<figure><img src="image_26.png" /><figcaption>**Figure 18.** Crystal structure of vAL-1(S) alginate lyase from chlorovirus CVK2. (a) Cartoon representation of the overall fold of the protein without ligand (PDB 3GNE). (b) Details of the CL2 complex with GlcA at pH 7.0 (PDB 3A0N), and (c) at pH 10.0 (PDB 3IM0). Depending on the pH, GlcA bound outside (pH 7, b) or inside (pH 10, c) the cleft located nearby A3–6 strands, as detailed by the interactions in place between the monosaccharide and the amino acids of the protein, that are not same in the two cases. Adapted with permission from ref 137. Copyright 2009 Elsevier.</figcaption></figure>

cleave alginate, an acidic polysaccharide produced by some
algae and bacteria made by D-mannuronic acid and L-guluronic
acid, in proportions that depend on the source used. The CL2
protein had an optimal pH of 10.5 and required Ca²⁺ ion for
activity as the addition of EDTA to the reaction mixture
inhibited its activity. CL2 was not active on other
polysaccharides such as chitin, chitosan, cellulose, or pectin.
Regarding its antialgal activity, CL2 degraded the cell wall of
*Chlorella* sp. NC64A, *Chlorella vulgaris* C-27, and *Chlorella vulgaris* C-207, suggesting all these green microalgae might
have alginate as a component of their cell wall.

**3.4.4. CVK2 Alginate Lyase.** By using an approach similar to that described for chlorovirus CVN1, it was found that chlorovirus CVK2 also encoded a PL enzyme. The gene encoding vAL-1 (349 aa, Table 2) has 93% sequence identity at the nucleotide level, with the gene encoding CVN1 alginate lyase CL2,¹⁴⁰ and 97% sequence identity with the gene encoding the PBCV-1 A215L, an uncharacterized protein.²²⁴ The vAL-1 gene was expressed early, at 60 min PI, and gradually decreased. At the end of the infection cycle, the vAL-1 protein was not incorporated into the viral particles but remained in the cell lysate, suggesting that its role was limited to the digestion of the cell wall prior to virion release.

Biochemical assays with the recombinant vAL-1 protein demonstrated that it had strong cell-lysing activity on different *Chlorella* species and that it was an alginate lyase.<sup>136,226</sup> Interestingly, vAL-1 had maximum activity at alkaline pH (about 10) and was minimally active at neutral pH, even though the enzyme could only digest the algal cell wall under physiological conditions. When the protein was stored at high protein and/or low salt concentration, it spontaneously degraded into vAL-1(S), by losing its N-terminal 105 amino acids resulting in a shift in the MW from 38 to 27 kDa. This N-terminal loss included 10 repeats of a Pro-Ala-Pro-Lys sequence that appeared to play an important role in the attachment of the protein to the algal cell wall.<sup>137</sup> As a result, vAL-1(S) lost the ability to degrade the algal cell wall, but its pH range of activity expanded from pH 7–10, compared to that of the native form, suggesting that the N-terminal domain regulated the lyase activity of the protein.

PLs can be endo- or exoenzymes, and vAL-1(S) could
switch from one to the other activity as a function of pH, with
endolytic activity at neutral pH, and exolytic activity at alkaline
pH.<sup>137</sup> The X-ray crystal structure of vAL-1(S) (PDB 3GNE)
determined that the enzyme belonged to the family 14 of PL
lyases and had a β-jelly roll fold composed of two antiparallel

β-sheets, named A and B, and two minors sets, named C and D (Figure 18a). vAL-1(S) was studied in complex with GlcA, an acidic monosaccharide used in place of mannuronic or guluronic acids, which were not available for the experiments. The vAL1(S)-GlcA complex at different pH values revealed that GlcA was bound outside of the active site cleft at neutral pH (Figure 18b) and in the cleft at alkaline pH (Figure 18c). These two different arrangements of the complex depended on the charge status of the different groups of the protein and of the ligand, and regulated the endo/exo activity of the enzyme in a unique way compared to other enzymes of the same class.<sup>137</sup>

**3.4.5. PBCV-1 A561L, a putative alginate lyase.** As noted above, chlorovirus PBCV-1 encodes five functional polysaccharide degrading enzymes, two chitinases, a chitosa- nase, a β-(1 → 3)-glucanase, and an alginate lyase. However, PBCV-1 does not use any of them to digest the host chlorella cell wall for virus entry because none of these enzymes are packaged in the virion. Re-examination of the 148 virus- encoded proteins that are packaged in the PBCV-1 virions led to the identification of A561L (649 aa, Table 2), a protein expressed late during the viral infection cycle.<sup>150,210</sup>

In silico analyses found that this protein is made of four
domains.¹⁵⁰ The first is an N-terminal transmembrane domain
1 (69 aa; A561L-D1) homologous to a transporter protein
encoded by *Listeria kieliensis*. The second domain, A561L-D2
(from aa 70 to 310), resembled the ToIA protein from
*Trichomonas vaginalis*, a protein similar to the Gram-negative
bacterial Tol proteins that are involved in the stability of the
outer membrane. The third, A561L-D3 (from aa 311 to 407),
had homology with the LEA 2 domain-containing protein
encoded by *Selaginella moellendorffii*, an important protein for
plants as it is involved in the hypersensitive response to plant
pathogen infection. The last, C-terminal domain (A561L-D4,
aa 408–649) shared 32% sequence identity over 211 amino
acids (50% similarity) with the C-terminal domain of A215L, a
close homologue of vAL-1, the chlorovirus CVK2 alginate
lyase (section 3.4.4).¹³⁷

Accordingly, the Phyre2 model of A561L-D4 corresponded
to alginate lyases in family 14 (87% coverage, 100%
confidence) with the *Aplysia kurodai* enzyme used as a
reference (PDB 5GMT). A561-D4 was produced as a
recombinant protein in *E. coli*, and when assayed on chlorella
ghost cells (algal cells without chlorophyll by treatment with
methanol) dyed with calcofluor-white, it induced the release of
the dye, thus demonstrating the protein ability to degrade the
algae cell wall. Optimal activity was at 37 °C and at pH 7.0–8.0; while the activity was not cation dependent, it was enhanced by Ca²⁺ and Mg²⁺ cations.

A561L extracted from PBCV-1 virions was shown to degrade the cell wall of different *Chlorella* isolates, thus demonstrating it was not host-specific. The *a561l* gene was conserved in all the 52 chloroviruses that have been sequenced, with domains D2 and D4 being the most conserved between all viral strains. The biochemical activity of A561L-D4 has not been determined, although its strong homology with the other proteins suggests that it has alginate lyase activity. Therefore, this enzyme is believed to be responsible for degrading the host cell wall during virus infection.

## 4. CHEMICAL SYNTHESIS OF GLYCANS INSPIRED FROM GIANT VIRUSES

Given their unusual structures, viral glycans have attracted the attention of synthetic chemists. To date, efforts to synthesize these molecules have focused on two areas: (1) derivatives of viosamine, a component of APMV glycans, and (2) the complex N-glycans produced by chloroviruses.

### 4.1. Viosamine

The presence of viosamine (6, Figure 19), in its N-acetylated, 2-O-methylated form (7), was recently identified in APMV.⁶⁰ However, this monosaccharide, with different acyl groups, had been identified earlier in bacteria.²²⁷,²²⁸ Prominent among these derivatives is anthrose (8), a focus of many synthetic investigations.²²⁹⁻²³⁶ Anthrose is a monosaccharide found in the *Bacillus anthracis* glycoprotein BcIA,²³⁷ the detection of which has been suggested to have potential in diagnosing anthrax.²³⁸ In addition, N-acylated forms of perosamine (9), the C-2 epimer of viosamine, are found in a number of microbial glycans.²³⁹,²⁴⁰ This section will focus not only on previous syntheses of viosamine but also anthrose because straightforward modification of the routes used for the preparation of the latter could be applied to the viosamine derivative present in APMV glycans.

<figure><img src="image_27.png" /><figcaption>**Figure 19.** Structures of Vio4N and related derivatives from viral and bacterial sources. Viosamine (6); 2OMeVio4NAc (7), the Vio4N derivative found in APMV glycan poly_2; anthrose (8), a Vio4N unit methylated at O-2 and with a complex acyl group at N-4; N-acylperosamine (9), the C-2 epimer of Vio4N.</figcaption></figure>

Over 50 years ago, Stevens and co-workers were the first to report the synthesis of viosamine.²⁴¹,²⁴² Starting from the mesylated methyl 6-deoxy-glucopyranoside derivative **10** (Figure 20), a three-step inversion, deprotection, and mesylation sequence provided a 64% overall yield of **11**. Conversion of this compound to amine **12** was carried out in 77% yield via displacement with sodium azide and reduction with lithium aluminum hydride. Hydrogenation (giving **13**), N-acetylation, and the acid hydrolysis provided viosamine (6)

in 62% overall yield. An alternate, lower-yielding, approach was also reported starting from ditosyl galactopyranoside **14**. Displacement with sodium iodide led to **15**, which was reduced and benzoylated to give **16** in 23% overall yield. Azide displacement, reduction, and cleavage of the benzoyl groups provided **13** in 50% overall yield. The low yield of the conversion of **14** into **15** was due to the formation of the 4,6-di-iodo-derivative **17** in 45% yield.

Since these early syntheses, a number of other approaches have been developed, which, for the most part, use the same general strategy: the conversion of a readily available monosaccharide to a viosamine derivative. Rather than detail all of these, the general strategies employed are instead shown in Figure 21. The common approaches have started with D-Gal (18),²²⁹⁻²³¹ D-Fuc (19),²³²⁻²³⁴ or D-Man (20)²³⁵,²³⁶ and, in several steps, arrived at a 4-azido-4,6-dideoxy derivative **21**. Depending on the final goal, **21** can be converted to a target molecule **22** through a combination of azide reduction, acylation, methylation, and glycosylation in an order depending on the desired molecule and/or convenience. A twist on this general approach has been reported by O'Doherty, who developed a de novo route to molecules of this type starting from furfural (23).²⁴³

### 4.2. Chlorovirus N-Glycans

The unique structures of chlorovirus N-glycans have piqued the curiosity of synthetic chemists not only as an intellectual and technical challenge but also to provide probe molecules that can be used to understand their biosynthesis and function. The major challenge in the synthesis of these molecules is the construction of the hyperbranched central Fuc residue, in which every hydroxyl group is glycosylated. Such highly branched glycans are rare in nature, and understanding how to assemble these types of systems efficiently remains relatively poorly understood. The discussion below focuses primarily on the approaches used to install this highly congested motif.

The first chlorovirus N-glycan to be synthesized was the smallest of the structures, the hexasaccharide motif from ATCV-1 (24, Figure 22a or Figure 8).²⁴⁴ An initial attempt to make 24 involved a 2 + 4 coupling between the disaccharide acceptor 25 and the tetrasaccharide donor 26 as the key step. The latter of the compounds was prepared starting from the *p*-methoxylphenyl fucoside 27, which was selectively elaborated by a series of glycosylations and deprotections and finally conversion to the activated imidate donor. The sequence used to install the monosaccharide substituents to the Fuc involved glycosylation of O-3, then O-2 and finally O-4.

Attempts to couple 25 and 26 (Figure 22b) failed to provide the desired hexasaccharide product; instead, two major products were generated: hemiacetal **28** (15%) and bicyclic product **29** (61%). The former was produced by hydrolysis of the donor, and the latter presumably by intramolecular trapping of the oxacarbenium ion intermediate generated from **26** by O-2 of the Gal residue and loss of a benzyl group. The formation of such products²⁴⁵ points to the hindered nature of the acceptor alcohol in disaccharide **25**. However, all efforts to change the outcome through the use of a different activatable group in the donor, alternative activation conditions, or a different acceptor molecule, failed to produce the desired glycosidic bond.

Fortunately, the target could be assembled using a stepwise approach, the key steps of which are illustrated in Figure 23. Disaccharide alcohol **30** was assembled using appropriate
<figure><img src="image_28.png" /><figcaption>**Figure 20.** First syntheses of viosamine (6) by Stevens and co-workers.<sup>241,242</sup></figcaption></figure>

<figure><img src="image_29.png" /><figcaption>**Figure 21.** General synthetic approaches to 2-O-methyl-N-acylated viosamine derivatives.<sup>229-236,243</sup></figcaption></figure>

monosaccharide precursors and then glycosylated with Fuc thioglycoside 31, which contains three protecting groups that can be cleaved independently. This reaction produced trisaccharide 32 in 83% yield. The key strategic decision at this point was the order of the addition of the monosaccharides to the Fuc. A number of sequences were investigated, many of which failed. The successful route involved the "counterclockwise" addition of the substituent monosaccharides (glycosylation of O-4, then O-3 and then O-2) via donors 33, 34, and 35, respectively. Using this strategy, the hexasaccharide 36 was produced in 35% yield over seven steps. Reduction of the azide, installation of the amino acid and then total deprotection gave the final target 24 (five steps, 70% overall yield).

The same "counterclockwise" strategy was subsequently applied to a more complex target, a nonasaccharide from virus PBCV-1 (37, Figure 24).<sup>246</sup> A number of fragments of 37 have

also been synthesized to probe the biosynthetic pathway by which these compounds are assembled by the virus.<sup>141</sup>

Soon after the first synthesis of the chlorovirus ATCV-1 N-glycan was described, the synthesis of a related molecule (38, Figure 25) was reported by Ye and co-workers.<sup>247</sup> As was the case for the synthesis of 24, more than one approach was explored, with some being unsuccessful. The successful approach involved first the chemoselective activation of 40 in the presence of 39, leading to the disaccharide thioglycoside 41.<sup>248</sup> Subsequent addition of the benzyl glycoside 42 to the reaction mixture produced trisaccharide 43, in 79% overall yield. Debenzoylation of 43 proceeded in 92% yield, affording diol 44. Interestingly, glycosylation of this diol with thioxyloside 45 led to preferential reaction at the presumably less reactive axial alcohol giving tetrasaccharide 46 in 61% yield. The authors attributed this selectivity to steric blocking of the C-3 hydroxyl group by the adjacent Gal residue. Also formed, in 15% yield was a pentasaccharide derived from the addition of two Xyl residues to 44. The remaining hydroxyl group in 46 was then glycosylated with donor 47 producing pentasaccharide 48 in good (78%) yield. Regioselective opening of the benzylidene acetal afforded an 85% yield of alcohol 49, which was then glycosylated in 47% yield, with 45 producing the hexasaccharide 50. Global deprotection of 50 afforded 38 in 73% yield.

More recently, Hotha and co-workers reported the synthesis of a fragment of the ATCV-1 N-glycan.<sup>249</sup> The target was a tetrasaccharide containing the hyperbranched Fuc moiety (51, Figure 26) in protected form. The strategy made use of a gold-catalyzed glycosylation method with alkynyl-carbonate donors.<sup>250</sup> Glycosylation of 52 with donor 53 in the presence of catalyst 54 and silver triflate afforded a 98% yield of 55. Moving on from this disaccharide, two approaches were explored, both of which could successfully provide target 51. In one route, the levulinate ester was removed from 55, and the resulting product was glycosylated with donor 56, a sequence that afforded trisaccharide 57 in 87% yield over the two steps. Cleavage of the *p*-methoxybenzyl ether and glycosylation of the product alcohol with donor 58 yielded tetrasaccharide 51 in 83% overall yield. Alternatively, the *p*-methoxybenzyl ether in 55 was removed and the product was glycosylated with 58, providing an 88% yield of trisaccharide 59. Finally, a 78% yield
<figure><img src="image_30.png" /><figcaption>**Figure 22.** (a) High-level retrosynthetic analysis of the chlorovirus ATCV-1 N-glycan. (b) Attempted synthesis of the ATCV-1 hexasaccharide via a 4 + 2 coupling of 25 and 26.²⁴⁴</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>**Figure 23.** Successful synthesis of the chlorovirus ATCV-1 N-glycan 24.²⁴⁴</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>**Figure 24.** Chlorovirus PBCV-1 nonasaccharide N-glycan 37 that has been synthesized.²⁴⁶</figcaption></figure>

of tetrasaccharide 51 could be produced from 59 in two steps: removal of the levulinate ester and glycosylation of the resulting product with donor 56.

In summary, although glycans from large viruses are a relatively recent discovery, there have been approaches developed to chemically synthesize those that have been described. A number of routes are available to prepare viosamine derivatives. With regard to the more complex chlorovirus N-glycans, it has been possible to assemble the key hyperbranched Fuc residue that characterizes these structures via different strategies. When the Fuc is linked to a simple aglycone, the assembly of this motif is possible using a variety of addition sequences: i.e., O-3→O-2→O-4 glycosylation,²⁴⁴
<figure><img src="image_33.png" /><figcaption>Figure 25. Synthesis of the chlorovirus ATCV-1 hexasaccharide 38.<sup>247</sup></figcaption></figure>

<figure><img src="image_34.png" /><figcaption>Figure 26. Synthesis of 51, a protected tetrasaccharide containing the hyperbranched fucose residue in chlorovirus ATCV-1 N-glycans.<sup>249</sup></figcaption></figure>

O-4→O-3→O-2 glycosylation,<sup>249</sup> and O-4→O-2→O-3 glycosylation.<sup>249</sup> However, when the Fuc is attached to a carbohydrate residue, greater difficulties are observed. In these latter cases, the successful strategies described have been the “counterclockwise” addition of the substituents (O-4→O-3→O-2 glycosylation) starting from a trisaccharide acceptor,<sup>244,246</sup> or an O-2→O-4→O-3 glycosylation strategy

producing a pentasaccharide where the proximal Xyl residue is added last.<sup>247</sup> Given their unique structures, the synthesis of chlorovirus N-glycans will undoubtedly continue to attract interest.

Several chlorovirus N-glycans of greater complexity<sup>13</sup> remain to be synthesized. Examples include even more highly branched structures as that produced by the chlorovirus NY-
<figure><img src="image_35.png" /><figcaption>**Figure 27.** Examples of additional chlorovirus N-glycans that have not been synthesized.¹³ The species **60** is equivalent to the oligosaccharide **NY-2A**₁ isolated from the chlorovirus **NY-2A** (Figure 9), **61** is common in chloroviruses TN603 and MT325, and the difference between the two viral glycans is the methylation pattern detailed in Figure 9.</figcaption></figure>

2A (**60**, Figure 27, or **NY-2A**₁ in Figure 9) and those containing unusual monosaccharides, e.g., xylulofuranose in its unsubstituted or methylated forms (**61**), present in the chloroviruses MT325 and TN603 (Figure 8). Synthetic access to these molecules will require overcoming challenges in the construction of sterically congested glycans and access to usual monosaccharide residues, in addition to clarifying the absolute configuration of the xylulose unit, which is yet to be defined. Another goal should be to provide key fragments to assay the activity of viral GTs as has been done for PBCV-1 A064R-D1, the GT that forms the $\beta$-L-Rha-(1→4)-$\beta$-D-Xyl glycosidic linkage.<sup>141,251,252</sup>

## 5. CONCLUSIONS AND FUTURE PERSPECTIVES

The story of large and giant viruses started ~40 years ago, and since that time these entities have continued to surprise the scientific community. Giant viruses are unusual, and their size ranges from 150 to 2000 nm (Figure 1, Table 1), and the genomes of some of them encode up to 1500 proteins. This number of proteins is just the tip of the iceberg because their annotation reveals many other surprises: a large proportion of these proteins have functions remaining to be discovered. Of the annotated proteins, many are uncommon in viruses, such as the genes involved in carbohydrate metabolism.

The past few years have seen a significant advances in the structural glycobiology of giant viruses, with experimental-based data collected primarily on two of them: PBCV-1 and APMV, belonging to *Phycodnaviridae* and *Mimiviridae* families, respectively (Table 1, section 2). The first report connecting giant viruses to sugar metabolism appeared in 1997 with the discovery that PBCV-1 encoded a functional HAS along with some of the genes necessary to produce the monosaccharides that make this polymer (section 3.3.1).¹¹ The second report followed few years later and demonstrated that this same virus

encoded a functional pathway for the production of GDP-α-D-Rha and GDP-β-L-Fuc (section 3.1.1.1).¹⁴⁸

Since that time, reports have steadily increased. New sugar biosynthetic routes have been characterized (section 3.1), and the enzymes discovered have triggered an interest in the scientific community, as noted by the crystallographic studies that have flourished in response. Another advance has been the characterization of the glycans that decorate the capsids of the giant viruses, with the first report appearing less than a decade ago. This report detailed the structure of the N-glycans of PBCV-1 (section 3.2.1),¹⁷⁷ and subsequently other studies have provided more information on the glycosylation patterns of chloroviruses (section 3.2.1)¹⁷⁷,¹⁷⁹,¹⁹²,¹⁹³ and on the fibril architecture of APMV (section 3.2.2).⁶⁵,¹⁹⁴

As always happens in science, every finding triggers new questions. As for PBCV-1, and chloroviruses in general, the finding of N-glycans that differ from those in any cellular organism opens many research avenues. For example, GTs involved in their synthesis are still being discovered. These enzymes are interesting because most are predicted to be soluble; consequently, they might be of interest for biotechnological applications.

Another intriguing question is how are these N-glycans added to the protein. Does this occur by a sequential addition of the sugars? Or, are they preassembled and transferred en block onto the protein? Perhaps the mechanism is a compromise of the previous two: a small core oligosaccharide is preassembled and transferred onto the capsid protein, and then the other sugars are added sequentially. Related to these questions are if any of the sugars, in particular the Glc moiety, is linked to a dolichol-like carrier. The N-glycosylated asparagine occurs in an atypical sequon, so that one can expect that the addition of these glycans occurs by a mechanism different from those previously identified. In support of this hypothesis is the fact that N-glycosylation in *Chlorella* is inhibited by tunicamycin, while the viral glycosylation continues undisturbed.

All of the points mentioned above are only a fraction of the stimuli that these viruses can trigger in the scientific community. Organic chemists have already taken up the challenge of the synthesis and structural chemistry of chlorovirus glycans, and their involvement is also key to studying the GTs involved by preparing the appropriate substrates to test.

However, other scientific areas can take this challenge by offering the tools appropriate to answer the many open questions. For instance, these viruses can be an attractive playground to develop new chemical biology probes.²⁵³ Such tools can offer shortcuts to trap biosynthetic intermediates or to follow the destiny of sugar analogues to learn about the capsid assembly mechanism, a fundamental step in viral replication.

Another interesting question concerns what happens to the host once infected. It is already clear that some chloroviruses modify the chlorella cell wall, which becomes covered with a thick layer of hyaluronan and/or chitin (section 3.3.1). However, nothing is known about the glycosylation pattern of the algal proteins, and this is an unexplored field of research.

All of the questions raised from chloroviruses apply, with the appropriate adaptations, to the other virus for which experimental data are available, APMV. The capsid of this virus, like almost all the others in the same family, is covered by a thick layer of fibrils, recently demonstrated to be made of
proteins and polysaccharides. Notably, APMV is the first virus to be glycosylated with polysaccharides.⁶⁵ Again, the mechanism by which these glycans are produced is unknown.

Another unique point for this virus is the involvement of collagenic proteins in the assembly of the capsid. Collagen is a molecule only present in the animal world, and APMV encodes seven collagen-like proteins, together with L230, a protein able to glycosylate the collagenic proteins by adding glucose instead of galactose (the monosaccharide present in mammalian collagen) to the hydroxylated lysine (section 3.3.3). A last intriguing point is the GlcNAc biosynthetic route that in APMV follows the eukaryotic strategy by using enzymes that are not necessarily from eukaryotes (section 3.1.2.2).

Collectively, data on PBCV-1 and APMV lead to deeper evolution questions: (i) How did these viruses acquire their glycosylation machinery? (ii) Why do they maintain it? (iii) What advantages does glycosylation confer? The answers to each of these questions and more remain to be addressed, and they just denote that the field of viral glycosylation is all but explored.

Moreover, answers to these questions may change depending on the virus considered. It should be noted that these questions were raised just by studying two viruses, PBCV-1 and APMV, while the number of giant viruses is constantly increasing, and information on their glycosylation machinery is only from in silico analyses; they are a new world to explore. For instance, one simple question already arises by considering that CroV and Fadolivirus encode Kdo biosynthetic machinery (section 3.1.2.4). This monosaccharide is a hallmark of Gram-negative bacteria, and outside this domain it is only found in plant pectin. This raises the question: Where do these two giant viruses stand from an evolutionary perspective? By extending this observation to all giant viruses, it appears that their glycosylation machinery mixes traits from the three forms of life. It should be noted that the evolutionary history of giant viruses has been proposed to predate the emergence of modern eukaryotic organisms.¹⁶ Maybe the suggestion in 2001 that glycosylation of the PBCV-1 major capsid protein occurred by an ancestral pathway that existed prior to ER and Golgi is not far out of line.²⁵⁴

Despite the many unanswered questions, one thing is clear: giant viruses are a new source of inspiration for chemists, biochemists, and other colleagues in life science because the understanding of their glycobiology is intriguing. These viruses provide major challenges but will offer many scientific rewards.

## AUTHOR INFORMATION

### Corresponding Author

Cristina De Castro - Department of Agricultural Sciences, University of Napoli, 80055 Portici, Italy; ● orcid.org/0000-0002-5147-1756; Phone: +39081674124; Email: decastro@unina.it

### Authors

Immacolata Speciale - Department of Agricultural Sciences, University of Napoli, 80055 Portici, Italy; ● orcid.org/0000-0003-4363-7450

Anna Notaro - Department of Agricultural Sciences, University of Napoli, 80055 Portici, Italy; Centre National de la Recherche Scientifique, Information Génomique & Structurale, Aix-Marseille University, 13288 Marseille, Cedex 9, France

Chantal Abergel - Centre National de la Recherche Scientifique, Information Génomique & Structurale, Aix-Marseille University, 13288 Marseille, Cedex 9, France

Rosa Lanzetta - Department of Chemical Sciences, University of Napoli, 80126 Napoli, Italy

Todd L. Lowary - Institute of Biological Chemistry, Academia Sinica, Nangang 11529 Taipei, Taiwan; ● orcid.org/0000-0002-8331-8211

Antonio Molinaro - Department of Chemical Sciences, University of Napoli, 80126 Napoli, Italy; ● orcid.org/0000-0002-3456-7369

Michela Tonetti - Department of Experimental Medicine and Center of Excellence for Biomedical Research, University of Genova, 16132 Genova, Italy

James L. Van Etten - Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska 68583-0900, United States; Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583-0722, United States

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.chemrev.2c00118

## Author Contributions

O.I.S. and A.N. contributed equally

## Notes

The authors declare no competing financial interest.

## Biographies

Immacolata Speciale is an organic chemist and currently is a postdoc in Carbohydrate Chemistry at the University of Naples Federico II. In 2017, she obtained her Ph.D. Europaeus in Chemical Science at the University of Napoli Federico II. Her research activity is focused on the structural determination of the carbohydrate content of different microbial sources, with the aim of understanding their biological impact. Most of her recent research activity concerns the unusual N-glycans produced by chloroviruses, which have been investigated from different perspectives, giving her the opportunity to gain experience in several fields, such as spectroscopic and spectrometric techniques, molecular modelling methods, and molecular biology up to organic synthesis and chemo-enzymatic reactions.

Anna Notaro is a biologist and currently is a postdoc in Carbohydrate Chemistry at the University of Naples Federico II. She received her Ph.D. degree in Chemistry at the University of Naples and her Ph.D. in Structural Biochemistry at Aix-Marseille University, both in 2019, in the frame of the Italy-France Vinci cooperation program. During her Ph.D., she was awarded in the frame of bando Star 2017 Linea 2 (Mobility of Young Researchers) for a six-month internship at Aix-Marseille University. Her research activity is focused on the glycobiology of giant viruses, from the structural elucidation of their glycans to the identification of the glycogenes and the validation of their function in vivo. She is also interested on the biosynthesis of the glycans from bacteria and archaea.

Chantal Abergel received her Ph.D. in Material Science at the Air Marseille University and is currently Head of the Structural and Genomic Information Laboratory, CNRS-AMU, Marseille (France). She did her Ph.D. in Structural Biology at NIH, NIDDK in Bethesda. She was recruited in 1995 at CNRS to start a new laboratory, with Jean-Michel Claverie, combining theory with experimental work. Together they discovered the four families of the largest giant viruses. Her research activity focuses on the isolation, experimental biology, and genomic studies of giant viruses, including members of the Mimiviridae, Pandoraviridae, Pithocedratviridae, and Marseilleviridae
families. Her research activity is translated in more than 110 papers in peer-reviewed journals and about 60 3D structures in the Protein Structure Database, including two cryo-EM structures. She received several awards for her research in the field of giant viruses. She is member of the Scientific CNRS Council, of the European Microbiological Society and of the World Society of Virology.

Rosa Lanzetta is a full Professor of Organic Chemistry and Head of the Department of Chemical Sciences of the University of Naples Federico II. She has been the recipient of awards, including the "Berti Medal" from the Italian Chemical Society for her contribution to research in the field of carbohydrates. Her scientific activity started in the field of chemistry of glycosylated natural compounds and followed up with bacterial and fungal polysaccharides as documented by over 250 scientific publications.

Todd Lowary received his B.A. in Chemistry from the University of Montana and his Ph.D. in Organic Chemistry from the University of Alberta. Postdoctoral appointments were at the University of Alberta and the Carlsberg Laboratory. In 1996, he started a position in the Department of Chemistry at The Ohio State University as an Assistant Professor, and in 2002, he was promoted to Associate Professor with tenure. He returned to the University of Alberta in 2003, retiring in 2021 as the R. U. Lemieux Professor of Carbohydrate Chemistry. In 2019, he became a Distinguished Research Fellow at the Institute of Biological Chemistry at Academia Sinica in Taipei, Taiwan, a position he continues to hold. Research interests are in carbohydrate chemistry and biochemistry, in particular as these fields relate to bacterial glycans. Areas of particular focus are glycans from mycobacteria, including *Mycobacterium tuberculosis*, the pathogen that causes tuberculosis, and *Campylobacter jejuni*, a gut pathogen that causes enteritis, and chlorella viruses.

Antonio Molinaro is a full Professor of Organic Chemistry at the University of Naples Federico II, Italy, and since 2015, the Special Appointed Professor of Organic Chemistry at the Faculty of Science of the University of Osaka, Japan. He has (co)authored over 280 peer reviewed papers on carbohydrate chemistry and structural glycoscience. He has been chairman and/or partner in several regional, national, and international projects on the structure and function of microbial glycoconjugates. He has been the coordinator of the interdivisional group of carbohydrate chemistry of the Italian Society of Chemistry and the President of the European Carbohydrate Organization. He has received various awards for his research in glycosciences, among which include the Chinese Academy of Science Visiting Fellowship under the CAS President's International Initiative and the Chemistry Award for Creativity in Life Science from the Italian Society of Chemistry, Division of Organic Chemistry. In 2021, he received the Emil Fischer award from the European Carbohydrate Organization to honor active carbohydrate scientists distinguished with contributions of excellence.

Michela Tonetti is full Professor of Biochemistry at the University of Genoa. Her scientific activity in recent years has been focused on the study of mechanisms of viral glycosylation and the role of glycosylation in cellular communication and adhesion. She is the author of about a hundred scientific publications in peer reviewed international journals and book chapters.

James L. Van Etten is the William D Allington Distinguished Professor in the Department of Plant Pathology at the University of Nebraska-Lincoln, where he has been a faculty member since 1966. His initial research focused on the physiology, biochemistry, and molecular biology of fungal spore germination. He also helped characterize the very unusual bacteriophage phi 6, which was the first virus to have an external lipid envelope and a dsRNA genome. In the

early 1980s, he and his colleagues reported the characterization of large, plaque-forming dsDNA viruses that infected chlorella-like green algae; his laboratory has spent the last 40 years studying these unique viruses, which are ubiquitous in fresh water from all over the world. He has published more than 225 peer-reviewed manuscripts on these chloroviruses and their gene products (over 320 total publications). His research has led to several honors, including being elected as a Fellow of the American Phytopathological Society, the American Association for the Advancement of Science, the American Academy of Microbiology, and the National Academy of Inventors. In 2003, he became a member of the United States National Academy of Sciences.

Cristina De Castro is Associate Professor of Organic Chemistry, University of Naples Federico II; she is actively working in the field of carbohydrate chemistry and biochemistry. Her research activity initially focused on the study of the carbohydrate polymers from microbes (both Gram-positive and Gram-negative) but has in the last years moved toward the understanding of the glycobiology and glycochemistry of the giant viruses. Accordingly, her research activity is translated in more than 140 papers in peer-reviewed journals. She is on the executive committee of the Interdivisional Group of the Chemistry of Carbohydrates of the Italian Chemical Society. Furthermore, she serves as Associate Editor of the *Carbohydrate Polymers* journal, and she is on the Editorial Boards of *Carbohydrate Research, Glycobiology*, and *Polysaccharides* journals.

## ACKNOWLEDGMENTS

C.D.C. gratefully acknowledges STARPLUS 2020 (project no. 21-UNINA-EPIG-042) and FRA promoted from the University of Napoli for financial support. A.N. acknowledges UIF Vinci program 2015 (contract C3_90) and 2019 (contract C4-180), and STAR2-2017 project for financial support. C.A. acknowledges the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 832601) for financial support. T.L.L. gratefully acknowledges Academia Sinica for financial support. The chlorovirus research in the Van Etten lab has been supported by grants from the National Institutes of Health, the National Science Foundation, the United States Dept of Agriculture, the United States Department of Energy, and the University of Nebraska over the past 40 years. Current support is provided by the National Science Foundation under grant no. 1736030. The authors apologize to the many colleagues who have influenced their thinking through work that was not explicitly cited in this review.

## ABBREVIATIONS

ASP = D-arabinose-5-phosphate

aa = amino acids

aaRS = amino-acyl tRNA synthetases

aa = amino acid

AaV = Aureococcus anaphagefferens virus

ADP = adenosine diphosphate

API = arabinose-5P isomerase

Asn = asparagine

Ara = arabinose

ATCV-1 = Acanthocystis surfacea chlorovirus 1

BsV = Bodo saltans virus

CBD = cellulose binding domain

CeV = Chrysochromulina ericina virus

CHS = chitin synthase

CKS = CMP-Kdo synthase

CMP = cytidine monophosphate
CroV = Cafeteria roenbergensis virus

CTP = cytidine triphosphate

Da = Dalton

dHex = deoxyhexose

dHexN = deoxyhexosamine

dTDP-D-glucose = thymidine-5'-diphospho-α-D-glucose

DTT = dithiothreitol

E. coli = Escherichia coli

EDTA = ethylenediaminetetraacetic acid

Fuc = fucose

FucT = fucosyltransferase

Gal = galactose

GalT = galactosyltransferase

GFAT = glutamine-fructose 6-phosphate amidotransferase

GlcA = glucuronic acid

GlcNAc = N-acetylglucosamine

GDP = guanosine diphosphate

GMD = GDP-D-mannose-4,6-dehydratase

GMER = GDP-4-keto-6-deoxy-D-mannose epimerase/re-
ductase

GNAT = GlcN-6P N-acetyltransferase

GST = glutathione S-transferase

GT = glycosyltransferase

HA = hyaluronan or hyaluronic acid

HAS = hyaluronan synthase

Hex = hexose

HexN = hexosamine

HexNAc = N-acetyl-hexosamine

Hys = histidine

HPLC = high pressure liquid chromatography

IPTG = isopropyl β-D-1-thiogalactopyranoside

Kdo = 3-deoxy-D-manno-oct-2-ulosonic acid

KdoPS = Kdo-8P synthase

KdoPase = Kdo-8P Phosphatase

Lys = lysine

Man = mannose

MBP = maltose-binding protein

MCP = major capsid protein

MW = molecular weight

NADP+ = nicotinamide adenine dinucleotide phosphate

NCLDV(s) = nucleocytoplasmic large DNA virus(es)

OG = oxoglutarate

ORF = open reading frame

PBCV-1 = Paramecium bursaria chlorella virus

PBS = phosphate buffer saline

PDB = Protein Data Bank

PEG = protein-encoding genes

PEP = phosphoenolpyruvate

PFU = plaque-forming units

PI = post infection

PL = polysaccharide lyase

PLP = pyridoxal phosphate

PMSF = phenyl-methylsulfonylfluoride

PNGase = peptide-N-glycosidase F

PSORT = protein subcellular localization prediction tool

Qui = 6-deoxy-Glc or quinovose

QuiNAc = 2-acetamido-2,6-dideoxy-Glc or 2-acetamido-
quinovose

QuiNAc4NAc = 2,4-diacetamido-2,4,6-trideoxy-Glc or 2,4-diacetamido-2,4-quinovose or di-N-acetyl-bacillosamine

Rha = rhamnose

2RHAMM = receptor for hyaluronan mediated motility

Ru5P = D-ribulose-5-phosphate

SAM = S-(5'-adenosyl)-L-methionine iodide

SDR = short-chain dehydrogenase/reductase enzyme

SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Ser = serine

TetV = tetraselmis virus

Thr = threonine

Tyr = tyrosine

UAP = UDP-GlcNAc pyrophosphorylase

UDP = uridine diphosphate

UGD = UDP-D-Glc (or GlcNAc) 4,6-dehydratase

UGDH or UDP-GlcDH = UDP-D-glucose dehydrogenase

UGER = UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/4-reductase

VF = viral factory

Xyl = xylose

XylT = xylosyltransferase

Xul = xylulose

WT = wild-type

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