologues in *Pandoraviridae* members.⁸⁸ The infectious cycle is similar to that of *Pandoraviridae*; it starts with the viral entry by
phagocytosis, membrane fusion, and delivery of the compacted genome into the host cytoplasm. The genome then travels to the nucleus and uncompacts, giving access to the host RNA polymerase for early transcription until the viral encoded enzyme is produced.⁸⁸ VF formation takes place in the cytoplasm, and there virion assembly is initiated by a membrane precursor resembling a cisterna, which later is coated with the thick tegument.⁹⁰ DNA-associated filaments fill the nascent viral particles in the late VF, and a late stage of maturation involves a change in morphology of the virions to produce the ostiole-like depression at the initiation pole. Neosynthesized virions can be released by either exocytosis or by cell lysis.⁸⁸,⁹⁰ Finally, M. sibericum is the only member of this proposed family to contain glyco-related genes, in particular its genome harbors two putative GTs, with one predicted as specific for glucosamine.

## 2.5. Proposed Family Pandoraviridae

Members of this family have been isolated from all over the world in all kinds of environments (fresh water, ocean,⁶ soil,⁹¹ waste,⁹² patients infected with *Acanthamoeba* sp.⁹³). Currently, this family includes 15 species (source NCBI/viruses)³⁸ divided into two clades (Table 1).⁶,⁹¹⁻⁹⁸ They are the most complex viruses known, endowed with linear GC-rich dsDNA genomes up to 2.5 Mb. Pandoravirus genome size can in part be explained by their ability to create new genes de novo from intergenic regions.⁹¹,⁹⁸ Pandoraviruses genomes encode up to 1500 proteins, and again, like in other NCLDV families, the vast majority of the genes encode ORFans.⁹⁸ However, they do encode a few putative carbohydrate enzymes: P. salinus, the prototype of clade A encodes a glucosamine transferase and the incomplete domain of a 6-phospho-glucosidase, while P. neoecaledonia, a representative of the B clade, has only a partial domain of a glucosamine transferase. Interestingly, the glucosamine transferase is encoded in all of the other sequenced pandoraviruses, together with one or two other glyco-related genes, as glycosidases or hydrolases.

The virions are amphora shaped, about 1 µm long and 500 nm wide, with an ostiole-like depression closed by a plug-like structure at one apex (Figure 1d). There is at least one lipidic membrane lining a thick tegument made of three layers, including one made of cellulose.⁹⁹

The infectious cycle starts, like all amoeba infecting viruses, by phagocytosis of the particles followed by opening of the plugged depression to allow fusion of the internal membrane with the phagosome membrane, which allows transfer of the genome and necessary proteins into the host cytoplasm. However, unlike many NCLDV members, pandoraviruses build their VF in the host cell nucleus. They encode an RNA polymerase that is not packaged in the capsids, and so they rely on the host cell for their early transcription until the virally encoded enzyme is translated. New virions start to assemble from the apex, and the neosynthesized virions are released either by exocytosis through membrane fusion with the plasma membrane when they are in vacuoles or by cell lysis.⁶,⁹²

## 2.6. Family Phycodnaviridae

The family *Phycodnaviridae* is in the order of *Algavirales*, which includes many other viruses that infect eukaryotic algae. The phycodnaviruses consist of a large genetically and morphologically diverse group of viruses with eukaryotic algal hosts from both inland and marine waters. The six genera in this family include *Chlorovirus*, *Coccolithovirus*, *Prasinovirus*, *Prymnesiovirus*, *Phaeovirus*, and *Raphidovirus*, all with genomes that range

in size from ~160 kb to ~450 kb, with some exceptions for viruses tentatively assigned to the genus *Prymnesiosvirus*. All six of these genera are reported by gene annotation to have enzymes involved in various aspects of carbohydrate metabolism,¹⁰⁰,¹⁰¹ although experimental proofs that these genes encode functional enzymes have only been provided for a few members of the *Chloroviruses*.

Moreover, the *Phycodnaviridae* family includes many viruses that are currently not classified into any of the genera mentioned above. Indeed, the taxonomical position of *Chrysochromulina ericina* and *parva* viruses (CeV and CpV, respectively),⁵⁴,⁶⁶,¹⁰² *Aureococcus anaphagefferens* virus (AaV),¹⁰² and *Tetraselmis virus* (TetV),¹⁰³ is currently being debated, and the alignment of their genome sequences along with those of recognized members of the *Prymnesiosvirus* genus, suggests that all these viruses should be placed in a new subfamily, called *Mesomimivirinae*, belonging to the *Mimiviridae* family.⁶⁶ In this review, all these viruses will be discussed together with the other *Prymnesiosviruses*.

### 2.6.1. Genus Chlorovirus.
As elaborated later on in this review, the chloroviruses provide the most information about virus-encoded genes involved in carbohydrate metabolism. Chloroviruses are plaque-forming, large icosahedral (190 nm in diameter), dsDNA-containing viruses (genomes of 290–370 kb) with an internal lipid-containing membrane (Figure 1a).¹⁰⁴ They exist in inland waters throughout the world, with titers occasionally reaching thousands of plaque-forming units (PFU) per mL of indigenous water. Known chlorovirus hosts, which are normally endosymbionts and are often referred to as zoolchlorellae, are associated either with the protozoan *Paramecium bursaria*, the coelenterate *Hydra viridis*, or the heliozoan *Acanthocystis tur facea*.¹⁰¹

Chloroviruses are divided in groups depending on their host specificity; viruses that replicate in *Chlorella variabilis* NC64A are referred to as NC64A viruses, viruses that infect (or replicate in) *Chlorella variabilis* Syngen 2–3 are referred to as Osy viruses, viruses that replicate in *Chlorella heliozoae* SAG 3.83 are referred to as SAG viruses, and viruses that replicate in *Micractinium conductrix* Pbi are referred to as Pbi viruses. The zoolchlorellae are resistant to virus infection when they are in their symbiotic relationships because the viruses have no way of reaching their host.

The genomes of 43 chloroviruses infecting these four hosts have been sequenced, assembled, and annotated.¹⁰⁵ Collectively, the viruses encode 1345 predicted protein families; however, any given chlorovirus only has 330–410 protein-encoding genes (PEGs). Thus, the genetic diversity among these viruses is large, and many of the proteins are unexpected for a virus. Except for homologues in other chlorovirus members, about 66% of their PEGs do not match anything in the databases.

The prototype of this genus, *Paramecium bursaria* chlorella virus type 1 (PBCV-1, Figure 1a), is an NC64A virus. PBCV-1 has an icosahedral capsid with a spike-like structure at one vertex and a few external fibers that extend from some of the capsomers.¹⁰⁶,¹⁰⁷ The outer capsid layer covers a single lipid bilayered membrane, which is essential for infection. The PBCV-1 MCP (named Vp54, coded by gene *a430l*) is a glycoprotein, and three Vp54s form the trimeric capsomere, which has pseudo-6-fold symmetry (section 3.2.1).¹⁰⁸ A proteomic analysis of highly purified PBCV-1 virions revealed that the virus contains 148 virus-encoded proteins and at least one host-encoded protein.¹⁰⁹
The PBCV-1 genome is a linear ~331 kb, nonpermuted dsDNA molecule with covalently closed hairpin termini. Identical ~2.2 kb inverted repeats flank each 35-nucleotide-long, incompletely base-paired, covalently closed hairpin loop.<sup>110</sup> The remainder of the PBCV-1 genome contains primarily single-copy DNA that encodes ~410 putative proteins and 11 tRNAs.<sup>109</sup> The G+C content of the PBCV-1 genome is 40%; in contrast, its host nuclear genome is 67% G +C. PBCV-1, and the other chlorovirus genomes contain methylated bases, which occur in specific DNA sequences. The methylated bases are part of chlorovirus-encoded DNA restriction and modification systems.<sup>111</sup>

As elaborated on below, the chloroviruses were the first viruses reported to code for sugar-nucleotide synthesizing enzymes (section 3.1.1), extracellular polysaccharide enzymes that make hyaluronan and chitin (section 3.3.1), and the GTs involved in the synthesis of the glycans attached to the major capsid glycoproteins of the virions (section 3.3.2).<sup>13</sup>

**2.6.2. Genus Coccolithovirus.** This viral genus includes only one species, *Emiliania huxleyi* virus (EhV), an enveloped virus of about 250 nm in size,<sup>112</sup> further divided into different strains all able to infect unicellular algae.<sup>38</sup> Of note, the alga *E. huxleyi* is known for its massive seasonal blooms that generally collapse within 5–8 days, and several studies have shown that bloom termination occurs upon viral infection, thus inferring a relevant role to this type of virus in maintaining the balance of the ecosystem.

Regarding the features of EhV, the strain EhV-86 is the one used as the reference,<sup>113</sup> and it has a circular dsDNA genome of 407 kb length, with a 40.2% G+C nucleotide composition, encoding 472 putative proteins, with about 80% annotated as hypothetical proteins. As for the others with an assigned function, there were five tRNAs, six RNA polymerases, eight proteases, as well as at least four genes that encode proteins involved in sphingolipid biosynthesis, along with four glyco-related genes: two GTs and two GHs.

**2.6.3. Genus Phaeovirus.** Viruses belonging to this genus have an icosahedral capsid of 120–150 nm, with linear dsDNA genomes of 150–350 kb in length. Phaeoviruses differ from the other genera in this family. First, phaeoviruses do not replicate in a unicellular algal host, but in filamentous brown algae, including members of the *Ectocarpus* and *Feldmannia* genera, and second, they are the only giant viruses whose replication occurs via a lysogenic infection cycle instead of the classical lytic mechanism. The phaeoviruses life cycle starts by infecting the free-swimming, wall-less gametes or spores of their hosts before they develop into mature algae. Hence, the viral DNA is first integrated into the spore genome, where it is replicated together with the host DNA and transmitted to the mature algae, where it is later expressed in the nuclei of the reproductive organs. At this stage, the host nucleus becomes disrupted, and the viral assembly continues in the cell until it is densely packed with viral particles. The new virions are then released by cell lysis into the surrounding water, and they are ready to start a new infection cycle.

Collectively, this genus consists of nine species, each of which can infect several algal isolates. The genome of only two phaeovirus species, *Feldmannia* and *Ectocarpus*, have been completely sequenced. Of these two species, only *Ectocarpus siliculosus* virus 1, which codes for about 230 proteins, has genes related to carbohydrate metabolism. Three such genes are predicted to encode a alginate epimerase, a sugar nucleotide dehydrogenase, and a GT.<sup>114</sup>

**2.6.4. Genus Prasinovirus.** Viruses assigned to this genus infect marine prasinophytes (a group of unicellular green algae) from three genera in the classes *Mamielloophyceae: Bathycoccus, Micromonas*, and *Ostreococcus*. Members of the *Ostreococcus* genus are the world's smallest free-living eukaryotes. With a small host size (less than 1 µm in diameter for *Ostreococcus*) and a virus capsid size of around 120 nm, it is estimated that there is physically room for no more than 100 virions at any one time. This is reflected in experimental data that suggest a typical burst size, i.e., number of infectious viral particles produced from one infected host cell, of 6–15 viruses per cell.<sup>115</sup>

Prasinoviruses are ubiquitous in seawater throughout the world and genomic sequences from inland waters suggest that some of them may also infect freshwater algae. Their genomes range in size from 180 to 215 kb, with a G+C content range from 35 to 50%, encoding 203–269 compactly arranged proteins (with an average intergenic distance of about 40 bp), 4–11 tRNAs, and terminal inverted repeats of 250–2150 bp. Prasinovirus particles are icosahedral, with diameters of 100–130 nm. Many of the prasinoviruses are sensitive to chloroform, which suggests that they have a lipid-containing membrane; however, others do not loose infectivity after exposure to chloroform.

Following viral infection, genome replication begins about 2 h PI, virions assemble in the cytoplasm from 6 to 20 h PI, after which cellular lysis occurs. The host cell nucleus, mitochondria and chloroplast remain intact throughout this period. According to the ICTV classification,<sup>15</sup> this genus includes two species, *Micromonas pusilla* virus and *Ostreococcus tauri* virus, although many others have been proposed to belong to this genus. In fact, more than 30 of these viruses have been completely sequenced.<sup>38</sup> They have clusters of genes predicted to code for the synthesis of sugar nucleotides and many GTs, suggesting the presence of complex glycosylation pathways in these viruses.<sup>73,116,117</sup> However, no one has investigated this aspect of their life cycle. In fact, it is not even known if their MCPs are glycosylated, although it is very likely they are.

**2.6.5. Genus Prymnesiovirus.** Members in this genus, such as *Chrysochromulina brevifilum* virus (CpV) and *Phaeocystis globosa* virus (PgV),<sup>52</sup> have all been isolated from marine environments where they infect unicellular eukaryotic algae. They have icosahedral capsids, which are about 150–400 nm in diameter, and dsDNA linear AT-rich genomes of at least 370 kb and up to 1.42 Mb for *Prymnesium kappa* virus RFO1 (PkV-RFO1).<sup>64</sup> Up to 45% of the viral encoded genes are ORFans and encode UV-DNA repair machinery and two copies of the RNA polymerase subunit 2. The VF is built in the cytoplasm, but it is unknown if the transcription machinery is packaged in the capsids, leading to an entirely cytoplasmic infectious cycle. They can encode a functional rhodopsin,<sup>52,118–122</sup> proteins involved in energy production, and up to two aaRSs.<sup>64,68,122</sup> Virophages have been isolated in association with CpV<sup>54</sup> and PgV,<sup>52</sup> and several other virophages were identified in metagenomic data with several members of this genus.<sup>123–127</sup> PkV-RFO1 has the largest genome and appears distantly related to other members of this subgroup with 40% ORFans. The PkV genome is enriched in putative genes encoding lipid metabolism and membrane biogenesis, carbohydrate transport, and metabolism and energy production and conservation. Interestingly, PkV RF01 encodes 48 putative GTs, the highest number so far
predicted in NCLDV, in addition to seven GHs, four carbohydrate esterases, and one PL.⁶⁴

As for the other members in this genus, based on genome annotation, CeV⁵⁴ encodes for 13 GTs, while AaV¹⁰² codes for sugar degradation and sugar binding proteins, and 10 GTs, while TetV¹⁰³ possesses a saccharide degradation enzyme α-galactosidase, a mannitol metabolism enzyme mannitol 1-phosphate dehydrogenase, and two key genes in algal fermentation pathways: pyruvate formate-lyase and pyruvate formate-lyase activating enzyme.¹⁰³ The presence of these latter two genes suggests that TetV has the unprecedented capacity to manipulate its host's fermentation pathway, which has intriguing implications for the ecology of the virus, such as its potential to spread in hypoxic/anoxic environments and/or the ocean biochemistry given that TetV infections may alter the production of fermentation products such as ethanol.¹⁰³

**2.6.6. Genus Raphidovirus.** Like Coccolithovirus, this genus includes only one species, *Heterosigma akashiwo* virus (HaV), named for its host, *Heterosigma akashiwo*,¹²⁸ a marine microalgae responsible for harmful algal blooms, and for this reason named with the equivalent of the Japanese word *akashivo*, "red tides". The HaV particle is icosahedral, with a diameter of about 200 nm, with a linear dsDNA of about 275 kb encoding about 250 proteins. HaV does not encode a DNA-directed RNA polymerase or polyA RNA polymerase, indicating that this virus depends entirely on the host's transcription machinery that it manipulates by using its own transcription initiation factors, mRNA capping enzyme subunits, and ribonuclease III.¹²⁹ Regarding the presence of glycogenes, HaV-1 encodes for four putative GTs, in addition to a putative GDP-Man dehydratase and an epimerase, that taken together collectively demonstrate that this virus has the ability to manipulate sugars at different levels.

**2.7. Proposed Pithocedratviridae Family**

Since the isolation of the first Pithovirus from a 30 000 years old permafrost sample,⁵ many additional members have been isolated and can be grouped into two distantly related genera *Pithovirus*,¹³⁰ and *Cedratvirus*,⁵⁵,¹³¹,¹³² here discussed together. These viruses have large amphora shaped capsids that can be up to 2 µm long and 600 nm wide. They are closed by one cork for Pithovirus (Figure 1i)⁵,¹³⁰ and two corks for Cedratvirus (Figure 1l),⁸⁵ made by proteins organized in a honeycomb array. Even though the virion's overall morphology resembles that of pandoraviruses (Figure 1d), the external tegument is different because they do not have cellulose and the tegument appears to be made of parallel strips, coated with short sparse fibrils.⁵,¹³³ The pithovirus and cedratvirus AT-rich genomes are circular or circularly permutated and ~610 kb and ~460–589 kb, respectively. Of note, members of Pithovirus and *Cedratvirus* genera have been defined as "empty vessels" because despite being the largest giant viruses in terms of virion size (Figures 1d,f), their genomes are smaller compared to those of other giant viruses such as pandoraviruses (2.5 Mb, amphora-shaped virion of 1000 nm × 500 nm) and Yasminevirus (2.1 Mb, icosahedron capsid of 330 nm, Table 1).

*Pithovirus* and *Cedratvirus* share 80% of their genes and encode up to 450 proteins, including some putative carbohydrate active enzymes.⁸⁵ For example, the Brazilian cedratvirus IHUMI strain IHUMI-277 encodes seven putative enzymes involved in carbohydrate metabolism, including three GTs and a protein with a rhamnan synthesis domain. Similarly,

Cedratvirus A11 encodes for five GTs, with one predicted as a N-acetylglucosaminyltransferase, a dTDP-D-Glc 4,6-dehydratase, and a NAD dependent epimerase/dehydratase. Pithovirus sibericum, the only species completely sequenced in the genus,³⁸ encodes four putative GTs, along with a NAD-dependent epimerase/dehydratase.

The infectious cycle of these two proposed virus genera proceeds like other amoeba infecting viruses, namely by phagocytosis followed by capsid opening and membrane fusion with the phagosome.⁴⁷ For the pithoviruses and cedratviruses, the viral encoded RNA polymerase is packaged in the virion, which initiates early transcription in the cytoplasm and the host nucleus remains intact throughout the replication cycle. The virions are assembled starting from one apex of the future virion, leading to a rectangular uncoated virion, then the tegument is built by patches from a reservoir in the cytoplasm and the virus morphology changes to amphora-shaped. The neosynthesized virions can exit by exocytosis and by cell lysis.⁵,⁸⁵

**3. BIOSYNTHETIC MACHINERY AND THE GLYCANS PRODUCED BY GIANT VIRUSES**

Before the discovery of giant viruses, the presence of glycan biosynthetic enzymes in the viral world was limited to a few eukaryotic viruses that encode for GTs as a strategy to increase their survival and propagation rate.¹² The first report dates back to 1960, with the discovery that bacteriophages T2, T4, and T6 of *Escherichia coli* encode a GT able to transfer a Glc residue to the hydroxymethylcytosines of their DNA to protect it from digestion by host restriction endonucleases.¹² Baculoviruses infecting *Lepidoptera* larvae are another example: these viruses express a GT that attaches Glc to the larval hormone ecdysone. Glucosylation inactivates the hormone, leading to the larva remaining in the pupation phase. This strategy allows baculoviruses to complete their infection cycle.¹² Bovine herpesvirus and Mixoma virus are two other examples; the former encodes a β-(1→6)-GlcNAc transferase,¹² while the latter encodes a α-(2→3)-sialyltransferase.¹³⁴ The viruses mentioned above were considered to be exceptions to the rule that viruses infecting eukaryotes exploit the host machinery to produce glycans.⁸ However, this belief must be reconsidered as it does not apply to giant viruses.

Some giant viruses are predicted to encode most, if not all, of the machinery required for the glycosylation of their structural proteins.¹⁴ However, we only have hard evidence to support this claim for a handful of systems (Table 2). As noted in the first part of this review, giant viruses encode many putative glyco-related genes, including those responsible for the synthesis of sugar nucleotides, sugars modification, and sugar assembly, along with other sugar or polysaccharide manipulating enzymes. Currently, the most studied carbohydrate manipulating enzymes are from chloroviruses and mimiviruses (Table 2), where the function of some of their glycogenes and the structure of their glycans have been determined. Accordingly, this section will describe first the works that demonstrate that these viruses do indeed encode proteins used in the production of their nucleotide sugars, then it will describe the structures of their glycans, and finally the GTs involved in the process. The last part of this section will examine other viral enzymes that instead of acting on the viral glycans seem to play a role in remodeling the host glycans.
<figure><img src="image_9.png" /><figcaption>**Figure 2.** Monosaccharides diversity in giant viruses. Structure of the monosaccharides detected in the glycans of giant viruses or synthesized by them as discussed in this review (the corresponding sections are enclosed in brackets). All monosaccharide units are in the pyranose form, unless explicitly indicated ("f" indicates the furanose form), and α configured at the anomeric center. Each structure is accompanied by the abbreviated name of the residue and by its symbol as defined by the rules of the Symbol Nomenclature of Glycans.<sup>156</sup></figcaption></figure>

## 3.1. Biosynthesis of Sugar Nucleotides

In 1997, chlorovirus PBCV-1 was the first virus reported to encode enzymes involved in the synthesis of sugar nucleotides and polysaccharides.<sup>11</sup> Indeed, PBCV-1 codes for a hyaluronate synthase (section 3.3.1) and for the two enzymes involved in the synthesis of the monosaccharides that comprise the polysaccharide (section 3.1.1, Table 2). The glycan biosynthetic ability of PBCV-1 was again recognized in 2003, when the pathways the virus uses to synthesize GDP-β-L-Fuc and GDP-α-D-Rha were identified and experimentally validated (section 3.1.1.1, Table 2).<sup>148</sup> This work advanced the hypothesis that PBCV-1 encoded a glycosylation machinery that was, at least in part, independent of the host. The pathways for other activated sugars that comprise the N-linked glycans of the PBCV-1 major capsid protein, Vp54 (L-Ara, D-Gal, D-Glc, D-Man, L-Rha, D-Xyl), are not present; we assume that the virus uses the sugar nucleotides produced from the host to build these glycans.

Years later, the biosynthetic pathways for sugar nucleotides were identified and experimentally verified for some members of the genus *Mimivirus* (section 3.1.2, Table 2).<sup>58,60–62,154</sup> Interestingly, *Mimivirus* clusters the genes encoding the sugar nucleotides enzymes (section 3.1.2.5) and these clusters are clade specific, while chloroviruses do not have this gene organization.

Finally, in silico analyses suggest that CroV and Fadolivirus encode the Kdo biosynthetic pathway, described later in this

review although not supported by experimental evidence due to the relevance that this monosaccharide has for plants and bacteria (section 3.1.2.4).<sup>59</sup>

The type of monosaccharides found in giant viruses, either involved in the construction of the glycans or for which the biosynthetic pathway has been elucidated, are summarized in Figure 2, along with the section(s) where they are discussed.

### 3.1.1. Chloroviruses.
3.1.1.1. GDP-β-L-Fuc and GDP-α-D-Rha Biosynthesis in PBCV-1.
Chlorovirus PBCV-1 encodes two enzymes, A118R and A295L, which are identified as a GDP-α-D-Man-4,6-dehydratase (GMD) and GDP-6-deoxy-4-keto-α-D-Man epimerase/reductase (GMER). These proteins are involved in the biosynthesis of GDP-α-D-Rha and GDP-β-L-Fuc, respectively (Figure 3).<sup>148</sup> The A118R protein (345 aa) catalyzes the two-step dehydration of GDP-α-D-Man at both C-4 and C-6 carbons, producing the GDP-6-deoxy-4-keto-α-D-Man product as an intermediate (Figure 3a), which is then used by the GMER enzyme, A295L (317 aa), to make GDP-β-L-Fuc (Figure 3b). Both A118R and A295L proteins are similar to those found in bacteria and eukaryotes (Table 2),<sup>157,158</sup> although the viral A118R has an additional NADPH-dependent activity.<sup>148</sup> Indeed, when the recombinant A118R was incubated with GDP-α-D-Man and both NADP<sup>+</sup> and NADPH, instead of NADP<sup>+</sup> alone, it produced GDP-α-D-Rha (Figure 3a), indicating that PBCV-1 GMD has an additional activity that occurs only when additional NADPH is present (Figure 3a). This double function is rare, and it has only been
<figure><img src="image_10.png" /><figcaption>**Figure 3.** Biosynthesis of sugar nucleotides encoded by chlorovirus PBCV-1. GDP-α-D-Rha and GDP-β-L-Fuc are produced by the chlorovirus PBCV-1 by using GDP-α-D-Man from the host. The PBCV-1 genome encodes both enzymes necessary to produce these two sugar nucleotides. A118R has the GMD activity, and A295L is the GMER protein. (a) Transformation of GDP-α-D-Man into GDP-α-D-Rha catalyzed by A118R. This enzyme is unique in its class because it has two activities.<sup>148</sup> In the first step, A118R transforms GDP-Man into its 6-deoxy-4-keto derivative. This transformation requires NADP<sup>+</sup> cofactor that is regenerated upon formation of the 6-deoxy-4-keto sugar nucleotide intermediate. This molecule is reduced by A118R in the presence of NADPH to give GDP-α-D-Rha. The A118R crystallographic structure is reported in the left lower part of the panel. Reproduced with permission from ref 160. Copyright 2006 Elsevier. A118R has a Rossman fold, and it crystallizes as a tetramer with NADP<sup>+</sup> (or NADPH) cofactors (red), promoting the association between two dimers. (b) Transformation of GDP-6-deoxy-4-keto-α-D-Man produced by A118R into GDP-β-L-Fuc by the NADPH-dependent reaction with A295L, a GDP-6-deoxy-4-keto-D-Man epimerase/reductase (GMER). The activities of the enzymes are indicated in parentheses.</figcaption></figure>

reported in *Aneurinibacillus thermoarophilus*,<sup>159</sup> whose recombi-
nant protein produces only a small amount of GDP-α-D-Rha,
suggesting that this process may not occur in vivo.

The crystal structure of A118R was solved at 3.8 Å
resolution (Figure 3a), and this protein belongs to the short-
chain dehydrogenase/reductase (SDR) superfamily, a group of
proteins involved in sugar nucleotide biosynthesis. PBCV-1
GMD consists of two domains: an N-terminal domain binding
the NADP(H) cofactor and a smaller C-terminal domain
containing the sequence Gly-X-X-Gly-X-X-Gly typical of the
SDR protein family, responsible for the binding of the sugar
nucleotide substrate.<sup>160</sup> The N-terminal domain has a modified
Rossman fold structure consisting of a seven-stranded parallel
β-sheet sandwiched between six α-helices, that create the
binding pocket used to accommodate NADP(H) next to the
residues <sup>125</sup>Thr, <sup>149</sup>Tyr, <sup>153</sup>Lys, and the catalytic triad
responsible for the activity of the enzyme. A118R can complex
both NADP<sup>+</sup> and NADPH, although the interaction with
NADPH is stronger and is essential to stabilize the protein
structure as a dimer. In contrast, A118R interaction with
NADP<sup>+</sup> is weaker, and this cofactor dissociates from the
complex once GDP-α-D-Man is transformed into GDP-6-
deoxy-4-keto-α-D-Man. The result is that the A118R dimer
turns into the monomeric form that is enzymatically
inactive.<sup>160</sup> NADPH maintains GMD in its dimeric form,
and it enables the protein to transform the GDP-6-deoxy-4-

keto-α-D-Man intermediate into GDP-α-D-Rha.<sup>135</sup> As for the
quaternary structure (Figure 3a), A118R exists in solution as a
dimer like other SDRs; however, two GMD dimers associate to
give a tetrameric structure through crystallographic symmetry
(Figure 3a).<sup>160</sup>

Hence, PBCV-1 encodes the genes necessary to synthesize
the activated precursor for two of the sugars necessary to
synthesize the N-linked glycans attached to its major capsid
proteins (section 3.2.1), L-Fuc and D-Rha. In this process, the
virus relies on the host for the GDP-α-D-Man substrate. Similar
genes encoding putative GMD and GMER enzymes, named
Z804L and Z282L, respectively, are also found in Acantho-
cystis turfacea chlorovirus 1 (ATCV-1).<sup>135</sup> Phylogenetic
analyses indicated that PBCV-1 and ATCV-1 GMERs were
more similar to one another than to 20 other taxa from all
forms of life and one virus.<sup>135</sup>

Regarding the similarities in GMD between the two
chloroviruses, the sequences from ATCV-1 and PBCV-1 only
had 53% identity, suggesting that the two enzymes diverged
early during viral evolution and that they might have different
properties. Indeed, ATCV-1 GMD biochemical character-
ization confirmed the expected dehydratase activity, but the
protein lacked the reductase activity characteristic of PBCV-1
GMD.<sup>135</sup> Moreover, by analyzing the cofactor content of the
two GMD proteins when expressed in E. coli, ATCV-1 GMD
had a high affinity for NADP<sup>+</sup> rather than NADPH, as reported
<figure><img src="image_11.png" /><figcaption>**Figure 4.** Biosynthetic pathway for UDP-β-L-Rha and UDP-α-D-Vio4NAc. (a) Comparison of NDP-β-L-Rha biosynthesis in bacteria and plants/viruses. The two pathways differ by the nucleotide used and the number of enzymes involved: three for bacteria and two in APMV. Plants have only one enzyme because UGD and UGER are part of the same protein. (b) Reaction of APMV R141 (or ATCV-1 Z544R) with UDP-α-D-Glc to give UDP-4-keto-α-D-Glc; this substrate is used by two APMV proteins L780 and L136 to synthesize UDP-β-L-Rha and UDP-α-D-Vio4N, respectively. This last product is N-acetylated by N-L142, leading to the formation of UDP-α-D-Vio4NAc. (c) Crystallographic structures of: L136 (PDB 7MFO) in complex with TDP-Vio4N linked as an external aldimine, R141 (PDB 6VL0) in complex with TDP and NAD, and L780 (PDB 7JID) with UDP-β-L-Rha bound; α-helices are colored in red, β-strands in cyan, and loops in gray. The N-L142 model was obtained by homology modeling using Neisseria gonorrhoeae PglB enzyme as a reference. The activities of the enzymes are indicated as acronyms in brackets.</figcaption></figure>

for the PBCV-1 enzyme. ATCV-1 GMD had both NADP⁺ and NADPH in variable ratios, while PBCV-1 protein only had NADPH.

### 3.1.1.2. Biosynthesis of the UDP-β-L-Rha Precursor in ATCV-1.
Analysis of the ATCV-1 genome identified the Z544R protein as homologous to UDP-α-D-Glc 4,6-dehydratase (UGD, Table 2),⁵⁸ the first enzyme involved in the production of UDP-β-L-Rha, according to the plant biosynthetic pathway (Figure 4a). Indeed, the ATCV-1 UGD protein preferred UDP-α-D-Glc as a substrate over dTDP-α-D-Glc and converted it to UDP-6-deoxy-4-keto-α-D-Glc, the intermediate transformed into UDP-β-L-Rha by the second enzyme in the pathway, an epimerase and reductase termed UGER (Figure 4a).¹⁶¹

ATCV-1 does not encode the second enzyme (UGER) needed to complete the UDP-β-L-Rha synthesis; therefore, the current hypothesis is that the virus encodes its own UGD enzyme to increase Rha production during infection that is brought to completion by using the host encoded UGER

enzyme. Furthermore, Z544R is expressed as a late gene product during virus replication. This could be due to the fact that high UGD activity during the initial phases of virus replication is avoided to prevent a reduction of UDP-α-D-Glc availability because this sugar nucleotide serves both as a donor for glucosyltransferases and as a precursor for other sugar nucleotides.⁵⁸

Biochemical characterization of Z544R demonstrated that the protein was cation independent and that its molecular weight was unaffected by the addition of NAD or NADP, in either oxidized or reduced forms, suggesting that the recombinant chlorovirus protein strongly bound the coenzyme (NAD⁺) in the binding pocket when expressed in *E. coli*.⁵⁸ ATCV-1 UGD associated as a dimer, and it had a specific activity of 23 nmol/min/mg of protein and a K_m for UDP-α-D-Glc of 18.4 ± 3.3 µM at the pH optimum of 7.5. Finally, incubation of the ATCV-1 UGD enzyme at temperatures higher than 20 °C resulted in a progressive loss of activity, and
its complete inactivation occurred at 30 °C in 30 min, indicating that the enzyme is thermolabile.⁵⁸

**3.1.2. Megavirinae.** Looking at the distribution of the glycogenes in the genomes of giant viruses, it appears that *Chlorovirus* and *Mimivirus* genera have a different organization. In PBCV-1, the prototype of the *Chloroviruses*, the glycogenes are mostly scattered throughout the genome, with a majority in the first 100 ORFs, making it difficult to define a specific region responsible for the glycosylation of the viral capsid protein. In contrast, glycogenes in the *Megavirinae* subfamily occur in specific regions of the genome, defining complex clusters of six to as many as 33 genes.⁸⁷ The next sections will describe the sugar nucleotide biosynthetic pathways that have been experimentally validated for *Megavirinae*, together with a few inferred from in silico analysis.

**3.1.2.1. APMV Production of UDP-β-L-Rha and UDP-α-D-Vio4NAc.** Unlike the chlorovirus ATCV-1, the mimivirus APMV encodes both the UGD and UGER enzymes necessary for UDP-β-L-Rha production (Figure 4b, Table 2).⁵⁸ Bioinformatic analysis of the APMV genome identified R141 as a UGD enzyme, and biochemical assays performed on the recombinant protein expressed in *E. coli* confirmed this prediction.⁵⁸

Compared to ATCV-1 UGD (Z544R, section 3.1.1.1), the binding of the cofactor NADH to R141 appeared less efficient. Indeed, recombinant Z544R copurified with its NADH cofactor, as demonstrated by the fact that it was insensitive to its addition in solution. Recombinant R141 instead did not copurify with the cofactor, as demonstrated by measuring its MW by size exclusion chromatography. With no addition of NADH or NAD⁺, the protein produced three forms at different MWs. However, upon incubation with either NADH or NAD⁺, the lower MW peaks were converted into the major one at ~100 kDa. Moreover, this experiment established that the protein had a 10-fold better affinity for NADH compared to NAD⁺, and none for NADP⁺ or NADPH. Notably, the apparent MW of 100 kDa exceeded that expected for the dimeric form of the enzyme (2 × 37 kDa), and thus it appears to adopt a higher degree of oligomerization, which was not determined.

Regarding its activity, R141 had maximal activity at pH between 7.5 and 8.5, and it required 100 µM NAD⁺ to transform UDP-α-D-Glc into UDP-6-deoxy-4-keto-α-D-Glc, with a specific activity of 4.2 nmol/min/mg of protein; a 60% feedback inhibition occurred with UDP-β-L-Rha.⁵⁸ Finally, R141 was cation independent, and loss of activity occurred at 30 °C in 30 min, like the analogous enzyme from ATCV-1.

As for UGER, the second enzyme in the UDP-β-L-Rha pathway, the APMV protein L780 had such activity; it had 40% amino acid identity with the C-terminal region of *Arabidopsis thaliana* RHM2 protein, the plant enzyme with the epimerase/reductase activity required to transform UDP-6-deoxy-4-keto-α-D-Glc produced from UGD into UDP-β-L-Rha. Biochemical studies on the recombinant APMV L780 protein revealed that (1) it needed NADPH (and not NADH) as a cofactor with a specific activity for UDP-6-deoxy-4-keto-α-D-Glc of 11 ± 2.5 nmol/min/mg and a $K_m$ of 183 ± 51 µM; (2) it had maximum activity at pH 7.5, (3) it was cation independent, and (4) it did not lose activity after 30 min incubation at 42 °C. Size exclusion chromatography indicated that L780 MW was 47 kDa, a value that did not fit with the monomeric form of the

protein (33 kDa) or its dimer, leaving this as an open point for further studies.

Recent studies have indicated that both R141 and L780 can use either UDP or dTDP-sugars as substrates, in contrast to their bacterial counterparts that specifically require dTDP-sugars. R141 can use either UDP-α-D-Glc or dTDP-α-D-Glc with the same catalytic efficiency;¹⁵² similarly, L780 used both UDP- and dTDP-6-deoxy-4-keto-α-D-Glc nucleotides, albeit it had a higher catalytic efficiency with the UDP-linked substrate.¹⁵⁵

Both R141 (PDB 6VLO) and L780 (PDB 7JID) belong to the SDR superfamily, and their structures have been defined by crystallographic studies (Figure 4c).¹⁵²,¹⁵⁵ In R141, the residues typically involved in the 4,6-dehydratation reaction are conserved and correspond to ¹²⁵Asp, ¹²⁶Gly, and ¹⁴⁹Tyr. The overall fold of R141 is comparable to that of the corresponding bacterial enzymes, with some differences: the catalytic ¹⁴⁹Tyr is ~12 Å away from the catalytic site, suggesting that the protein undergoes a large conformational change during substrate binding, which is unusual for SDR proteins.

The L780 crystallographic structure had a good match with the *A. thaliana* RHM2 protein, in agreement with the fact that the viral enzyme is bifunctional and has both 3,5-epimerase and 4-reductase activities, different from those from bacteria and fungi, where the same transformation requires two different enzymes. Concerning the catalytic mechanism of L780, comparison of this enzyme with a different sugar nucleotide synthase, the GDP-β-L-fucose synthase from *E. coli*,¹⁶² led to the identification of ¹⁰⁸Cys and ¹⁷⁵Lys as key catalytic residues, as also confirmed by mutagenesis experiments.¹⁵⁵

The UDP-β-L-Rha and the UDP-α-D-Vio4NAc pathways are interconnected;⁵⁸,⁶² the intermediate UDP-6-deoxy-4-keto-α-D-glucose produced by R141 and used by L780 to make UDP-β-L-Rha, is also used by L136, a pyridoxal phosphate (PLP) dependent-aminotransferase or transaminase (Table 2), to synthesize UDP-α-D-Vio4N (Figure 4b). The last enzyme in the UDP-α-D-Vio4NAc pathway, is an N-acetyl transferase corresponding to the N-terminal domain of L142 (N-L142), a protein with two domains (Table 2).⁶⁰

The two enzymes involved in the UDP-α-D-Vio4NAc biosynthetic pathway, L136 and N-L142, were characterized biochemically, and the three-dimensional structure of L136 was determined by X-ray crystallography (PDB 7MFQ),¹⁵¹ while the N-L142 structure was derived by homology modeling studies using the *Neisseria gonorrhoeae* PglB enzyme as a reference, a transferase able to acetylate the amino function at C-4 of bacillosamine.⁶⁰

L136 is dimeric in the crystal structure, and has the same typical type I fold of the superfamily of aminotransferases (Figure 4c).¹⁵¹ L136, like both R141 and L780, accepts both UDP and dTDP-sugars, with similar catalytic efficiencies.¹⁵¹ Recombinant L136 maintains its cofactor, PLP, during all the purification procedures, and it uses glutamate as the amino donor for the transaminase reaction, that is not reversible. Glutamine, the amino acid used as donor in bacterial enzymes,⁶² instead inhibits L136 activity.

The reaction product of L136 is UDP-α-D-Vio4N, that in turn is acetylated on the amino group by L142. The N-terminal domain has the acetyltransferase activity, and it uses acetyl-CoA as the donor of the acetyl group.⁶⁰ N-L142 activity was only tested with UDP-α-D-Vio4N, although we speculate that
<figure><img src="image_12.png" /><figcaption>**Figure 5.** UDP-α-D-GlcNAc biosynthetic pathway. The biosynthesis of UDP-α-D-GlcNAc occurs by different routes, with that from bacteria requiring fewer enzymatic steps. Both pathways start from Fru-6P. Members of the five clades of the *Mimivirus* genus encode all the enzymes in the eukaryotic biosynthetic pattern, except for the GlcNAc-6P to GlcNAc-1P phosphomutase, which is likely encoded by an unknown viral enzyme or by the host (ND: not defined). APMV (prototype of A clade) is the only virus for which the activity of each enzyme (annotated in bold) has been experimentally validated:⁶¹ L619, L316, and R689. L619 and L316 are close to their eukaryotic counterpart, while R689 appears to be of prokaryotic origin. The activities of the enzymes are indicated in parentheses. As for the representatives of the other clades, the genes involved in this pathway have been assigned by in silico analysis,⁸⁷ and they are listed in the following order: clade B, Moumouvirus australiensis (Ma) and Moumouvirus maliensis (Mm); clade C, Megavirus chilensis (Mg); clade D, Tupanvirus deep ocean (L or R depending on the coding sense of the gene); clade E, Cotonvirus japonicus (ORF).</figcaption></figure>

it might also accept a dTDP-nucleotide as found for L136, the previous enzyme in the pathway. N-L142 was predicted to be a homotrimer, with ¹³⁶His playing a key role in the catalysis, as shown by mutagenesis experiments.⁶⁰ In silico analysis of the full length L142, predicted that the second domain (C-L142) was a GT, leading to speculation that it could be involved in the transfer of the viosamine onto its acceptor during the biosynthesis of poly_2, one of the two polysaccharides decorating the fibrils of APMV (section 3.2.2.2).⁶⁰

In conclusion, the existence of UDP-β-L-Rha and UDP-α-D-Vio4NAc biosynthetic pathways seem essential for APMV to build the polysaccharides of its fibrils because these activated sugars are not encoded by the amoeba host. However, it should be noted that the prevalent viosamine form in poly_2 is methylated at O-2, implying the existence of a methyltransferase specific for this sugar. Currently, the identity of such an enzyme has only been predicted (section 3.1.2.5)⁸⁷ and awaits experimental verification.

### 3.1.2.2. APMV UDP-α-D-GlcNAc Pathway.
N-acetyl-D-glucosamine is present in all domains of life. It is essential for the synthesis of the peptidoglycan layer in bacteria,¹⁶³ of the lipid A moiety of lipopolysaccharides in Gram-negative bacteria,¹⁶⁴ for the assembly of glycosaminoglycans in eukaryotes,¹⁶⁵ for the regulation of cellular functions by the reversible O-GlcNAc glycosylation of eukaryotic proteins,¹⁶⁶ and because it is the monosaccharide directly attached to proteins in most of the N-linked glycans.¹⁶⁷

The biosynthesis of D-GlcNAc varies between organisms and the APMV pathway has a combination of prokaryotic and eukaryotic traits. Three of the four enzymes required for UDP-α-D-GlcNAc production were identified and experimentally

characterized in APMV (Table 2).¹⁵⁴ The phospho-mutase that converts GlcNAc-6P into GlcNAc-1P is the only enzyme not encoded in the genome. Therefore, it is possible that either APMV encodes an unidentified enzyme or it uses a host enzyme to complete the synthesis of GlcNAc-1P (Figure 5).

The first enzyme in the pathway is L619 (606 aa), a glutamine-fructose 6-phosphate transaminase (GFAT) that converts the Fru-6P to GlcN-6P, using glutamine as an amine donor (Figure 5). This enzyme has a N-terminal domain with glutaminase activity, while the C-terminal domain is an isomerase. The conversion of Fru-6P to GlcN-6P occurs in two independent steps. The first step involves the catalytic activity of the C-terminal domain that converts Fru-6P into Glc-6P, which in turn, is transformed into GlcN-6P by the transaminase activity located at the N-terminal domain of the enzyme. This enzyme is well conserved among other members of the *Mimivirus* genus; it has 61% identity with a Moumouvirus species GFAT (AEX62494), and 59% identity with Megavirus chilensis Mg745 protein, while it has some limited similarity (~37%) with those from some protista, including its host *Acanthamoeba castellani*. In contrast to the eukaryotic enzyme, the viral enzyme is shorter and lacks an insertion of 40–70 aa between the N- and C-terminal regions, as also happens for the bacterial enzymes.¹⁶⁸ However, L619 does not exhibit a feedback inhibition by the final product UDP-α-D-GlcNAc, a trait common with the bacterial enzymes. Of note, GFAT has also been found in several chloroviruses (section 3.3.1), where this enzyme seems necessary for some chloroviruses to increase the GlcNAc supply required for the biosynthesis of hyaluronic acid and/or chitin during the infection process (section 3.3.1).¹¹,¹⁴⁶
The second reaction in the pathway to synthesize UDP-α-D-GlcNAc is catalyzed by L316 (148 aa), a GlcN-6P N-acetyltransferase (GNAT) that adds an acetyl group from the acetyl-CoA to the free amino group, leading to GlcNAc-6P (Figure 5). L316 sequence aligns with putative acetyl transferases encoded by other *Mimivirinae* members, it has 57% identical residues with a Moumouvirus species GNAT (YP_007354484) and 56% with the Megavirus chilensis Mg590 (Figure 5). Its closest homologues in cellular organisms are the GNAT proteins from *Trichomonas vaginalis* G3 (41% identity, XP_001303745) and from the archaean *Candidatus nitrosopumilus* (50% identity, ZP_1039775). L316 acts on both GlcN-1P and GlcN-6P with a marked preference (5-fold) for GlcN-6P, in agreement with the results reported for GNAT proteins of eukaryotic origin and contrary to bacterial enzymes that only act on GlcN-1P due to a different biosynthetic pathway (Figure 5).¹⁶⁹

The last step in the pathway is the addition of a uridyl moiety from UTP to the phosphorylated sugar to generate UDP-α-D-GlcNAc, carried out by R689 (255 aa). This enzyme is a UDP-α-D-GlcNAc pyrophosphorylase (UAP) that belongs to the GTA-type GT superfamily with some resemblance to the N-terminal domain of *E. coli* GlmU. Its closest homologues are in other members of the *Megavirinae* subfamily, with 66% identity with the Megavirus chilensis Mg240 protein and 68% identity with the Moumouvirus protein (YP_007354151), albeit here the comparison was limited to the 172 amino acids of this smaller protein. The closest homologues to R689 are the N-terminal domains of Cyano- and Proteobacteria GlmU proteins, with 36 and 38% identical residues, respectively. Biochemical assays indicated that R689 had both uridyl-transferase and pyrophosphorylase activities and that it was Mg²⁺ dependent. The highest efficiency was observed when using UTP as donor nucleotide and GlcNAc-1P as the substrate both for the forward and reverse reaction. The reaction occurred with lower efficiency when Glc-1P was used as a substrate or when dTTP and GTP were used as nucleotide donors, while no reaction was observed with ATP and CTP.

The identification of three of the enzymes involved in the UDP-α-D-GlcNAc pathway in APMV was unexpected, as this monosaccharide is already synthesized by the host, contrary to Rha and Vio4NAc (section 3.1.2.1). We hypothesize that APMV synthesizes this sugar nucleotide to be independent of the host supply, especially during the replication cycle when large amounts are required for the synthesis of the glycans that decorate the fibrils (section 3.2.2).

Regarding the strategy adopted by the virus, it should be noted that it follows the eukaryote route,¹⁷⁰ although it uses enzymes that have different origins as denoted by the fact that the closest match to R689 is a bacterial protein, while the other two seem to be of eukaryotic origin.¹⁶⁹ These results lead to an intriguing question on the evolutionary history of this pathway in relation to that of the cellular world.

Homologues to the APMV enzymes used to produce UDP-α-D-GlcNAc have been identified in members of the other clades (Figure 5), during investigation of the clustering of their glycogenes (section 3.1.2.5). Indeed, GFAT is encoded by Moumouvirus australiensis (Ma652), Moumouvirus maliensis (Mm595), Tupanvirus deep ocean (R59), and *Cotonvirus japonicum* (ORF951); GNAT occurs in *Moumouvirus australiensis* (Ma514), Moumouvirus maliensis (Mm467), Tupanvirus deep ocean (L412), and *Cotonvirus japonicum* (ORF781); UAP is found in Moumouvirus australiensis (Ma192),

Moumouvirus maliensis (Mm152), Tupanvirus deep ocean (L1108), and Cotonvirus japonicum (ORF381).

Notably, three of the four genes involved in the UDP-α-D-GlcNAc pathway are conserved in all members of the *Mimivirus* genus. These genes are scattered along the genome and not clustered together as found for those involved in fibril synthesis and their glycosylation in clades A–C or clades D and E, for which experimental data are not available yet (section 3.1.2.5).⁸⁷

#### 3.1.2.3. UDP-6-deoxy-β-L-HexNAc Pathway in Megavirus chilensis
Analysis of the Megavirus chilensis genome revealed the presence of a gene cluster with several ORFs potentially involved in glycosylation, with three of them (*mg534*, *mg535*, and *mg536*) involved in the production of two different sugar nucleotides (Figure 6, Table 2). The protein products of two of them have been characterized (Mg534 and Mg535), and they encode a functional pathway for UDP-β-L-RhaNAc synthesis.⁶¹ Mg536 was predicted to produce UDP-β-L-QuiNAc, and its activity was later demonstrated by the finding of QuiNAc when the monosaccharide composition of the virion was determined (section 3.1.2.5).⁸⁷ Notably, both RhaNAc and QuiNAc were sugars previously found only in the bacterial world, and their production begins with UDP-α-D-GlcNAc, in turn also synthesized by Megavirus chilensis (section 3.1.2.2).

The first enzyme in the UDP-6-deoxy-HexNAc pathway, Mg534 (323 aa), has two catalytic activities. Mg534 first performs the 4,6-dehydration of UDP-α-D-GlcNAc using a NADP⁺ cofactor like UGD-type enzymes, then the enzyme epimerizes C-5 in the intermediate to give UDP-6-deoxy-4-keto-β-L-IdoNAc (Figure 6). The three-dimensional structure of Mg534 (PDB 4TQG) revealed two subdomains, a Rossman-like fold binding NADP and a smaller domain for the substrate binding. The protein is found as a hexamer made of a trimer of dimers, and it belongs to the SDR superfamily, in agreement with the presence of a catalytic triad containing the motif ¹⁴⁰Tyr-X-X-X-¹⁴⁴Lys (with X any amino acid), close to a disordered loop (aa 173–195), and for this reason is not visible in the crystallographic structure.⁶¹

The first reaction product of Mg534, UDP-4-keto-α-D-QuiNAc is epimerized at C-5 to give UDP-6-deoxy-4-keto-β-L-IdoNAc, the substrate of Mg535 (270 aa), a NADPH dependent enzyme that yields UDP-β-L-RhaNAc following a two-step mechanism. First, UDP-6-deoxy-4-keto-β-L-IdoNAc is epimerized at C-3, then the carbonyl function is reduced to produce the final product (Figure 6).

Regarding the Mg535 reaction mechanism, it can be compared to enzymes involved in NDP-β-L-Rha synthesis. In plants and viruses, the RmlD reductase domain and the 3,5 double epimerase are in the same protein, while in bacteria, these are different proteins (Figure 4a). At odds with plant and other giant viruses enzymes such as L780 (section 3.1.2.1), Mg535 maintains the reductase Rmld-like domain and only the C-3 epimerase activity. This enzyme lacks the C-5 epimerase activity as the C-5 stereochemistry is already inverted by the previous enzyme in the pathway, Mg534 (Figure 6). Interestingly, studies on a different sugar-nucleotide manipulating enzyme, a GDP-β-L-Fuc synthase, enabled the identification of the catalytic residue involved in the C-3 epimerization of the intermediate, a cysteine conserved in Mg535, despite the low sequence homology between the two proteins.¹⁶²
<figure><img src="image_13.png" /><figcaption>**Figure 6.** Biosynthetic pathway encoded by Megavirus chilensis to produce two UDP-2,6-dideoxy-β-L-HexNAc. UDP-β-L-RhaNAc is produced by four reactions carried out by two bifunctional enzymes, Mg534 and Mg535, both resembling prokaryotic enzymes. The crystallographic structure of Mg534 is reported and colored according to its secondary structure elements (α-helices in red, β-sheets in cyan, and loops in gray). UDP-β-L-RhaNAc is produced from UDP-β-L-RhaNAc by C-2 epimerization promoted by Mg536. The activities of Mg534 and Mg535 have been experimentally validated, while that of Mg536 was predicted in silico, and it is supported by the monosaccharide analysis of Megavirus chilensis fibrils.<sup>87</sup></figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 7.** Putative Kdo biosynthetic pathway in CroV and Fadolivirus (*Mimiviridae* family). The Kdo pathway follows the same path in bacteria and plants, and in silico analysis suggests that these two giant viruses encode all the enzymes required to produce this monosaccharide. The arrangement of the genes is given for CroV only (lower left part of the figure), and the arrows (in black) indicate the direction of the coding strand.</figcaption></figure>

Regarding Mg536, it is annotated as a UDP-α-D-GlcNAc 2-epimerase, but it also displays 50% and 47% identity, respectively, with WbvD from *Vibrio cholerae* O37,<sup>171</sup> and WbjD from *Pseudomonas aeruginosa*,<sup>172</sup> two proteins involved in C-2 epimerization of 2-acetamido-2,6-dideoxy-L-hexoses. This finding suggested that Mg536 could act on UDP-β-L-RhaNAc produced by Mg535 to transform it into UDP-β-L-QuiNAc (Figure 6). This hypothesis has been confirmed by analyzing the monosaccharide content of the virion,<sup>87</sup> validating the tentative bioinformatic prediction of the original study (section 3.1.2.5).<sup>61</sup>

### 3.1.2.4. Putative Kdo Biosynthetic Pathway in Cafeteria Roenbergensis Virus and Fadolivirus.
Fischer and coauthors have studied the genomic features of CroV, and described a 38

kb gene cluster involved in carbohydrate metabolism.<sup>59</sup> This cluster has 34 ORFs (crov242–275), of which 14 show significant homology with bacterial enzymes; seven are clearly related to carbohydrate metabolism. Interestingly, this cluster is predicted to encode proteins involved in the biosynthesis of Kdo (Figure 7), an eight carbon atoms sugar that is essential for the construction of the LPS in Gram-negative bacteria and which also occurs in plant pectins.<sup>164</sup> The Kdo pathway is well conserved in bacteria and plants (Figure 7).<sup>173,174</sup> First, D-ribulose-5-phosphate (Ru5P) is isomerized to D-arabinose-5-phosphate (A5P) via an isomerase (API). Then a synthase (KdoPS) condenses ASP with phosphoenolpyruvate (PEP) to give Kdo-8P, which is hydrolyzed to free Kdo by a phosphatase (KdoPase). Finally, the free Kdo reacts with CTP to produce
<table><caption>Table 2. Principal Features of the Glyco-Related Enzymes from Giant Viruses Whose Activity Is Based on Experimental Data<sup>a,b</sup></caption><thead><tr><th>genus</th><th>species</th><th>protein</th><th>protein ID</th><th>aa</th><th>domains</th><th>activity</th><th>homology</th><th>PDB</th><th>section</th><th>ref</th></tr></thead><tbody><tr><td rowspan="10">Chlorovirus</td><td rowspan="2">ATCV-1</td><td>Z544R</td><td>YP_001427025</td><td>350</td><td>1</td><td>UGD</td><td>E</td><td></td><td>3.1.1.2</td><td>58</td></tr><tr><td>Z804L</td><td>YP_001427285</td><td>352</td><td>1</td><td>GMD</td><td>B</td><td></td><td>3.1.1.1</td><td>135</td></tr><tr><td rowspan="4">CVK2</td><td>vAL-1</td><td>BAB19127</td><td>349</td><td>1</td><td>PL</td><td>E</td><td>3GNE<sup>c</sup></td><td>3.4.4</td><td>136, 137</td></tr><tr><td>vChta-1</td><td>BAA20342</td><td>328</td><td>1</td><td>GH</td><td>B</td><td></td><td>3.4.1.2</td><td>138</td></tr><tr><td>vChti-1</td><td>BAA78554</td><td>836</td><td>3</td><td>D1: GH<br>D2: GH<br>D3: ND</td><td>B<br>B<br>ND</td><td></td><td>3.4.1.2</td><td>139</td></tr><tr><td>CL2</td><td>BAA83789</td><td>333</td><td>1</td><td>PL</td><td>E</td><td></td><td>3.4.3</td><td>140</td></tr><tr><td rowspan="15">PBCV-1</td><td>A061L</td><td>NP_048409</td><td>209</td><td>1</td><td>methyl-transferase</td><td>B</td><td></td><td>3.3.2.2</td><td>141</td></tr><tr><td rowspan="3">A064R</td><td rowspan="3">NP_048412</td><td rowspan="3">638</td><td rowspan="3">3</td><td>D1: Rha-transferase</td><td>B/E</td><td>1LL2</td><td>3.3.2.2</td><td>141–143</td></tr><tr><td>D2: Rha-transferase</td><td>B</td><td></td><td></td><td></td></tr><tr><td>D3: methyl-transferase</td><td>B</td><td></td><td></td><td></td></tr><tr><td>A094L</td><td>NP_048442</td><td>364</td><td>1</td><td>GH</td><td>B</td><td></td><td>3.4.2</td><td>144</td></tr><tr><td>A098R</td><td>NP_048446</td><td>568</td><td>1</td><td>HAS</td><td>E</td><td>7SP6<sup>c</sup></td><td>3.3.1</td><td>11, 145</td></tr><tr><td>A100R</td><td>NP_048448</td><td>595</td><td>1</td><td>GFAT</td><td>B</td><td></td><td>3.3.1</td><td>146</td></tr><tr><td>A111/114R</td><td>NP_048459</td><td>860</td><td>3</td><td>D1: Gal-transferase<br>D2: Xyl-transferase<br>D3: Fuc-transferase</td><td>B<br>B<br>B</td><td></td><td>3.3.2.3</td><td>147</td></tr><tr><td>A118R</td><td>NP_048466</td><td>345</td><td>1</td><td>GMD</td><td>B</td><td></td><td>3.1.1.1</td><td>135, 148</td></tr><tr><td rowspan="3">A181/182R</td><td rowspan="3">NP_048529</td><td rowspan="3">830</td><td rowspan="3">3</td><td>D1: CBD</td><td>B</td><td></td><td>3.4.1.1</td><td>149</td></tr><tr><td>D2: GH</td><td>B</td><td></td><td></td><td></td></tr><tr><td>D3: NCR</td><td>B</td><td></td><td></td><td></td></tr><tr><td>A260R</td><td>NP_048613</td><td>505</td><td>1</td><td>GH</td><td>E</td><td></td><td>3.4.1.1</td><td>149</td></tr><tr><td>A292L</td><td>NP_048646</td><td>328</td><td>1</td><td>GH</td><td>B</td><td></td><td>3.4.1.1</td><td>149</td></tr><tr><td>A295L</td><td>NP_048649</td><td>317</td><td>1</td><td>GMER</td><td></td><td></td><td>3.1.1.1</td><td>148</td></tr><tr><td rowspan="4">A561L</td><td rowspan="4">NP_048917</td><td rowspan="4">649</td><td rowspan="4">4</td><td>D1: NCR</td><td>B</td><td></td><td>3.4.5</td><td>150</td></tr><tr><td>D2: NCR</td><td>E</td><td></td><td></td><td></td></tr><tr><td>D3: NCR</td><td>E</td><td></td><td></td><td></td></tr><tr><td>D4: PL<sup>d</sup></td><td>E</td><td></td><td></td><td></td></tr><tr><td>A609L</td><td>NP_048965</td><td>389</td><td>1</td><td>UGDH</td><td>B</td><td></td><td>3.3.1</td><td>146</td></tr><tr><td rowspan="15">Mimivirus</td><td rowspan="10">APMV</td><td>L136</td><td>YP_003986628</td><td>352</td><td>1</td><td>aminotransferase</td><td>B</td><td>7MFQ</td><td>3.1.2.1</td><td>62, 151</td></tr><tr><td>R141</td><td>YP_003986633</td><td>323</td><td>1</td><td>UGD</td><td>E</td><td>6VLO</td><td>3.1.2.1</td><td>58, 152</td></tr><tr><td>L142</td><td>YP_003986634</td><td>490</td><td>2</td><td>D1: GT<br>D2: acetyl-transferase</td><td>B</td><td></td><td>3.1.2.1</td><td>60</td></tr><tr><td rowspan="2">L230</td><td rowspan="2">YP_003986726</td><td rowspan="2">895</td><td rowspan="2">2</td><td>D1: Glc-transferase</td><td>E</td><td></td><td>3.3.3</td><td>24, 153</td></tr><tr><td>D2: NCR</td><td>E</td><td>6AX6</td><td></td><td></td></tr><tr><td>L316</td><td>YP_003986819</td><td>148</td><td>1</td><td>GNAT</td><td>A</td><td></td><td>3.1.2.2</td><td>154</td></tr><tr><td>L619</td><td>YP_003987136</td><td>606</td><td>2</td><td>GFAT</td><td>E</td><td></td><td>3.1.2.2</td><td>154</td></tr><tr><td>R689</td><td>YP_003987216</td><td>255</td><td>1</td><td>UAP</td><td>B</td><td></td><td>3.1.2.2</td><td>154</td></tr><tr><td>R707</td><td>YP_003987235</td><td>281</td><td>1</td><td>Glc-transferase</td><td>E</td><td></td><td>3.3.3</td><td>25</td></tr><tr><td>L780</td><td>YP_003987312</td><td>289</td><td>1</td><td>UGER</td><td>E</td><td>7JID</td><td>3.1.2.1</td><td>58, 155</td></tr><tr><td rowspan="6">Ma</td><td>Ma458</td><td>AVL94844</td><td>324</td><td>1</td><td>UGD and epimerase</td><td>ND<sup>e</sup></td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Ma459</td><td>AVL94845</td><td>270</td><td>1</td><td>reductase</td><td>ND<sup>e</sup></td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Ma460</td><td>AVL94846</td><td>376</td><td>1</td><td>epimerase</td><td>ND<sup>e</sup></td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Ma465</td><td>AVL94851</td><td>384</td><td>1</td><td>aminotransferase</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Ma466</td><td>AVL94852</td><td>209</td><td>1</td><td>acetyl-transferase</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Ma467</td><td>AVL94853</td><td>278</td><td>1</td><td>UGD</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td rowspan="3">Mg</td><td>Mg534</td><td>YP_004894585</td><td>323</td><td>1</td><td>UGD and epimerase</td><td>B</td><td>4TQG</td><td>3.1.2.3; 3.1.2.5</td><td>61</td></tr><tr><td>Mg535</td><td>YP_004894586</td><td>270</td><td>1</td><td>epimerase and reductase</td><td>B</td><td></td><td>3.1.2.3; 3.1.2.5</td><td>61</td></tr><tr><td>Mg536</td><td>YP_004894587</td><td>370</td><td>1</td><td>epimerase</td><td>B</td><td></td><td>3.1.2.3; 3.1.2.5</td><td>61</td></tr><tr><td rowspan="3">Mm</td><td>Mm419</td><td>QGR53988</td><td>278</td><td>1</td><td>reductase</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Mm421</td><td>QGR53990</td><td>321</td><td>1</td><td>reductase</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr><tr><td>Mm422</td><td>QGR53991</td><td>279</td><td>1</td><td>UGD</td><td>B</td><td></td><td>3.1.2.5</td><td>87</td></tr></tbody></table>
Table 2. continued

ᵃProteins are named as in the original publication, and their ID number is provided for a quick retrieval in the NCBI database. The homology column refers to the organism (all but viruses) whose protein had the best match with the viral protein: bacterium (B), archaea (A), or eukaryote (E). Moumouvirus maliensis, Mm; Moumouvirus australiensis, Ma; Megavirus chilensis, Mg. ᵇNCR, not related to carbohydrate/glycan manipulation; ND, not determined or reported in the publication. ᶜOne of the crystallographic coordinates available in the publication, others PDBs are the same protein in complex with other molecules and/or at different pH. ᵈPutative activity. ᶜAssignment based on homology with proteins from other giant viruses.

<table><caption>Table 3. Number of Viruses in the *Mimivirus* Genus Used to Define the Glycogene Clusters of Each Clade, Along with Information Regarding the Type of Sugars Encoded and the Size of the Clustersᵃ,⁸⁷</caption><thead><tr><th>clade</th><th>no. of viruses</th><th>prototype</th><th>cluster (no. of genes)</th><th>monosaccharide marker</th></tr></thead><tbody><tr><td>A</td><td>28</td><td>APMV</td><td>12</td><td>L-Rha, D-Vio4NAc</td></tr><tr><td rowspan="2">B</td><td rowspan="2">7</td><td>Moumouvirus australiensis<sup>b</sup></td><td>12</td><td>L-RhaNAc, L-QuiNAc, D-QuiNAc4NAc</td></tr><tr><td>Moumouvirus maliensis</td><td>6</td><td>D-QuiNAc, D-FucNAc</td></tr><tr><td>C</td><td>17</td><td>Megavirus chilensis</td><td>6</td><td>L-RhaNAc, L-QuiNAc</td></tr><tr><td>D</td><td>2</td><td>Tupanvirus deep ocean</td><td>33</td><td>D-QuiNAc or D-FucNAc, D-QuiNAc4NAc, D-GlcA</td></tr><tr><td>E</td><td>1</td><td>Cotonvirus japonicus</td><td>21</td><td>L-RhaNAc, L-QuiNAc, D-QuiNAc4NAc</td></tr></tbody></table>

ᵃMonosaccharide composition is based on experimental data, except for clades D and E, where it is provided by *in silico* analysis. ᵇMoumouvirus australiensis is considered as an outlier of the clade based on the characteristics of its glycogene cluster. See text for discussion.

CMP-β-Kdo, the activated form used by GTs, in a reaction catalyzed by CMP-Kdo synthetase (CKS). The identity of the four bacterial enzymes, together with their plant counterparts, is known, albeit the nature of the phosphatase involved in the production of free Kdo in plants is still unknown.

The predicted Kdo biosynthetic pathway in CroV involves three proteins: crov265, crov266, and crov267. The C-terminal domain of crov265 matched with API and catalyzed the first biosynthetic step of Kdo synthesis; the second enzyme in the pathway was assigned to the N-terminal domain of crov267 due to its resemblance to the KDO-8P synthase (KdoPS). The third reaction is catalyzed by N-terminal domain of crov265, which has the Kdo-8P phosphatase (KdoPase) activity. Finally, the fourth step is the activation of Kdo as a CMP nucleotide, and this activity was tentatively assigned to the N-terminal domain of crov266, although phylogenetic analysis associated this putative protein to a N-acylneuraminate cytidylyltransferase (CMP-NeuAS) and not to CKS (Figure 7). Notably, all of the genes involved in the Kdo pathway encode bifunctional enzymes. Both domains of crov265 are involved in different steps in the synthesis of the Kdo. On the other hand, the C-terminal of crov267 is predicted to encode a dTDP-6-deoxy-L-hexose 3-O-methyltransferase, while the C-terminal domain of crov266 is annotated as a demethylmenaquinone methyl-transferase.⁵⁹ Currently, none of the enzymatic activities of this putative Kdo pathway have been validated, nor have the structure of the CroV glycans been determined, which would support the in silico prediction.

Interestingly, CroV is not the only giant virus that has been predicted to encode the Kdo pathway. Fadolivirus, a member of the *Mimiviridae* family but in a different subfamily (Table 1), has all the genes necessary to produce this monosaccharide. Indeed, the protein with ID QKF94409 (Fadolivirus_1_951, or fdv951 for simplicity) is annotated as a bifunctional protein with 3-deoxy-D-manno-octulosonate 8P phosphatase/sugar isomerase activity at the N- and C-terminal domains, respectively; assuming that the sugar isomerase domain performs the API function, this gene would have the same predicted function as crov265 (Figure 7). The protein ID QKF94408.1 (fdv950) is annotated as a 3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS), while protein ID QKF94406 (fdv948) is annotated as a 3-deoxy-8-phosphooc

tulonate synthase (KdoPS). Regarding Fadolivirus, information about the putative Kdo pathway does not go beyond in silico analysis,³⁸ while the analysis of CroV enzymes have examined the potential evolutionary source of the pathway, i.e., from plants or bacteria. However, the authors could not reach a definitive conclusion about the origin of this gene cluster. They speculated that it could have been acquired from a bacterium, although they noted that some of these proteins (crov267 and crov265) have features common to both plants and bacteria. Thus, the prediction that CroV and Fadolivirus encode Kdo biosynthetic enzymes leads to many questions about how the viruses use this monosaccharide and about the origin of the enzymes.

### 3.1.2.5. *Mimivirus Glycogenes Are Organized in Clade-Specific Clusters.*
A recent study on several members of the *Mimivirus* genus (Table 3) found that most of the glycogenes were organized in clusters whose size is clade-dependent.⁸⁷ The study also reported that each clade uses specific sugars, with some clades having few pathways in common (Table 3, Figure 8). This study also analyzed the genome of 55 fully sequenced viruses in five clades and combined the results of the bioinformatic predictions with the experimental data, either acquired in the same work or taken from previous studies.⁸⁷ The protein sequences of well-annotated glycogenes were used as input to search for homologous proteins in the genome of each virus. Multiple alignments were performed to assess the conservation level of the active sites and consequently the functionality of the enzymes. The analytical results for each clade are summarized at the end of this section, while the main conclusions of the study follow hereafter.

Overall, the data suggest that sugar composition in the genus *Mimivirus* is clade-specific, and it is governed by complex gene clusters (Figure 8) responsible for the synthesis of sugars mostly restricted to the bacterial world. How these gene clusters originated is an open question. Notaro and coauthors proposed that the cluster organization of the glycogenes resembles the operon system in the bacterial world. However, in these giant viruses, most of the glycogenes have their own promoter, thus placing them between bacteria and eukaryotes. Moreover, the GC content of these glycogenes and the sequence of the promoter regions are similar to the core genes (genes conserved in members of the family), suggesting that
<figure><img src="image_15.png" /><figcaption>**Figure 8.** Organization of glycosylation genes in the *Mimivirus* genus. Genes in bold indicate that their activity has been experimentally validated, otherwise they are written in plain text. The activity of each gene is color coded as reported in the legend or is explicitly mentioned (in brackets) in the schemes of the reactions. The arrows below the gene names indicate the direction of the coding strand. Importantly, sugar fibril composition has only been determined experimentally for the viruses in the A, B, and C clades; for the other clades (D and E), sugar composition is predicted from in silico analysis. (a) Clade A: APMV glycogene cluster (12 genes) corresponds to UDP-β-L-Rha and UDP-α-D-Vio4NAc production, along with methyl-, pyruvyl-, and glycosyltransferases and other enzymes either manipulating sugars or involved in the construction of the fibrils. (b) Clade B: *Moumouvirus australiensis* glycogene cluster (12 genes) corresponds to the biosynthetic pathway for L-QuiNAc and d-QuiNAc4NAc, and several GTs and other sugar manipulating enzymes including those of sulfate metabolism. (c) Clade B: *Moumouvirus maliensis* cluster (six genes) corresponds to D-QuiNAc and D-FucNAc production: notably, Mm419 and Mm421 are two 4-reductases, and it is not clear which one produces D-QuiNAc or D-FucNAc. (d) Clade C: *Megavirus chilensis* cluster (six glycogenes) corresponds to L-QuiNAc and L-RhaNAc production; notably the activity of Mg534 and Mg535 has been determined experimentally, while that of Mg536 was only predicted. (e) Clade D: Tupanvirus deep ocean cluster (33 genes in total, only the 25 related to carbohydrate metabolism are displayed) is predicted to correspond to the biosynthesis of D-QuiNAc4NAc and to that of a yet unidentified 6-deoxy-D-HexNAc (either D-QuiNAc or D-FucNAc) along with several other glycogenes. (f) Clade E: *Cotonvirus japonicus* cluster (21 genes) is predicted to correspond to L-QuiNAc and D-QuiNAc4NAc production, along with enzymes involved in sulfate metabolism and others.</figcaption></figure>

giant viruses might have acquired their own glycosylation machinery before the evolution of the first eukaryotes. Finally, the existence of this great variability in the glycosylation machinery of the different clades suggests that glycans contribute to the fitness of the viruses in the environment, where they have to compete with bacteria and other giant viruses for the same host.⁸⁷

Clade A. Analysis of this clade involved 28 different strains, with APMV as the prototype. This choice was made as it is the virus for which the function of many of the gene products have been experimentally demonstrated⁵⁸,⁶⁰,⁶² and found to be consistent with the fibril glycans (section 3.2.2.2).

Originally, the APMV gene cluster included nine genes:⁶⁰ those involved in the biosynthesis of UDP-α-D-Vio4NAc and UDP-β-L-Rha (*R141*, *L136*, *N-L142*, sections 3.1.2.1), a
putative pyruvyl transferase (L143), five putative GTs (L137, L138, R139, L140, and C-L142), and R135, a GMC-type oxidoreductase known to be a component of the fibrils, that in this virus are glycosylated (section 3.2.2.2).⁴⁵ The inclusion of three other genes in the cluster was prompted by the finding that Vio4NAc, a component of the polysaccharide of the fibrils, was partially methylated at O-2. Accordingly, R132 was identified as a SAM-dependent methyltransferase and suggested to be responsible for this modification (Figure 8a), thus expanding the original cluster from 9 to 12 genes by including L133 and L134, both corresponding to hypothetical proteins (Figure 8a).⁸⁷

Importantly, the *Mimivirus* 12 gene-cluster was preserved inside the A clade, and it was not shared by members of other clades, suggesting that Rha and Vio4NAc are the markers for this clade (Table 3, Figure 8a). Other glyco-related genes were scattered elsewhere in the genome: GlcNAc biosynthesis (section 3.1.2.2) and L780, which completes UDP-β-L-Rha production that is started by R141 (section 3.1.2.1).

*Clade B*. Analysis of this clade included seven viruses, and the composition of their fibrils was determined for two, Moumouvirus australiensis (Figure 1g) and Moumouvirus maliensis (Figure 1h). These viruses have different fibril morphology (thick and compact for maliensis and sparse for australiensis) and differences in their respective glyco-related clusters.

Moumouvirus australiensis fibrils have D-GlcNAc, L-QuiNAc, and D-QuiNAc4NAc and the biosynthetic pathways for the corresponding sugar nucleotides were assigned in silico. As for UDP-β-L-QuiNAc (Figure 8b), UDP-α-D-GlcNAc is converted to 6-deoxy-4-keto-β-L-IdoNAc by the bifunctional enzyme Ma458 (4,6-dehydratase, 5-epimerase), and the intermediate is epimerized at C-3 and reduced at the 4-keto function by Ma459. The resulting UDP-β-L-RhaNAc is finally epimerized at C-2 by Ma460 to produce UDP-β-L-QuiNAc. For UDP-α-D-QuiNAc4NAc, the predicted biosynthesis starts from UDP-α-D-GlcNAc, with C-4 oxidation catalyzed by Ma467, followed by transamination of the C-4 keto function promoted by Ma465. Finally, Ma466 attaches the acetyl group to the newly generated amino group at C-4 (enzymes characteristics are reported in Table 2).

The Moumouvirus australiensis gene cluster includes 12 genes, with 10 having glyco-related activity. Two of the encoded enzymes (Ma457 and Ma461) are involved in sulfate metabolism, suggesting that the sugars could be decorated with a sulfate group. Others include genes encoding for single (Ma463) or multidomain GTs (Ma468) and yet more related to sugar nucleotide biosynthesis (Ma458, Ma459, Ma460, Ma655, Ma466, and Ma467), whose functions are yet to be identified. The other two genes included in the cluster (Ma462 and Ma464) have papain-like domains (Figure 8b). The glycogenes defined for Moumouvirus australiensis are not shared by the other members of the clade, making this virus an outlier of the B clade. Moreover, the Moumouvirus australiensis glycosylation gene cluster had features common to those of clades C, D, and E (Table 3). Indeed, Moumouvirus australiensis has pathways to make the same sugars as members of clade E (L-RhaNAc, L-QuiNAc, and D-QuiNAc4NAc) along with the enzymes involved in sulfate metabolism, while it has L-RhaNAc and L-QuiNAc in common with clade C and D-QuiNAc4NAc with clade D (Table 3, Figure 8b).

In Moumouvirus maliensis (Figure 8c), the fibrils have D-QuiNAc and D-FucNAc, and only traces of D-GlcNAc. The synthesis of UDP-α-D-QuiNAc and UDP-α-D-FucNAc are interconnected, as they begin with the conversion of the UDP-α-D-GlcNAc into UDP-4-keto-α-D-QuiNAc by Mm422, a 4,6-dehydratase enzyme (UGD). This intermediate is converted to UDP-α-D-QuiNAc or to its C-4 epimer UDP-α-D-FucNAc, depending on the stereospecificity of the 4-reductase enzymes involved. The cluster has two enzymes with this predicted activity, Mm419 and Mm421, but in silico analyses could not attribute the precise activity to either of them due to their strong homology (enzymes characteristics are reported in Table 2). Next to the genes involved in the production of D-QuiNAc and D-FucNAc, the cluster includes three GTs (Mm417, Mm418, and Mm420), for a total number of six genes (Figure 8c). Importantly, the Moumouvirus maliensis six-gene cluster is conserved in all the members of this clade (except Moumouvirus australiensis) so that D-QuiNAc and D-FucNAc can be considered as the markers of clade B, with one of them also being encoded by *Tupanvirus* (clade D, Figure 8e).

*Clade C*. Analysis of this clade included 17 viruses and identified a cluster of six well-conserved genes (Figure 8d). Megavirus chilensis served as prototype for the clade, and analysis of the fibrils detected D-GlcNAc, L-RhaNAc, 4OMe-L-RhaNAc, and L-QuiNAc. The identification of RhaNAc was in agreement with the pathway previously described for the nucleotide, UDP-β-L-RhaNAc (section 3.1.2.3, Figures 5 and 7d),¹⁵⁴ while that of L-QuiNAc corroborated the hypothesis that the gene *mg536* could encode a 2-epimerase (section 3.1.2.3, Figures 5 and 7d).⁶¹ The region next to the genes encoding UDP-β-L-RhaNAc and UDP-β-L-QuiNAc (*mg534*, *mg535*, and *mg536*) contained three other genes involved in sugar manipulation: one putative single GT domain (*mg537*), one with three GT domains (*mg539*), and a putative pyruvyl transferase (*mg538*). Therefore, the glycosylation gene cluster of Megavirus chilensis consists of six genes (from *mg534* to *mg539*) as for Moumouvirus maliensis, although it lacks the transferase that methylates O-4 of RhaNAc (Figure 8d). This methyltransferase is yet to be identified. L-RhaNAc and L-QuiNAc are considered to be markers of clade C.

*Clade D*. Analysis of this clade identified a cluster of 33 conserved genes for the two members analyzed (Table 3). Tupanvirus deep ocean was used as a reference, and the cluster corresponds to a 49 kb DNA region made of 25 genes involved in carbohydrate metabolism (Figure 8e). Within this cluster, the biosynthetic pathway of UDP-β-D-QuiNAc4NAc (L515, L509, and L514, Figure 7b,e) was identified along with that for a UDP-6-deoxy-α-D-HexNAc (L515 and L518, Figures 7c,e), either UDP-α-D-QuiNAc or UDP-α-D-FucNAc. The identity of the HexNAc could not be predicted because L518, an enzyme predicted as a 4-reductase, had a tight match with both Moumouvirus maliensis Mm419 and Mm421 (Figure 8c), whose stereospecificity is undetermined.

Interestingly, Tupanvirus encodes a putative UDP-α-D-Glc-dehydrogenase (UGDH) able to convert UDP-α-D-Glc to UDP-α-D-GlcA (R520), and Glc-1P-uridyltransferase (N-terminal domain of L502) that produces UDP-α-D-Glc from Glc-1P, an additional tool to manipulate the host cell glycosylation machinery. Regarding the GTs, this virus encodes 11 single-domain GTs (L498, R501, R503, R504, L505, R507, L511, L512, R513, R516, R519), and two others that are predicted to be multidomain proteins (R496 and L510), with
four and two GT domains, respectively (Figure 8e). Of the remaining genes, there were methyltransferases (L506 and L517), a SAT/APS kinase involved in sulfate metabolism (R499), six ORFans (R497, R500, R522, R524, L525, L526), a putative bifunctional glutamate/proline tRNA-synthetase (L521), and a histidine tRNA synthetase (L523).

Notably, Tupanvirus deep ocean encoded the highest number of GTs within the *Megavirinae* subfamily, suggesting a complicated glycosylation pattern for its fibrils. Indeed, an intriguing question is whether the fibrils in the tail and in the capsid (Figure 1b,c) have the same glycans or if these are different depending on which part of the virion they decorate. Finally, the monosaccharide markers of clade D, based on in silico analyses are D-QuiNAc4NAc, D-QuiNAc (or D-FucNAc), and D-GlcA (Table 3).

*Clade E.* This clade has only one member, Cotonvirus japonicus, for which no experimental data are available on its carbohydrate composition. In silico analyses identified a cluster of 21 genes (Figure 8f) having some analogies with that of Moumouvirus australiensis (Figure 8b). Indeed, it encodes a putative biosynthetic pathway for UDP-β-L-QuiNAc (ORFs 708, 709, and 710) and UDP-α-D-QuiNAc4NAc (ORFs 718, 720, and 719), along with enzymes involved in sulfate metabolism (ORFs 724 and 725) and two papain-like proteins (ORFs 721 and 723) that are not related to glycosylation. Contrary to Moumouvirus australiensis, Cotonvirus encodes a higher number of GTs, six in total: ORFs 711, 712, 713, 717, 722, and 728. Finally, this cluster also has genes involved in DNA/RNA metabolism (ORFs 714, 726, and 727) and one hypothetical protein (ORF 716). Regarding the sugars that can be considered as markers of the clade, the assignment of L-RhaNAc, L-QuiNAc, and D-QuiNAc4NAc are tentative because they rely on a unique strain (Table 3).

## 3.2. Glycans Structures

**3.2.1. Chloroviruses.** In 1993, the MCP (called Vp54) of the prototype NC64A chlorovirus PBCV-1 was isolated,¹⁷⁵ and the initial chemical data suggested that it was glycosylated with an unusual glycan due to the presence of different types of sugars, namely Ara, Gal, Glc, Rha, Man, and Xyl. This was confirmed by the fact that classic procedures used to cleave N-linked glycans from the glycoproteins, such as the use of endoglycosidase PNGase F, did not remove the glycan from Vp54. This assumption was later supported in 2002 by crystallographic studies¹⁷⁶ that detected four N-linked oligosaccharides attached to asparagine residues located in atypical sequons, none were the typical sequence NX(T/S) (with X being any amino acid but proline) used in all forms of life. Regarding the structure of the Vp54 glycans from the crystallographic study, the information was not complete and was limited to the sugar units next to the protein backbone. However, at that time, the interpretation of the data only considered the structure typical for the eukaryotic N-linked glycans because the structure of these glycans was still unknown.

Determination of the structure of the PBCV-1 N-glycans¹⁷⁷ occurred 20 years after their initial identification,¹⁷⁵ when approaches commonly used for bacterial glycans were adapted to PBCV-1.¹⁷⁸ Purified PBCV-1 particles were disrupted by heating to 60 °C, and the capsid protein recovered from the soluble fraction by precipitation with ice-cold acetone, which produced ~90% purity. The chemical characterization of the monosaccharide constituents was either performed directly on

the glycoprotein or on the glycopeptide blend released by enzymatic digestion with a protease. Notably, the N-glycan has Rha units with opposite configurations (Figure 9), and the evaluation of which residue was in a certain position was possible by incorporating the information on the linkage pattern into the method used to determine their absolute configuration.¹⁷⁷

NMR spectroscopic studies were only conducted on the glycopeptides, because when the glycans were linked to the MCP, the NMR signals of the glycans were broad due to the low mobility of the macromolecule, which in solution spontaneously associates with two other copies to form trimers, termed capsomers.¹⁷⁶ Accordingly, for NMR studies, Vp54 was digested with proteinase K, a highly efficient but unspecific protease that releases short glycopeptide fragments, sometimes consisting of only one (or two) amino acid(s). To determine the site of N-glycosylation, the enzymatic treatment has to release glycopeptides of sufficient length to identify their location in the sequence of the protein. Thus, for PBCV-1, thermolysin, which provided longer protein fragments, was used,¹⁷⁷ while for other chloroviruses, the glycopeptide mixture produced by proteinase K was sufficient to identify most, if not all, of the glycosylation sites.¹⁷⁹ Regardless of the protease used, the digestion products were a mixture of N-glycopeptides and peptides, and the glycopeptide components were purified via size exclusion chromatography (generally Bio-Gel P10). The fraction enriched in glycopeptides was suitable for both NMR spectroscopic and mass spectrometric analyses.¹⁷⁷,¹⁸⁰

This protocol led to the identification of four N-linked glycoforms of PBCV-1 (denoted as PBCV-1-4, Figure 9), which have several remarkable features. First, the attachment of the glycans to the protein occurs via a β-Glc-Asn linkage, which is rare although it has been found in the three forms of life: the surface glycopeptides and flagellins of the halophilic archaea *Halobacterium halobium*¹⁸¹ and *Halobacterium volcanii*,¹⁸² in the glycoproteins from *Haemophilus influenzae*¹⁸³,¹⁸⁴ and *Actinobacillus pleuropneumoniae*,¹⁸⁵ and in the B2 chain of rat kidney laminin, the only known example of this type of linkage in eukaryotes.¹⁸⁶ Another feature that makes this type of N-glycosylation unique is the location of the Asn units linked to PBCV-1 glycans: three of the four sites share the sequence NTXT (the sequons are ³⁰²NTGT, ³⁹⁹NTET, and ⁴⁰⁶NTAT), whereas the fourth is ²⁸⁰NIPG. None are in the typical eukaryotic sequon NX(T/S), and collectively they denote a new pattern of N-glycosylation with implications and open questions about their biosynthesis.

Regarding the PBCV-1 Vp54 glycan, the main glycoform is a nonasaccharide (PBCV-1<sub>1</sub>, Figure 9) with a Fuc unit fully substituted at all available positions, not terminal as typically occurs in most N-linked glycans.¹⁸⁷ Notably, a hyperbranched Fuc has only been reported in a complex phosphoglycan from *Trypanosoma cruzi*,¹⁸⁸ but with a substitution pattern different from that found in PBCV-1. The Vp54 N-glycan contains both D- and L-Rha residues, a feature that is rare and so far only found in bacteria, along with a terminal L-Rha unit at the nonreducing end of the main chain capped with two methyl groups. This is an uncommon type of termination and so far only reported for glycolipids produced from *Mycobacterium hemophilum*¹⁸⁹ and *Mycobacterium leprae*.¹⁹⁰

The four glycoforms of Vp54 glycans occur because Man and Araf are not stoichiometric substituents. The two glycoforms, PBCV-1<sub>3</sub> and PBCV-1<sub>4</sub> (Figure 9), both devoid of Man and differing by the presence or absence of the Araf
<figure><img src="image_16.png" /><figcaption>**Figure 9.** Structure of the N-glycans from all chloroviruses characterized to date. The structures from the same virus are arranged in a row, and the different viruses are grouped according to their host specificity, indicated at the bottom margin of the grouping boxes. Methyl groups enclosed in brackets denote a nonstoichiometric substitution. All of these N-glycans share a common core oligosaccharide that is indicated as a dashed box in the structure of PBCV-1. All sugars are in the pyranose form except where specified.</figcaption></figure>

unit, were present in traces, and their existence was only inferred by MALDI spectrometry studies. PBCV-1₁ and PBCV-1₂ were the main constituents of the glycopeptide

mixture, and they were identified by NMR spectroscopy. MALDI mass spectrometry confirmed the NMR data and added further details. Apart from detecting the two minor
glycoforms mentioned above, MALDI found that the N-glycosylation pattern was not the same at the different glycosylation sites. Indeed, ²⁸⁰Asn was glycosylated almost exclusively by **PBCV-1₂**, while the glycosylation pattern at the other three Asn units was more heterogeneous, with **PBCV-1₁** as the main glycan, along with variable proportions of the other three.¹⁷⁷

The purification protocol used for PBCV-1 enabled the identification of the glycan structures of other chloroviruses (Figure 9), classified either in the same group of viruses as PBCV-1 or in one of the other three chlorovirus groups (section 2.6.1).¹⁷⁹,¹⁹¹⁻¹⁹³ Of note, the chlorovirus NY-2A was predicted to have two MCPs with a high level of identity between them,¹⁷⁷ and when this method was applied to NY-2A, both glycoproteins were extracted and the N-glycans derived from both proteins were studied together. Collectively, these structural studies established that the chlorovirus MCPs are always N-glycosylated and also revealed other interesting features. First, all chloroviruses glycans include the same oligosaccharide motif and, for this reason, termed conserved core oligosaccharide, marked in **PBCV-1₁** with a dashed box (Figure 9). Initially, this conserved oligosaccharide core consisted of five monosaccharide units:¹⁷⁹ the four highlighted in Figure 9 together with a so-called distal Xyl residue. However, this view has recently changed after determining the glycan structure of chlorovirus MA-1D,¹⁹³ a NC64A virus that produces three different N-glycans, all missing the distal Xyl unit. Accordingly, the conserved core region for all chloroviruses now consists of four sugar units (Figure 9).¹⁹³

All chlorovirus N-glycans have a Rha unit linked at the third position of the hyperbranched Fuc, which is a semiconserved element because its absolute configuration is D for all the viruses within the NC64A and Osy groups, while it is L and always methylated at the O-3 position in the Pbi and SAG groups.¹⁷⁹ The substitution pattern of the conserved core region inclusive of the semiconserved Rha unit appears to be virus-specific, and it can be considered as a glycan-based molecular signature for each virus. Indeed, several neutral monosaccharides can be part of this variable region of the N-glycan, such as xylulofuranose (Xul/), Araf, or Rha and Xyl units substituted with various methyl or acetyl groups (Figure 9). Finally, these N-glycans are always branched, and their size varies from five, as MA-1D₂, to 10 monosaccharide units, like the PBCV-1₂ (Figure 9).

Regarding the position of the glycosylated Asn, MALDI MS investigation identified the N-glycosylation sites for some of them (NY-2A, CVM-1, and MA-1D).¹⁷⁹,¹⁹³ For all these viruses, the location of the glycosylated asparagine residue was in an atypical sequon when compared to that from all forms of life.

In chlorovirus NY-2A, the capsomer consists of two similar MCPs, B585L and B617L, both sharing a high level of homology with PBCV-1 Vp54. During the study of the two NY-2A N-glycans, the two MCPs could not be separated; therefore, five of the six glycosylation sites found (⁵⁴NKVS, ²⁸⁰NIPG, ³⁰²NTGT, ³⁹⁹NTET, ⁴⁰⁶NTAT, and ²⁹¹NVAT), could not be assigned exclusively to one of the two MCPs, while the sequon ²⁹¹NVAT was assigned to B617L because it was not conserved in B585L.

With regard to MA-1D, another chlorovirus in the NC64A group, like NY-2A and PBCV-1, in silico analysis suggested the presence of two MCPs (named 609L and 635L),¹⁹³ again with a very high level of homology between them and with the

MCPs of both PBCV-1 and NY-2A. The two MA-1D MCPs were predicted to have the same, in terms of sequence and position, glycosylation sites as the other two viruses, and MALDI MS analysis confirmed three of them: ²⁸⁰NIPG, ³⁰²NTGT, and ⁴⁰⁶NTAT. The fourth position predicted to be glycosylated, ³⁹⁹NTET, was not detected; therefore, its glycosylation status is unknown.

Four glycosylation sites were detected in chlorovirus CVM-1, ⁴⁷NGSV, ²⁷⁹NLTA, ²⁸⁵NVGY, and ²⁹³NTAV, making this virus the only one with a N-glycan attached to the canonical NX(T/S) consensus sequon. Of note: ⁴⁷Asn is conserved in PBCV-1 and other MCPs, but it was only glycosylated in CVM-1.¹⁷⁹

PBCV-1 is the only chlorovirus for which the structure of the capsid, along with the structure of the glycans has been determined.³⁶,¹⁰⁸,¹⁷⁶ The PBCV-1 capsid has been thoroughly characterized by crystallographic and cryo-EM studies to 3.5 Å resolution, which revealed that it is composed of a set of 15 different proteins, with Vp54 as the most abundant and estimated to be present in ~5040 copies per virion. Accordingly, the capsid includes 60 copies of the penton protein which is also a glycoprotein, presumably glycosylated by the same N-glycans as Vp54, while the collective number of the remaining 13 minor capsid proteins reaches ~1800 copies.³⁶ One of the 12 vertices of the capsid has a spike structure attached to it, and the proteins involved in the spike structure are currently unknown.

This complex set of proteins is organized into two main motifs, the trisymmetrons and the pentasymmetrons (Figures 10a,b),¹⁰⁷,¹⁷⁶ present in 20 and 11 number of copies, respectively. Each of these is composed of many copies of the capsomer (Figure 10b-d): 66 for each trisymmetron and 30 for each pentasymmetron. Each of the 11 pentasymmetrons has a pentamer made of the penton protein in its center (Figure 10a). An additional pentasymmetron, the twelfth, differs from the others by having the spike structure in its center (Figure 10b). Each capsomer is composed of three copies of the MCP (Figures 10d,e), arranged with a pseudohexagonal symmetry resembling the shape of a doughnut (Figure 10c). In addition, one capsomer in each trisymmetron has a fiber that extends from the particle (Figure 10b, note the small violet fiber on the left side). The fiber is always located in a capsomer that is in the middle of the second row of capsomers.¹⁰⁶

As for the Vp54 glycoprotein, the crystallographic experiments established that it consists of two consecutive domains held together by an α-helix, each of about 200 aa, with a jelly roll fold (D₁, 2–212 aa; D₂, 225–437 aa; Figure 10f). The first 24 amino acids are organized in two α-helices connected by a linker, and they are interconnected in the lower face of the capsomer (Figure 10e). In this structure, the N-terminal region of one Vp54 unit of the capsomer makes contacts with the nearby unit, thus stabilizing the trimeric complex. This interaction occurs on the side of the capsomer that is not glycosylated and that points toward the inside of the viral particle.

Domain D₂ is glycosylated at positions ²⁸⁰Asn, ³⁰²Asn, ³⁹⁹Asn, and ⁴⁰⁶Asn, and the crystallographic data revealed only some of the sugar units (Figure 10f), namely those less flexible and located next to their respective glycosylation sites; the missing sugars were reconstructed by molecular modeling.¹⁰⁸ This approach revealed that all glycans were located on the same side of the protein and that they pointed outward from

W
<figure><img src="image_17.png" /></figure>

**Figure 10.** PBCV-1 capsid organization. (a) Top view of the cryo-EM reconstruction of the virus PBCV-1 capsid showing the trisymmetron (cyan, one explicitly contoured with a black line) and pentasymmetron (green) structures. Adapted with permission from ref 36. Copyright 2019 Springer Nature. Eleven of the 12 pentasymmetron have at their centers a pentamer made of the penton protein, drawn in violet. The twelfth pentasymmetron has a spike structure at the vertex (b). Both pentasymmetron and trisymmetron (a total of 20 in the capsid) structures are built by 66 and 30 capsomer units (c,d), respectively, with one highlighted by a red dotted circle, and expanded in (c–e). (b) different visualization of PBCV-1 capsid. Here each trisymmetron is represented with a different color; pentasymmetrons are always in yellow, except that with the spike protein at its center, drawn in orange, and placed at the apical vertex of the capsid. Reproduced with permission from ref 101. Copyright 2020 Multidisciplinary Digital Publishing Institute. (c) Cryo-EM reconstruction of the capsomers. Reproduced with permission from ref 101. Copyright 2020 Multidisciplinary Digital Publishing Institute. (d) Top view of the capsomer structure from X-ray crystallography and molecular modeling: the complex is made by three units of the major capsid protein (Vp54). The peptide backbone of the three glycoproteins is presented in ribbon representation, each with a different color. The peptide part is partially hidden by the N-linked glycans, represented in the space filling-mode with their atoms colored according to the residue they are attached to (Asn-280, green; Asn-302, gray; Asn-399, red; Asn-406, blue). (e) Lateral view of the capsomer, with the first 24 amino acids of each MCP drawn in pink to highlight their location on the lower face of the complex. (f) Lateral view of Vp54 as a single monomer, with its two jelly roll domains, D1 (aa 2–212) and D2 (225–437) colored in green and red, respectively; the N-glycans follow the same notation as in (b), and this figure reports only those detected by X-ray crystallography. (d–f) Reproduced with permission from ref 108. Copyright 2018 Proceedings of the National Academy of Sciences of the United States of America.

the viral particle, with their outermost part free to move and able to assume multiple conformations.¹⁰⁸ The role of the glycans in the interactions with the external environment or in the packaging process is an open question, and further experiments are needed to establish their importance. We deem that the oligosaccharide common core is essential for the stability of the particles, as no chlorovirus lacking glycans has been found to date.

**3.2.2. APMV Glycans Structures.** The hallmark of the *Mimivirus* genus is the presence of a fibril layer around the icosahedral capsid (Figure 1e,j), including the tail in Tupanviruses (Figure 1b,c). Since the discovery of the first mimivirus APMV,⁴¹ the prototype of the clade A group (section 2.3.2.2, Table 1), it has been postulated that the fibrils are highly glycosylated due to the positive response of the viral particles to Gram staining. This first hypothesis has been experimentally validated for some of the viruses in three of the five clades by analyzing the monosaccharide composition of their fibrils (Table 3, section 3.1.2.5).⁶²,⁶⁵,⁸⁷ APMV is the most studied virus of this genus, and recently the structures of its glycans have been resolved, as reported in the next two sections.⁶⁵,¹⁹⁴

**3.2.2.1. APMV Surface Proteins Are Decorated with O-Glycans.** The occurrence of sugars associated with the fibrils

was first confirmed in 2012,⁶² and this discovery prompted a study of the surface of the APMV particles, assuming that these sugars were linked to one or more proteins by N- and/or O-type linkages. In the first study by Hülsmeier and Hennet,¹⁹⁴ the surface glycoproteins were extracted by applying a periodate oxidation method for the carbohydrates followed by biotinylation to isolate the glycosylated proteins and to establish their identity. This protocol led to the enrichment of 40 proteins, most of which were annotated as ORFans. These results were in agreement with those of others,⁴⁵,¹⁹⁵ except that other work predicted that the L425 protein was glycosylated,¹⁹⁶ but it was poorly enriched by this procedure.

A sugar compositional analysis carried out on the pool of the extracted proteins revealed the presence of Rha, GlcNAc, Vio4NAc, and a methylated form of Vio4NAc, in agreement with previous data from Piacente et al.,⁶² along with traces of Xyl and Fuc. Additional structural information was obtained by performing a β-elimination reaction on the extracted proteins under reductive conditions, so that the O-glycans were converted to the corresponding reducing end alditols, which were isolated and investigated via chemical analysis and MALDI MS.

MALDI analysis of the methylated or of the perdeuteriomethylated oligosaccharide alditols detected 26 O-glycans,
<figure><img src="image_18.png" /><figcaption>**Figure 11.** Glycan structures from APMV, the prototype of the clade A of the Mimivirus genus. (a) Structures of the APMV O-glycans. The numbers from 1 to 6 indicate the structural group to which the O-glycan belongs. The first group is the only one with a branched architecture, and it has a hexose (Hex) at the reducing end. The other groups are linear polymers of a hexose and differ in the nature of the monosaccharide at the reducing end: group 2 has a dimethylated dHex, group 3 has a methylated dHexN, group 4 has a dimethylated hexose, group 5 has a hexose, and group 6 has a HexN. On the basis of the carbohydrate chemical composition, the Hex is assigned to Glc, the dHex to Rha, dHexN to Vio4N, and HexN to GlcN. (b) The APMV fibrils contain two distinct polysaccharides, poly_1 and poly_2. The first has anionic features due to the presence of pyruvic acid linked as a ketal. The other, poly_2, is neutral, and the viosamine unit is always N-acetylated and partially methylated at position 2. All monosaccharides are represented according to the SNFG system and the “o” inside the symbol indicates that unit is an alditol. Nonstoichiometric substituents are indicated with the linkages as a broken line (a), or in parentheses (the methyl group in b).</figcaption></figure>

divided into six structural groups based on the nature of the first sugar attached to the protein. Using the compositional analysis data, the monosaccharides that composed these O-glycans were assumed to be Glc, GlcNAc, GlcN, Vio4N, and Vio4NAc, along with their methylated forms, but discussion of the MALDI structures will make use of the generic names (dHex, dHexN, Hex, HexN, and HexNAc) like in the original publication.¹⁹⁴

The first group encompassed four branched O-glycans, made of 4-7 monosaccharide units, all sharing a methylated HexNAc at the nonreducing end, and a substituted Hex at the reducing end. Moreover, the two largest species had a terminal pentose (probably Xyl) linked to O-3 (or O-4) of the HexNAc unit attached to the Hex at the reducing end (Figure 11a). The glycans of all the other groups had a linear architecture, with a variable number of hexose units (Figure 11a).

Group 2 included three linear O-glycans all with a double methylated dHex at the reducing end. MALDI analysis of the perdeuteromethylated products suggested that the terminal Hex unit was attached to O-4 (or to O-6) of the tetrasaccharide (n = 2, Figure 11a) or at O-3 (or O-4) of the pentasaccharide (n = 3, Figure 11a).

The third group was the most abundant and encompassed 11 glycans of 2-12 monosaccharide units, all with a methylated dHexN at the reducing end. In this case, analysis of the MALDI spectra of the perdeuteromethylated alditols of the three oligosaccharides with n = 1 or 2 (Figure 11a) suggested a 4- or a 6-linkage between the Hex-Hex units, while the mode of attachment of the Hex to the dHexN could not be determined.

The fourth and fifth groups contained three and two glycans, respectively, and a double or an unmethylated hexose at the reducing end, respectively (Figure 11a). Regarding the nature of the glycosidic linkage between two consecutive Hex units, MALDI analysis of the perdeuteromethylated oligosaccharide alditols of group 4, suggested that the terminal nonreducing Hex was attached to O-3 or O-4 of the penultimate Hex, while the other glycosidic connections could also involve O-6. As for group 5, the same spectrometric approach suggested that the

Hex-Hex glycosidic linkages involved either O-4 or O-6 of the next unit. Finally, the sixth group included three O-glycans with a methylated HexN at the reducing end. In addition, the MALDI-TOF-MS analysis suggested that the glycans of this group had 4-linked consecutive Hex units.

The authors also performed linkage analysis on the whole oligosaccharide mixture. They found 4-linked and terminal non substituted Glc units as major components, which supported the MALDI results and led to the hypothesis that some O-glycans had an amylose-like arrangement of the Glc units. To prove this hypothesis, the APMV particles were treated with α-amylase and their infectivity compared to that of the untreated virions, but no difference was observed between the two groups, suggesting that these glycans were not essential for viral infection.

Regarding their biosynthetic mechanisms, at the time of this study information about the GTs encoded by APMV were limited to the discovery of a bifunctional enzyme, L230, that hydroxylates the lysine residues of a collagen-like viral protein and transfers a Glc unit to it (see section 3.3.3).²⁴ Therefore, the authors speculated that APMV highjacked the host enzymes to add the pentose unit onto the HexNAc unit and/or to methylate some of the monosaccharide units based on the information that some *Acanthamoeba* species have N-glycans with terminal sugars methylated or pentosylated. There was no information provided about the GTs involved in the assembly of the other glycosidic linkages.¹⁹⁷ This study was extremely important because it was the first to provide clear insight into how complicated the structure of the APMV fibrils are.

3.2.2.2. APMV Fibrils Are Covered with a Glycocalyx Consisting of Two Polysaccharides. Notaro et al.⁶⁵ recently analyzed APMV glycan fibrils using an experimental approach different from that of Hülsmeier and Hennet.¹⁹⁴ The fibrils were released from the virions by heat treatment under denaturing conditions realized with dithiothreitol. The NMR spectroscopic data collected on the crude product revealed the presence of two distinct polysaccharides (Figure 11b). Polysaccharide 1 (poly_1) was an acidic polysaccharide with a linear repeating unit made of 3)-α-L-Rha-(1→3)-β-D-
GlcNAc-(1→, with GlcNAc having a pyruvic acid linked at positions 4,6 as a ketal with the S configuration at its stereocenter. Poly_2 instead (Figure 11b) was a neutral polysaccharide with a branched repeating unit: the linear backbone consisted of a disaccharide repeating unit with the sequence (2)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→, with Rha further substituted at O-3 with β-D-2OMe-Vio4NAc (80%) or with β-D-Vio4NAc (20%). From a structural point of view, these two polysaccharides are unique but have some resemblance to those of bacterial origin. In a particular, poly_1 resembles those produced by some strains of Klebsiella pneumoniae,¹⁹⁸ Serratia marcescens,¹⁹⁸ and Agrobacterium tumefaciens,¹⁹⁹ albeit these all lack the pyruvic acid moiety.

The two polysaccharides were purified by anion exchange chromatography, and their molecular weights (MW) were 100 and 25 kDa for poly_1 and poly_2, respectively. Importantly, the enrichment of the two polysaccharides in pure form was possible only by digesting the fibrils with proteinase K, indicating that they were attached to the same polypeptide backbone. In agreement with this hypothesis, the MW of the intact fibrils was about 400 kDa, a value much higher than that of the single polysaccharides. After protease treatment, the mass of the recovered material dropped considerably, leading to the conclusion that the two polysaccharides constituted about 13% (w/w) of the intact fibrils, also allowing their respective MW determination.

The authors collected preliminary information about the identity of the protein/s acting as carriers, albeit the nature of the glycosylation sites could not be determined due to the difficulties in applying consolidated proteomics methods to glycoproteins with high MW glycans. However, this approach defined the proteome of the fibrils, and this ensemble of proteins was refined through a bioinformatic screening that identified L894/L893 as the most promising candidate to carry one or more copies of the two polysaccharides. This protein addressed different requirements: it was highly conserved among all the members of the A–C clades of the Mimivirus genus, its PI expression time matched that of other proteins known to be involved in the sugar-nucleotide metabolism, and it had several serine residues predicted to be glycosylated.

The finding that polysaccharides form a glycocalyx around APMV has changed the view that viruses of any type (giant or not) are only covered with oligosaccharides of discrete size. This discovery officially marks the entry of polysaccharides into the viral world, illustrating that these macromolecules are not limited to the cellular world. Moreover, these glycans are attached to one or more carrier proteins, thus placing the phenotype of APMV glycocalyx at the interface between the microbial and eukaryotic world.

On the basis of the information available for the M4 isolate, a mutant of APMV devoid of the external fibrils,¹⁹⁵ it appears that APMV mimics bacteria with its polysaccharide coating. The authors hypothesize that this fibril architecture provides an advantage both during phagocytosis and in the competition with bacteria and other viruses on which the amoebae feed. This hypothesis is supported by the finding that uptake of M4 by the host is less efficient compared to that of the wild-type virus, even though the lack of fibrils does not impair the ability of the virus to replicate once it infects the host.²⁰⁰ To demonstrate the role of the glycans of the fibrils in the infection process, the authors performed competition experiments by using the wild-type virus, APMV, together with some monosaccharides. The authors found that GlcNAc and Man

reduced the ability of APMV to adhere to the amoeba and observed a decreased infection rate. Finally, the authors found that APMV exploits the glycans of its fibrils to adhere to other organisms, like fungi and insects, without infecting them. This finding prompted us to hypothesize that these organisms act as dispersers and contribute to the spread of the virus in the environment.²⁰⁰

These studies with APMV have clearly expanded the scope of viral glycobiology studies and have demonstrated that there is still much to learn about giant viruses and their glycans. By looking at the fibril architecture, it appears that the viruses possess a complex glycosylation pattern that consists of oligosaccharides of discrete length (Figure 11a) and polysaccharides (Figure 11b). The way these glycans are linked to their protein carriers is an unanswered question, as are other challenging questions such as: How do these glycans contribute to the fitness of the virus in the environment? How do they promote the ability of the virus to infect the host? How do they shield the virus against virophages.

### 3.3. Glycosyltransferases

As mentioned in section 2, giant viruses encode many glycosylated genes, with many identified as GTs. How these viruses use the GTs has long been an intriguing question, and currently it is possible to give an answer for only some representatives of the chloroviruses and mimiviruses.

The first experimental results indicated that some chloroviruses remodel the cell wall of the host during infection (section 3.3.1). Then, determination of the glycan structures attached to the MCP of the chlorovirus PBCV-1 advanced the research on its GTs (section 3.2.2). Other information has been obtained about two GTs encoded by mimivirusAPMV (section 3.3.3). The next subsections will discuss what is known about the viral-encoded GTs and also mention some open questions.

#### 3.3.1. Chloroviruses Remodel the Host Cell Surface.

The first connection between giant viruses and glycobiology occurred in the late 1990s, when it was discovered that PBCV-1 encoded a functional hyaluronan synthase (HAS, gene *a098r*).¹¹ At that time, HAS had already raised the interest of enzymologists due to the fact that it had broken the dogma that one enzyme could have only one GT function. On the contrary, HAS had two GT specificities, one for each of the two sugars, glucuronic acid and *N*-acetyl glucosamine, that are required for hyaluronan synthesis. PBCV-1 added to the story in that a virus encoded such an enzyme.¹¹ Moreover, the activity of the PBCV-1 A098R protein was supported by two other virally encoded enzymes necessary to produce the sugars composing hyaluronan (Figure 12a): UDP-Glc dehydrogenase (UGDH, 389 aa, gene *a609l*) synthesizes UDP-α-D-GlcA, and glutamine:Fru-6P aminotransferase (GFAT, 595 aa, gene *a100r*) is involved in GlcNAc synthesis (Figure 12a, Table 2). The closest homologues of A100R and A609L enzymes are from bacteria, *Aquifex aeolicus* (41% identity over 602 aa) and *Vibrio cholerae* (56% identity over 390 aa).¹⁴⁶

HAS enzymes are divided into two classes, I and II, with class II having HAS from the bacterium *Pasteurella multocida* as a unique member.²⁰¹ PBCV-1 HAS belongs to class I based on its amino acid sequence and structure, it is closer to eukaryotic enzymes (45% sequence similarity with the human HAS2)¹⁴⁵ than to the bacterial counterparts,¹⁴⁶ and its structure was recently determined by a combination of cryo-EM, molecular modeling, and functional studies.¹⁴⁵
<figure><img src="image_19.png" /><figcaption>**Figure 12.** PBCV-1 hyaluronic acid biosynthesis. (a) Activities of the viral enzymes A609L and A100R, involved in the production of the two nucleotide sugars, UDP-α-D-GlcA and UDP-α-D-GlcNAc, respectively. (b) Synthesis of hyaluronic acid catalyzed by A098R; notably, this enzyme requires GlcNAc as primer to add the other monosaccharide units, thus elongating the polysaccharide chain from the nonreducing end. (c) Electron microscopy images of the *Chlorella* cell wall: top, uninfected algae; middle, image of the cell wall taken 240 min PI; bottom, cell wall of the infected algae treated with hyaluronidase. “Cyto” indicates the cytoplasm of the cell, while “C” the cell wall. (d) Adapted with permission from ref 205. Copyright 1999 Elsevier.</figcaption></figure>

The PBCV-1 HAS enzyme was unexpected because, until then, hyaluronan had only been found as a component of the extracellular matrix of vertebrates and of the extracellular capsule of some pathogenic bacteria.²⁰² Hyaluronan is a linear polysaccharide belonging to the family of glycosaminoglycans²⁰³ composed of alternating 4-linked β-D-GlcA and 3-linked β-D-GlcNAc residues, with MWs up 10⁶ Da (Figure 12b). As a fundamental component of the extracellular matrix in vertebrates, it interacts with several proteins such as CD44, RHAMM, and fibrinogen. Thus, it is involved in several cellular processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion.²⁰⁴

PBCV-1 HAS is 568 aa long with high similarity to both vertebrate and bacterial HASs.¹¹,²⁰⁵ The gene was expressed in *E. coli*, and biochemical assays demonstrated that the recombinant protein requires manganese for activity,¹¹,¹⁴⁵ opposite to the streptococcal and mammalian HASs that prefer magnesium.²⁰¹ The structure consists of six transmembrane helix and three interface helix domains which enclose a cytoplasmatic region endowed with a GT-A fold and that possesses the catalytic activities necessary to produce hyaluronan. To this end, A098R requires GlcNAc monosaccharide as a primer for the polymerization reaction, which occurs by the sequential addition of GlcA and GlcNAc, by using the corresponding UDP nucleotide sugars as activated donors of the reaction. Thus, the polysaccharide chain elongation occurs on the nonreducing end of the polymer, like HAS class II enzymes, and opposite to what was reported in an earlier study (Figure 12b).²⁰⁴ Interestingly, the high sequence similarity (about 45%) between the viral HAS and the human HAS2,¹⁴⁵ one of three human encoded enzymes and the only one so far proved as essential, suggests that the same mechanism applies to HAS2, if not to many other class I enzymes.

Expression of the three viral genes, *a098r*, *a100r*, and *a609l*, has major consequences on the morphology of the host

*Chlorella* (Figure 12c). The three genes are expressed early in viral infection, and hyaluronan fibers appear on the infected cell surface within 30 min PI. The fibers continue to accumulate on the chlorella cell wall surface for at least 4 h and cover the entire surface of the infected algae, as revealed by quick-freeze, deep etch microscopy analysis (Figure 12c). The fibers can be removed by treatment with hyaluronidase, restoring the phenotype of the cell wall back to that of the uninfected algae (Figure 12c).²⁰⁵

Because PBCV-1 produces three enzymes involved in hyaluronan biosynthesis, this polysaccharide would seem to have an important function in the PBCV-1 life cycle. However, this hypothesis was soon challenged as the *a098r* gene is not present in all chloroviruses that infect *Chlorella* NC64A,²⁰⁵ indicating that this enzyme is not always essential for replication. One explanation is that the *has*-lacking viruses might encode an enzyme(s) that produces an alternative polysaccharide on the external surface of the infected *Chlorella* cells.

This hypothesis was confirmed by Kawasaki et al.,²⁰⁶ who reported that some chloroviruses have a gene encoding a functional chitin synthase (CHS) instead of HAS and that these viruses produced chitin fibers surrounding the external surface of infected *Chlorella* cells. Furthermore, some chloroviruses (e.g., CVIK1 and CVHA1) contained both *has* and *chs* genes. These viruses therefore produce both hyaluronan and chitin polysaccharides simultaneously on the surface of the infected host cells,²⁰⁶ suggesting that there is no functional incompatibility between the two genes or their products. Even more interesting is the fact that some chloroviruses lack both *has* and *chs* genes and do not produce any extracellular polysaccharide on the surface of their host after infection.

Like the *has* gene, the *chs* gene is expressed as early as 10 min PI, producing chitin, which is rare in algal cell walls, although it has been reported in some green algae.²⁰⁷ Further
support for the presence of chitin on the external surface of the
infected chlorella cells was provided by the observation that
treating the algae with chitinase abolished the dense fibrous
network.²⁰⁶ The CHS enzyme belongs to the GT2 family of
processive polymerizing GTs, which produces chitin, a
homopolymer of 4-linked β-D-GlcNAc, that may act both as
a structural element and as a signaling molecule, similar to the
hyaluronan.

Comparative genomic analysis between viruses CVK2 and PBCV-1 revealed high identity except for a few differences. Among them, the region corresponding to the PBCV-1 *has* gene was absent in CVK2 and replaced by a 5 kb region containing fragmented ORFs, some of which had similarity to *chs* genes from other species, together with *udp-gdh2* (a gene encoding a second UGDH).²⁰⁶ Comparison of the predicted amino acid sequence of CVK2 *chs* with those from various organisms indicated that it had a high similarity to the CHS3-type enzyme from yeast and fungi, even though its size was much smaller than the fungal enzymes (516 amino acids compared to ~1200 amino acids) with the sequence homology restricted to the C-terminal region, where the catalytic site occurs. The smaller size of the CVK2 CHS protein could be explained by its simpler regulatory and processing mechanism or by a different location in the cell.²⁰⁸ Kawasaki et al. also demonstrated that the newly synthesized chitin was efficiently secreted across the *Chlorella* membrane and cell wall to the outside of the infected cell, meaning that the CVK2 CHS is probably located in the cell plasma membrane, where the chitin molecules are synthesized and pushed through the cell wall.²⁰⁶ The *chs* gene has also been found in NY-2A and AR158 chloroviruses as well as in other large dsDNA viruses, such as Ectocarpus siliculosus virus EsV-1 (*Phaeovirus* genus of the *Phycodnaviridae* family, Table 1),¹¹⁴ and Fadolivirus (*Klosneuvirinae* subfamily, in *Mimiviridae* family, Table 1).

Interestingly, all chloroviruses contain the *gfat* gene required for the synthesis of GlcNAc, a sugar common to both hyaluronan and chitin polysaccharides.¹³ In contrast, some chitin-synthesizing viruses loose *ugdh* used for the synthesis of UDP-α-D-GlcA, necessary for hyaluronan synthesis.¹³ Therefore, it could be hypothesized that for viruses that produce chitin, the components of HA synthesis become obsolete and are lost.²⁰⁸

The synthesis of hyaluronan and/or chitin requires a huge amount of energy, and the destiny of the host cell at the end of the infection process is lysis to release the progeny viruses. Therefore, why the chloroviruses synthesize hyaluronan and/ or chitin is unknown. Another interesting question is how do the hyaluronan and chitin fibers pass through the cell wall. In fact, when the chlorovirus *has* gene was expressed in a higher plant, hyaluronan did not pass through the cell wall but collected between the plasma membrane and the cell wall.²⁰⁹ The ability of chloroviruses to manipulate the host by modifying its cell surface with other polysaccharides is remarkable, although the reason for this manipulation and the advantages gained for the virus await to be discovered.

**3.3.2. Chloroviruses Encode the GTs that Synthesize Glycans Attached to Proteins. 3.3.2.1. Historical Background.** It was predicted in 1993 that the chloroviruses might encode most, if not all, of the machinery to glycosylate their MCP(s) and that the process occurred in the cytoplasm.¹⁷⁵ For the chlorovirus PBCV-1 MCP (Vp54, gene *a430l*), this conclusion originally arose from antibody studies.¹⁷⁵ Rabbit polyclonal antiserum prepared against intact PBCV-1 particles

inhibited virus plaque formation by agglutinating the virions. However, spontaneously derived, antiserum-resistant plaque forming PBCV-1 antigenic variants occurred at a frequency of 10⁻⁵ to 10⁻⁶. Polyclonal antiserum prepared against members of each of these antigenic classes reacted primarily with the Vp54 equiv from the viruses used for each immunization. The Vp54 proteins from the antigenic variants migrated faster on SDS-PAGE than that from WT. However, the nucleotide sequence of the *a430l* gene in each of the variants was identical to the WT gene, which verified that the polypeptide portion of Vp54 was not altered in the mutants. Western blot analyses of Vp54 proteins isolated from the variants, before and after removing the glycans with triflic acid or altering the glycan with periodic acid, also supported the notion that the antigenic variants reflected differences in the Vp54 glycans, not the Vp54 polypeptide.¹⁷⁵ Notably, the antigenic variants were grown in the same host so that the glycan differences were not due to the host but to the genes encoded by the virus, thus supporting the idea that the virus has its own glycosylation machinery. These results indicated that the differences between the different capsid proteins resided in the size of the glycans.

Subsequent DNA sequencing and annotation revealed that the PBCV-1 genome encoded eight putative GTs genes (*a064r*, *a071r*, *a075l*, *a111/114r*, *a219/222/226r*, *a301r*, *a473l*, *a546l*), some of which were predicted to be involved in MCP glycosylation, in addition to the hyaluronate synthase gene *a098r* (section 3.3.1).¹³,¹⁰¹ The cellular protein localization program PSORT predicted that most of these GTs were soluble and located in the cytoplasm, i.e., they lacked an identifiable signal peptide that would target them to the ER and Golgi.¹³ The exceptions were A473L and A219/222/226R, which were predicted to contain six and nine transmembrane domains, respectively.¹³ Moreover, these eight genes are expressed early during PBCV-1 infection,²¹⁰ suggesting that they were good candidates for adding sugars to the Vp54 MCP during viral replication.

Many of the PBCV-1 antigenic variants had mutations in *a064r*, a gene encoding a protein with three catalytic domains, or had the gene deleted and/or deletions elsewhere in the genome (Figure 13). The variants are organized into six antigenic classes, A to F, based on their different reactions to polyclonal antiserum, and representatives from all the antigenic classes (except E) have been studied. The antigenic class B had a mutation in A064R domain 1; the class A domain 1 was intact but the second domain had mutations. Class C included viruses in which no mutations occurred in the *a064r* gene (except E11), but mutations occurred elsewhere in the genome (Figure 13). Viruses that had large deletions in the region nearby and inclusive of *a064r* were members of the D antigenic class. Finally, class F is characterized by an early truncation of the third domain of the A064R protein and includes only one virus.¹³,¹⁴³ The structure of the N-glycans of some representatives of each antigenic class have been determined and related to the type of mutation in the virus, thus leading to the identification of the potential functions of some of the virus-encoded GTs.

**3.3.2.2. The Proteins A064R and A061L (Table 2).** The *a064r* gene encodes a 638 amino acid protein, and in silico analysis predicted that it had three domains of ~200 amino acids each. The N-terminal domain (A064R-D1, from 1 to 212 aa) was the first to be studied due to its resemblance to a GT, and its crystallographic structure was determined before its GT specificity was known (inset in Figure 14).¹⁴² This domain has
<figure><img src="image_20.png" /><figcaption>**Figure 13.** The N-glycans of PBCV-1 antigenic variants. Most of the antigenic variants have a mutation in the *a064r* gene that encodes the protein A064R with three domains (D1, D2, and D3), as sketched near the structure of the N-glycan representative of each antigenic class, including the wild-type PBCV-1. A red mark indicates a point mutation, a blue mark indicates an insertion, and the length of the segment is proportional to the level of truncation of the translated protein. All sugars are in the pyranose form except where specified; linkages drawn with a broken line denote a nonstoichiometric substitution.</figcaption></figure>

a mixed α/β fold containing a central, six-stranded β-sheet flanked by α-helices and a small, two-stranded β-sheet. The fold is similar to catalytic GT domains in the GT-A group, in subfamily 34, although the amino acid sequence similarity is very low. This GT subfamily has a retaining mechanism and the typical DXD motif that coordinates the sugar nucleotide phosphate together with a divalent cation.¹⁴² Furthermore, this study established the A064R-D1 specificity for UDP- rather than other sugar nucleotides, recognizing UDP-α-D-Glc as the preferred sugar donor and Mn²⁺ as the bivalent cation.¹⁴² It took several years to determine that not all of these initial assumptions were correct. Domain 2 (A064R-D2, 213–405 amino acids) resembled a few proteins of unknown function in GenBank, and the C-terminal (A064R-D3, 439–638 aa) was predicted to be a methyltransferase. The activities of each of the A064R domains became clear once the N-glycan structures of the antigenic variants were determined (Figure 13).¹⁴³

First, the *a064r* gene of the two representatives in the antigenic class B (EPA1 and EPA2) led to proteins either with a point mutation or a truncation in the first domain (A064R-D1), and the N-glycans of the two mutants were identical and truncated at the level of the distal Xyl, with β-L-Rha missing compared to the N-glycan of wild-type PBCV-1 (Figure 13).

In contrast, variants belonging to the antigenic class A (P91, P9L1, P9L10) had the first A064R domain intact, and their N-glycans were truncated at the level of the β-L-Rha unit, suggesting that the A064R-D1 function was related to the formation of the β-L-Rha-(1→4)-Xyl linkage.¹⁴³ A064R from the CME6 mutant had a truncation in the third domain (Figure 13), and the corresponding N-glycan had the terminal α-L-Rha unit, but it was not methylated like wild-type PBCV-1.¹⁴³ These findings indicated that the second domain of A064R was a α-L-Rha transferase, while the third domain was a methyltransferase, although it was not clear if the third domain had a single or a double transferase activity.

All of these hypotheses were validated by expressing both the entire A064R protein and/or each domain individually and testing their activities in vitro with appropriate substrates.¹⁴¹ All proteins were expressed in *E. coli* cells as fusion proteins and UDP-β-L-Rha was used as a donor substrate for both A064R-D1 and A064R-D2 (Figure 14). This sugar-nucleotide was prepared by using the APMV enzyme L780 (section 3.1.2.1). The experiments were conducted in the presence of either Mn²⁺ or Mg²⁺; EDTA was used as a chelating agent to investigate the cation dependency of the reaction, and the reaction products were analyzed via NMR spectroscopy (Figure 14).¹⁴¹

The synthetic oligosaccharide 1 (Figure 14) was used as a simplified version of the EPA1 N-glycan as substrate for A064R-D1. This experiment confirmed that the first domain (amino acids 1–212) was the β-L-rhamnosyltransferase that transferred the Rha unit from the donor onto the fourth position of Xyl in 1 to give product 2. Notably, this enzyme could also use Xyl as the acceptor monosaccharide, while it could not transfer Glc, the donor hypothesized in the crystallographic study. Moreover, the reaction occurred via a retaining mechanism and in the presence of Mn²⁺ cation, in agreement with the previous work.¹⁴²

A064R-D2 was assayed by using 2 as a substrate (Figure 14); the assay confirmed that D2 was the α-L-rhamnosyl-transferase that links the α-L-Rha unit to O-2 of the β-L-Rha unit to give 3 via an inverting mechanism without the need for a cation. The cloning of this enzyme also revealed the limits of the in silico analyses. The size of the first construct (amino acids 191–405) matched that of the in silico analyses but was not active; however, the protein with an additional 35 amino acids at the C-terminus was. Of note, A064R-D2 was produced in two versions, D2L (amino acids 191–438) and D2L₂ (amino acids 213–438); both were active and could also glycosylate free rhamnose in addition to 2.
<figure><img src="image_21.png" /><figcaption>**Figure 14.** Biochemical assays of A064R and its individual domains. Substrate 1 was used as an analogue of the N-glycan from a class B variant (Figure 13), and it was transformed into 2 by A064R-D1 and, in turn, converted into 3 by A064R-D2. Substrate 1 was used to assess the activity of the complete A064R and validated domain 3 as responsible for adding a methyl group to the O-2 position of the terminal L-Rha. Product 4 (but not 3) was the substrate for A061L, which added the methyl to O-3 of the terminal L-Rha. The inset at the center displays the crystallographic structure of A064R-D1 and the sequence of the secondary structural motifs. Adapted with permission from ref 142. Copyright 2007 Elsevier. \*A064R-D1 could also use Xyl as a substrate. \*\*A064R-D2 could also use Rha as a substrate.</figcaption></figure>

The discovery of A064R-D2 activity was significant because this domain has no sequence homology with annotated rhamnosyltransferases (<30% with only 15% coverage), making it an outlier in a phylogenetic analysis. BLASTp investigation revealed that the A064R-D2 domain shares partial homology with many hypothetical and uncharacterized bacterial proteins, including one GT-family protein from *Lacipirellula parvula* (31.4% identity with 95% coverage) and two homologues (a hypothetical protein and a GT10 fucosyltransferase) in *Pithovirus*. Hence, A064R-D2 represents a new GT family.

The activity of the A064R-D3 domain was validated on the entire A064R enzyme together with acceptor 1, with two equivalents of UDP-β-L-Rha and SAM, in the presence of both Mn²⁺ and Mg²⁺ (Figure 14). The reaction produced product 4, thus confirming the activities of the first two domains of A064R and demonstrating that the third domain added only one of the two identified methyl groups, that at the O-2 position of the terminal α-L-Rha unit. The activity of the third domain was confirmed by a pBLAST search, returning many class-I SAM-dependent methyltransferases from both bacteria and viruses as the best matches, as well as several bacterial hypothetical proteins. This finding also suggested that there was another enzyme involved in the methylation at the O-3 position.¹⁴¹

PBCV-1 genome screening identified the *a061l* gene as encoding a 209 amino acid protein that shared high sequence similarity with several well-characterized class I SAM-dependent methyltransferases from bacteria and algae.¹⁴¹ A061L was

expressed as a recombinant protein with a GST-tag, its activity was assayed using SAM as the donor of the methyl group, both Mn²⁺ and Mg²⁺ as bivalent cations, and two different acceptors (Figure 14): one having the terminal α-L-Rha methylated at O-2 (4) and the other lacking the O-2 methylation (3). The experiment established that A061L was the methyltransferase that added the second methyl group to the terminal Rha. Moreover, this work showed that it had a specific substrate requirement, attaching the methyl group only if Rha was already methylated at O-2. Indeed, 3 was not transformed into a different product, while 4 gave the final product 5 (Figure 14).

Hence, these experiments determined a total of four activities by analyzing two different proteins. More importantly, the original predictions of A064R activity were correct and defined the first viral encoded enzyme involved in the synthesis of its own glycan. The three activities of A064R build the terminal part of the PBCV-1 N-glycan (Figure 15), and it should be noted that this enzyme is one of the few described to date that has three transferase domains, two of which are GTs.¹⁴¹,²¹¹ Once A064R has synthesized the terminal part of the N-glycan, A061L completes the decoration of the ultimate α-L-Rha with the second methyl group (Figure 15).

#### 3.3.2.3. PBCV-1 Proteins A075L, A071R, and A111/114R.
Regarding the other putative GTs mentioned in section 3.3.2.1, there is no experimental evidence to support their role in the synthesis of the Vp54 glycan. However, by combining in silico analyses with the structural determination of other chloroviruses N-glycans, initial predictions can be made.
<figure><img src="image_22.png" /><figcaption>**Figure 15.** Comprehensive view of PBCV-1 GTs. (a) Structure of PBCV-1 N-glycan with known and predicted GTs activities. Text in black indicates that the activity has been experimentally validated, otherwise (text in gray) the activity was deduced by indirect evidence. (b) Crystallographic structure of B736L (PDB 3OY2) from the chlorovirus NY-2A, which shares high conservation with A546L of PBCV-1, whose activity is yet to be uncharacterized. Reproduced with permission from ref 213. Copyright 2010 American Society for Microbiology.</figcaption></figure>

**Gene a075l.** The genome of PIL6, a member of the antigenic class D (Figure 13), has a large deletion from gene *a014r* to *a078l*, and its N-glycan is restricted to the conserved core structure, suggesting that the gene encoding the enzyme involved in the addition of the distal D-Xyl is in this region. In parallel, the distal Xyl is also missing in all class C antigenic variants (E1L3 and E11, Figure 13), which all have mutations in the *a075l* gene.143 These observations suggest that A075L is the xylosyltransferase that links D-Xyl to L-Fuc (Figure 15), although it was previously annotated as exostosin, a glycosyltransferase involved in heparin/heparan biosynthesis.212 This hypothesis is further supported by the analysis of the other chloroviruses, whose MCP glycan structures have been determined (Figure 9).179,191,192 Indeed, all these chloroviruses encode orthologues of the PBCV-1 A075L protein, and all have the distal D-Xyl attached to the L-Fuc, like PBCV-1. Further evidence suggesting that A075L links D-Xyl to L-Fuc is provided by the recent finding that chlorovirus MA-1D has a mutation in the *a075l* orthologous gene, and its N-glycan lacks this residue (Figure 9).193

**Gene a071R.** The N-glycans of the antigenic classes D (Figure 13) also differ in another monosaccharide, D-Rha. Because PIL6 (class D variant) has no mutations in its genome other than the large deletion that extends from gene *a005r* to *a077l*, the gene encoding this D-rhamnosyltransferase must be located within these genes. This gene must be conserved in other NC64A and OsyNE5 chloroviruses, which have a D-Rha attached to the L-Fuc, and it must be absent from SAG and Pbi viruses because both lack D-Rha at that position (Figure 9).179,191,192 The only PBCV-1 gene that fulfills all of these prerequisites is *a071r*. This gene encodes a 354 aa protein previously not annotated as a GT, but whose sequence resembles that of A064R-D2, which also escaped from in silico analysis due to a lack of homology with any known GT. Accordingly, A071R should be the GT that attaches D-Rha to O-3 of L-Fuc (Figure 15).

**Gene a111/114r.** This gene is conserved in all the sequenced chloroviruses and PBCV-1 variants. The gene product is a 860 aa protein that has been proposed to be involved in the synthesis of the conserved four-sugar oligosaccharide core shared by all chloroviruses studied to date. This prediction has been supported by a combination of bioinformatic and preliminary biochemical analysis, which indicated that A111/114R, like A064R, is a three-domain GT likely involved in the attachment of three of the four monosaccharides of the core N-glycan.147 HHpred analysis

combined with the three-dimensional protein models built by Phyre2, suggested that the N-terminal domain (from 1 to 260 aa, A111/114R-D1) is a galactosyltransferase, the central domain (261–559 aa, A111/114R-D2) a xylosyltransferase, and the C-terminal domain (560–860 aa, A111/114R-D3) a fucosyltransferase (enzymes characteristics are reported in Table 2). Domains 2 and 3 both have the DXD motif necessary to bind bivalent cations.

The intact PBCV-1 A111/114R protein and each domain were expressed as fusion proteins with a maltose-binding protein (MBP) tag at the N-terminus, and bioluminescent UDP/GDP-Glo assays were performed on the recombinant proteins to detect free UDP/GDP released by GT-mediated hydrolysis of the sugar nucleotides.147 These experiments revealed the preferred nucleotide donor for each domain: UDP-α-D-Gal, UDP-α-D-Xyl, and GDP-β-L-Fuc for A111/114R-D1, -D2, and -D3, respectively (Figure 15). These results agree with the HHpred analysis for the function of each domain and also determined their correct boundaries, that, like A064R-D2, were not accurately predicted in silico. Currently, all these preliminary data remain to be experimentally validated.

**Other PBCV-1 GTs.** Not much is known about the other predicted GT proteins: A301L (241 aa), A219/222/226R (677 aa, putative GT2), A473L (517 aa, GT2), and A546L (396 aa, a GT-B type). We can speculate that among them one should be involved in the linkage of Glc to Asn or to a lipid carrier (this is unresolved). In addition, we are still missing the GTs that link Man to D-Rha and Araf to L-Rha.

The PBCV-1 A546L protein presents 81% sequence identity with a predicted GT, B736L from chlorovirus NY-2A (405 aa) whose crystallographic structure is available.213 Out of the tested nucleotides, GDP-α-D-Man bound best to the protein, and B736L crystals were obtained both with and without GDP-α-D-Man (PDB 3OY7 and 3OY2, respectively). The structure has two Rossmann-like folds (from 1 to 163 aa and 164 to 355 aa) separated by a cleft, exhibiting the typical GT-B type structure. Residues 356 to 392 formed a long α-helix that spanned both the N-terminal and the C-terminal domains, and no metal ions were visible in the cleft of B736L, despite Mg²⁺ cations being added to the crystallization buffer. Structural comparison of B736L with other GDP-α-D-Man binding GTs indicated that crucial amino acids in the catalytic site were preserved. These results led to the proposal that B736L enzyme is a mannosyltransferase. However, because the N-glycans attached to NY-2A MCP lack Man (Figure 9), this
<figure><img src="image_23.png" /><figcaption>**Figure 16.** Crystallographic structure of the C-terminal domain of L230. L230 is a bifunctional enzyme with GT and lysyl hydroxylase activities. (a) Representation of the secondary motifs of L230 C-terminal domain and its arrangement as a dimer. The small inset highlights amino acids involved in Fe²⁺ cation (orange) coordination. (b) Surface representation of the L230 active site: the residues inside the binding pocket conserved in the human enzyme LH2 are in yellow and otherwise in white. Residues outside the binding pocket are in purple. Reproduced with permission from ref 153. Copyright Springer Nature.</figcaption></figure>

suggests that either: (i) the acceptor substrate might not be the virus MCP but some other protein, or (ii) that there is another preferred sugar nucleotide donor, or (iii) the enzyme is not functional.

**3.3.3. Megavirinae GTs: a Focus on APMV GTs.** As mentioned previously, several mimiviruses have been analyzed for glyco-related genes, and these studies revealed that most of them are organized in clusters.⁸⁷ The presence of genes encoding putative GTs next to those encoding sugar nucleotides of the monosaccharides present in the fibrils (section 3.1.2.5, Figure 8) led the authors to speculate that these enzymes were involved in the formation of the glycans that decorate the fibrils. Currently, APMV is the only *Mimiviridae* member for which the glycan structures have been determined (section 3.2.2),⁶⁵,¹⁹⁴ and this information has been used to determine the role of the GTs identified in the cluster. Two additional GTs have been characterized, L230 and R707 (enzymes characteristics are reported in Table 2),²⁴,²⁵ located outside the glycogene cluster and with no evident relationship with the structure of the fibril glycans. In 2011, Luther et al.²⁴ studied the gene l230, which encodes a bifunctional enzyme with the N-terminal domain (1–194 aa) bearing a GT activity and the C-terminal domain (537–895 aa) with a lysin-hydroxylase activity, which can modify collagen. Collagen is an animal protein, and homologues have been found in APMV: L71, R196, R239, R240, R241, L668, and L669, all of which contain the Gly–X–Y repeat typical of collagenic proteins, where X and Y are often Pro and Lys, respectively. However, these viral proteins were rather low in proline content in contrast to their mammalian counterparts.

The N-terminal domain of L230 presents 27.7% amino acids identity (61% similarity) over 285 amino acids with a human GT, GLT25D1, which adds Gal to collagen.²¹⁴ The C-terminal part of the protein has 35% identity (68% similarity) over 388 amino acids, with the human lysyl hydroxylase PLOD1 enzyme. Both activities of the viral enzyme were experimentally demonstrated, and mutagenesis experiments showed that each domain activity was independent of the other.

The residues ⁸²⁵Hys (histidine) and ⁸²⁵Asp in the C-terminal domain of L230 were essential for lysyl hydroxylase activity. Experiments performed with different peptide substrates showed that this domain could hydroxylate Lys in both

human- or viral-like sequences, with the peptide derived from the APMV R240 protein being preferred above the others. The crystallographic structure of this domain (680 to 895 aa) was determined (PDB 6AX6, Figure 16).¹⁵³ It formed a dimer with an overall fold resembling that of Fe²⁺ and 2-oxoglutarate (2-OG) dependent oxygenases, like human LH2, which is also a bifunctional enzyme.¹⁵³ The typical residues of the oxygenase domain involved in Fe²⁺ coordination (⁸²⁵Hys, ⁸²⁷Asp, and ⁸⁷⁷Hys) and in the 2-OG binding (⁸¹⁴Thr and ⁸⁸⁷Arg) were conserved and their mutation completely abrogated the protein activity. Their dimer formation involved both ⁸⁷³Leu and Fe²⁺ ions, as L873D mutation of the residue coordinating Fe²⁺, or demetalation, prevented dimerization. Dimer formation increased the ability of the enzyme to bind the collagen chains, and it was required to hydroxylate the substrate.

Regarding the GT activity, L230 added Glc instead of Gal to the substrate and it also had a certain permissivity in terms of substrate, as it was active both on human- and viral-like peptides, as observed for the hydroxylase domain. However, mutation experiments meant to define the key amino acids for catalysis, did not produce clear results. Mutation of ¹⁰⁶Glu, ¹⁰⁷Asp, ¹⁰⁸Asp (the putative DXD motif involved in the coordination of the cation with the sugar-nucleotide), ⁷⁸Leu, ⁸⁰His, ⁹⁵Asp, ⁹⁹Asp, and ¹³¹Asp, all produced unstable proteins that could not be studied.

The activity of full length L230 protein was validated on peptides derived from both viral collagen-like protein L71 and human collagen, with the result that L230 reacted with the Lys residues in all the substrates and added a Glc unit onto the newly formed hydroxylysine units.²⁴ Of note, analysis of the APMV particles confirmed the presence of hydroxylated lysine residues in the collagenic proteins, albeit not their glucosylation.²⁴

L230 was the first collagen-glycosylating enzyme found outside the animal world, and its GT activity is specific for Glc and not for Gal, the sugar attached to the collagen in all animal sources. The attachment of Glc to viral collagen makes it unique, thus raising the question on the role it could have played in the evolution of collagen biology.

In 2016, a second GT from APMV, R707, was characterized.²⁵ A BLAST search revealed that R707 is a
<figure><img src="image_24.png" /><figcaption>**Figure 17.** Electron microscopy images detailing some of the phases of PBCV-1 infection. (a) Attachment of the virus on the host cell (infection time zero). Reproduced with permission from ref 216. Copyright 1991 American Society for Microbiology. (b) Degradation of the wall at the site of attachment, typically 1–3 min PI. Reproduced with permission from ref 215. Copyright 1984 Elsevier. (c) PBCV-1 particles in the so-called virus assembly centers or viral factories in the cytoplasm of the host, ~5 h PI, the DNA containing particles are dark, otherwise the capsids are empty. Reproduced with permission from ref 217. Copyright 1986 Elsevier. (d) Lysis of the host cell plasma membrane and cell wall and release of progeny viruses at ~7 h PI. Reproduced with permission from 218. Copyright 1981 Elsevier.</figcaption></figure>

homologue of the animal glycogenin-1, with which it shares 26% amino acid sequence identity and 42% similarity, which increased to 58% identity when focusing on the amino acid residues in the GT catalytic pocket, thus suggesting a similar function. Indeed, a model of R707 generated with Phyre using glycogenin as reference confirmed that R707 shared the typical glycogenin DXD motif (¹⁰¹Asp, ¹⁰²Leu, and ¹⁰³Asp). Additional experiments demonstrated that the protein coordinated UDP-α-D-Glc in the presence of Mn²⁺. The mutation of ¹⁰³Asp or of ²³²Lys, two residues important for UDP-α-D-Glc recognition, led to an inactive enzyme.

Interestingly, R707 could use both pNP-α-Glc and pNP-α-Xyl as acceptors, although it preferred the first. Using pNP-α-Glc produced disaccharides with Glc linked via α-(1→6), β-(1→6), or α-(1→4) linkages. Thus, R707 showed a limited selectivity in terms of site of attachment (4 or 6) and of configuration of the newly formed glycosidic linkage (α or β), with this last feature being unique among all the GTs studied.

In contrast to glycogenin, R707 cannot self-glucosylate even though it has the same conserved Tyr residue; mutation of this residue (²¹⁵Tyr) had no effect on the stability and activity of the protein. It was therefore speculated that R707 was not involved in glycogen formation as glycogenin self-glycosylates itself at one Tyr residue to prime the synthesis of the polymer. Indeed, APMV uses energy from the host, and it would not be expected that this viral enzyme would be involved in energy storage. Instead, R707 could play a role in protein glycosylation by synthesizing the linear polymers of Glc identified in the O-glycans (section 3.2.2.1).¹⁹⁴

### 3.4. Glycosyl Hydrolases (GHs) and Lyases (PL)

An important part of the infection process is how viruses interact with the host cells; in particular, those hosts that have a carbohydrate-rich cell wall. This interaction is required as the point of entry of the virus to start the infection process as well as during the release of new progeny at the end of it.

As demonstrated for chloroviruses, these two check points target the glycans of the host, which are degraded by the action of some viral enzymes. Indeed, chlorovirus PBCV-1 infects the *Chlorella* NC64A host by (i) attaching to the cell wall (Figure 17a), (ii) degrading the wall at the point of attachment (Figure 17b), (iii) releasing viral DNA into the host cytoplasm while leaving an empty capsid at the top of the cell wall, (iv) replicating in the assembly centers or viral factories (Figure 17c), and (v) breaking the host cell wall to release the neosynthesized virion progeny (Figure 17d).²¹⁵,²¹⁶

Regarding the degradation of the host cell wall, this process occurs during the chlorovirus entry (Figure 17b), and again during the virion release (Figure 17d). In the first case, the

enzyme(s) is already packaged in the capsid to make a breach in the host (Figure 17b); it is subsequently produced at a certain time during the infection process to break the cell wall and to let the particles out of the host (Figure 17d). Accordingly, these cell wall degrading proteins have different roles and are not expressed at the same PI time. Their production is not continuous during the infection process, and it is tightly controlled because if it occurs too early, only incomplete virus particles would be released, which would lead to its demise.

A characteristic of virus-sensitive *Chlorella* microalgae is a rigid cell wall with a complex architecture likely involving different types of glycans.²¹⁹ The overall sugar composition of the cell wall is 7–15% glucosamine and also contains uronic acids and neutral sugars. Chloroviruses penetrate this cell wall, and early hypotheses suggested that enzymes capable of degrading polymers of glucosamine, like chitosan and chitin, were involved in the viral infection process. Overall, in silico analyses of the chloroviruses genomes led to the discovery of both hydrolases and lyases able to degrade glycans, and the studies reported in the next sections focus on those enzymes that have been experimentally characterized.

#### 3.4.1. Chlorovirus Chitinase and Chitosanase.
In silico analyses of chloroviruses indicated that chitinase and chitosanase genes are widespread, suggesting that they are important for viral replication. Chitinolytic enzymes are classified in the CAZY database into three different families, namely GH 18, GH 19, and GH 20, depending on their overall fold and mechanism of action.²²⁰,²²¹ GH families 18 and 19 act on chitin, while GH family 20 includes enzymes that catalyze the cleavage of GlcNAc from the disaccharide chitobiose or glucosamine from glycoconjugates. Regarding GH families 18 and 19, members of the latter are mostly from plants, while those of the former are from different sources (bacteria, fungi, insects, and plants).

As for chitosanases, these enzymes catalyze the endohydrolysis of chitosan and belong to families GH 46, GH 75, and GH 80 according to CAZY database.²²¹,²²² Members of the GH46 family are the most characterized. They are found in eubacteria and can be divided into five clusters, A to E. Chloroviruses encode chitosanases belonging exclusively to cluster C,²²³ and they all have an additional N-terminal domain of unknown function that is unique to chloroviruses. It can then be assumed their function is closely linked to a particular trait of the biology of these viruses.

##### 3.4.1.1. PBCV-1 Chitin and Chitosan Hydrolases.
The PBCV-1 gene a181/182r encodes a large protein (A181/182R, 830 aa, Table 2), and in silico analyses found that it was composed of three distinct domains.¹⁴⁹ An N-terminal domain
or domain 1 (aa 6–101) has homology to cellulose-binding domains, with its best homologue in *Cellulomonas fimi* (32% over 74 aa). The closest homologue of domain 2 (aa 101–411) corresponds to an endochitinase from *Aeromonas* sp. (36% identity over 168 aa). Domain 3 (aa 553–825) has 35% identity over 201 amino acids with a protein from *Ewingella americana*. Domains 2 and 3 are separated by a Pro-rich linker of 140 amino acids.

Originally, it was thought the two chitinases were encoded by two separate genes, *a181r* and *a182r*,²²⁴ but later reevaluation of the genomic sequence demonstrated that they were part of a single gene.¹⁴⁹ Of the two recombinant proteins, only A181R had detectable chitinase activity suggesting that the two domains could function independently. The two chitinase domains share 40% sequence identity, suggesting that they were the result of a duplication event, and both domains were classified into the chitinase family 18. Biochemical assays on the recombinant full-length protein, showed its optimum pH value was between 4 and 6 and was active up to 50 °C. Transcriptomic analysis revealed that *a181/182r* was expressed early, at 30 min PI and that its transcription was maintained throughout the entire replication cycle, although the protein was not packaged in the virion.

In contrast to A181/182R, A260R is a single domain chitinase within the GH family 18,²²¹ with 32–36% amino acid identity with bacterial and fungal chitinases (Table 2). This protein is endowed with both endo- and exochitinase activities and has an optimum pH between 5 and 9, up to 50 °C. Transcription of *a260r* started at 60 min PI, reached its maximum at 90–120 min PI, and it was maintained until the host cells lysed. Like A181/182R, A260R was not packaged in neosynthesized PCBV-1 virions.¹⁰⁹

The A292L protein is a chitosanase that belongs to the GH family 46,²²¹ with 97% amino acids sequence identity with the chitosanase from chlorovirus CVK2,¹³⁸ and about 29% identity with bacterial chitosanases (Table 2). The enzyme had a pH optimum value in the range 5 to 8 and was active up to 50 °C.¹⁴⁹ *a292l* exhibited the same expression pattern as *a260r*; it first appeared at 60 min PI and remained until the lysis of the cells with a maximum production at 90–120 min PI. Like the two PBCV-1 encoded chitinases, A292L is not packaged in the virion.¹⁰⁹

### 3.4.1.2. CVK2 Chitin and Chitosan Hydrolases.
Like PBCV-1, chlorella virus strain CVK2 also encodes chitinase (*vChti-1*), and chitosanase (*vChta-1*) enzymes (Table 2).¹³⁸,¹³⁹ *vChti-1* (836 aa) has 98% identity with A181/A182R from PBCV-1, with two chitinase domains assigned to the GH family 18. The first domain (144–413 aa) has the most similarity with *Saccharopolyspora erythrae* (30% identity), while the second domain (aa 560–836) has a match (35%) with the enzyme from *Ewingella americana*. Like A181/182R of PBCV-1, it was suggested that the two chitinase domains of *vChti-1* are the result of a duplication event in their common ancestor. Such events were also proposed for the hyper-thermophile archaeon *Pyrococcus kodakaraensis* KOD1 and for the plant pathogenic bacterium *Flexibacter* sp. FL824A.²²⁵

Regarding the timing of transcription, analysis of the viral RNA during the infectious cycle revealed that *vChti-1* was a late gene because it appeared after 120 min PI and remained up to 360 min PI, gradually decreasing afterward. Like its orthologue A181/182R in PBCV-1, the protein was not packaged in the virions, and it was proposed that *vChti-1* chitinase was not involved in the initial infection events, while

its presence in the host cytoplasm served to digest the host cell wall to enable viral release at the final stage of infection.¹³⁹

As for the chitosanase, *vChta-1* (328 aa) has 95% sequence identity with PBCV-1 A292L, with a predicted MW of 36.8 kDa. The recombinant protein was used to produce antibodies to monitor the presence of this protein in virus-infected cells.¹³⁸ This identified two proteins of 37 and 65 kDa, respectively, which were detected at 240 min PI. The larger protein was later packaged in the virion, while the other remained in the cell lysate. In-depth analysis of the CVK2 genome suggested that these two chitosanases arose from the same gene, and it was hypothesized that the *a292l* stop codon was fused with the next downstream gene by a read-through mechanism to produce the 65 kDa protein. It was proposed that the 65 kDa protein packaged in the virion was involved in cell wall degradation at the beginning of infection and the 37 kDa enzyme produced late during infection contributed to cell lysis at the final step of infection. However, subsequent experiments indicated that the 65 kDa protein was not packaged in the virion. Interestingly, *Chlorella* NC64A cells treated with both chitinase (*vChti-1*) and chitosanase (*vChta-1*) did not show relevant morphological changes, indicating that additional enzymatic activities are required to digest its complex cell wall.¹³⁹

### 3.4.2. PBCV-1 β-Glucanase.
PBCV-1 also encodes a functional *β*-(*1*→*3*)-glucanase,¹⁴⁴ A094L (Table 2), sharing 26–30% sequence identity with GH family 16 of endo-*β*-(*1*→*3*)-glucanases from several bacteria, with conservation of residues important for activity: ²³⁴Glu, ²³⁶Asp, and ²³⁹Glu. The recombinant protein A094L expressed in *E. coli* was able to hydrolyze laminarin, a linear polysaccharide made of *β*-(*1*→*3*) glucose units, better than other *β*-glucans, like lichenan and barley *β*-glucan, both having mixed *β*-(*1*→*3*)/*β*-(*1*→*4*)-linkages. A094L was active over a pH range of 4–10, with an optimum at about 8. Its activity increased from 25 to 65 °C, and was inhibited by 1 mM silver, cobalt, copper, or manganese ions.¹⁴⁴

Interestingly, this gene is expressed 15 min PI and disappeared after 60 min PI, so that it was classified as an early gene. For this reason, no role during the late phase of viral infection was proposed.¹⁴⁴,²¹⁰ Accordingly, this protein was not predicted to be involved in breaking down the host cell wall. Instead, it was hypothesized that it might degrade *β*-glucans stored by the host to produce the energy required for viral replication. Indeed, laminarin is a common reserve polysaccharide for many algae, although there is no experimental evidence that *Chlorella* NC64A also stores this polysaccharide. The search for this gene in other chloroviruses revealed it was not conserved,¹⁴⁴ suggesting it is not essential for their replication, although many of the viruses devoid of A094L homologue contained another gene, *bgl2*, also predicted to encode an endo-*β*-(*1*→*3*)-glucanase.²⁰⁸

### 3.4.3. CVN1 Alginate Lyase.
Chloroviruses can remodel the host cell wall also by using polysaccharide lyases (PL), as described for CVN1.¹⁴⁰ Screening of the CVN1 viral DNA library cloned into an *E. coli* expression vector, with the resulting *E. coli* strains assayed on *Chlorella* NC64A, identified the CL2 protein (333 aa, Table 2) encoded by *orf2* as capable of lysing different *Chlorella* species. The recombinant CL2 protein had 65% sequence identity with PBCV-1 A215L, a protein of unknown function, with a weak homology (20% sequence identity) with the mannuronate lyase from the mollusk *Turbo cornutus*.¹⁴⁰ Indeed, CL2 was a PL able to