# Evolutionarily conserved grammar rules viral factories of amoeba-infecting members of the hyperdiverse Nucleocytoviricota phylum

Sofia Rigou, Alain Schmitt, Anaísa Moreno, Audrey Lartigue, Lucile Danner, Lotte Mayer, Claire Giry, Feres Trabelsi, Lucid Belmudes, Natalia Olivero-Deibe, et al.

► To cite this version:

Sofia Rigou, Alain Schmitt, Anaísa Moreno, Audrey Lartigue, Lucile Danner, et al. Evolutionarily conserved grammar rules viral factories of amoeba-infecting members of the hyperdiverse Nucleocytoviricota phylum. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (35), pp.e2515074122. 10.1073/pnas.2515074122. hal-05370745

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## Main Manuscript for

### Evolutionarily conserved grammar rules viral factories of amoeba-infecting members of the hyper-diverse *Nucleocytoviricota* phylum

Sofia Rigou¹†‡, Alain Schmitt¹†, Anaísa B. Moreno¹†, Audrey Lartigue¹, Lucile Danner¹, Lotte Mayer², Claire Giry¹, Feres Trabelsi¹, Lucid Belmudes³, Natalia Olivero-Deibe⁴, Hugo Le Guenno⁵, Yohann Couté³, Mabel Berois⁶, Matthieu Legendre¹, Sandra Jeudy¹, Chantal Abergel¹*, Hugo Bisio¹*.

1 Aix-Marseille University, Centre National de la Recherche Scientifique, Information Génomique & Structurale (IGS), Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479), IM2B, IOM, 13288 Marseille Cedex 9, France.

2 University of Vienna, Centre for Microbiology and Environmental Systems Science, Division of Microbial Ecology, Djerassiplatz 1, 1030 Vienna, Austria

3 Univ. Grenoble Alpes, INSERM, CEA, UA13 BGE, CNRS, CEA, FR2048, 38000 Grenoble, France.

4 Immunovirology Lab, Institut Pasteur de Montevideo, 11400, Montevideo, Uruguay.

5 Aix Marseille Univ, CNRS, IMM, Microscopy Core Facility, 13009 Marseille, France.

6 Sección Virología, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo 11600, Uruguay.

† These authors contributed equally to this work

‡ Present addresses: University of Vienna, Centre for Microbiology and Environmental Systems Science, Division of Microbial Ecology, Djerassiplatz 1, 1030 Vienna, Austria

* Corresponding author.

Email: hugo.bisio@igs.cnrs-mrs.fr, chantal.abergel@igs.cnrs-mrs.fr

PNAS strongly encourages authors to supply an ORCID identifier for each author. Do not include

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## Author Contributions:

Conceptualization: SR, AS, ABM, CA, HB
33 Methodology: SR, AS, ABM, HB

34 Investigation: SR, AS, ABM, AL, LD, LM, CG, FT, LB, NOD, MB, SJ, HB

35 Visualization: SR, AS, ABM, LM, YC, HB

36 Funding acquisition: YC, CA, HB

37 Project administration: HB

38 Supervision: YC, MB, ML, SJ, CA, HB

39 Writing – original draft: SR, AS, HB

40 Writing – review & editing: SR, AS, ABM, NOD, YC, MB, ML, SJ, CA, HB

41 Competing Interest Statement: The authors declare no competing interests.

42 Classification: Biological Sciences/Evolution

43 Keywords: Nucleocytoviricota, mimivirus, phase separation, biomolecular condensate, giant

44 virus.

45

46 This PDF file includes:

47 Main Text

48 Figures 1 to 5
49

## Abstract

Despite sharing fewer than 10 core genes, the hyper-diverse *Nucleocytoviricota* phylum (ranging from poxviruses to giant viruses) universally assembles viral factories resembling biomolecular condensates. Regardless, it is unclear how these viruses achieve such a level of functional conservation without clear conserved genetic information. We demonstrate that the viral factories produced by amoeba-infecting viruses have liquid-like properties and identify a conserved molecular grammar governing viral factory scaffold protein: charge-patterned intrinsically disordered regions that drive phase separation independently of sequence homology. This grammar predicts functional scaffold proteins across the 15 viral families, revealing evolutionary constraints invisible to sequence or structural analysis. Strikingly, viral factories exhibit sub-compartmentalization analogous to nuclei, segregating transcription and mRNA processing (inner condensates) from replication (interphase zones) and translation (host cytoplasm). Our work establishes phase separation as a fundamental organizational principle bridging extreme genomic diversity, explaining how biological complexity emerges without gene conservation. This grammar is likely also conserved in non-amoeba-infecting members of the phylum and thus, may represent a primordial solution for organelle-like organization, with broad implications for antiviral targeting.

## Significance Statement

Viral factories are organelle-like compartments that are conserved across the *Nucleocytoviricota* phylum despite extensive genomic diversity. How such phenotypic conservation is maintained remains unknown. Here, we show that viral factories of amoeba-infecting viruses emerge from a conserved organizational molecular grammar, condensed within intrinsically disordered regions, thereby efficiently bypassing the need for gene conservation. This compact mode of functional inheritance likely provides a robust platform for genome diversification.

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## Main Text

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## Introduction

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The phylum *Nucleocytoviricota* exemplifies the remarkable diversity of the virosphere, encompassing viruses with a wide range of morphologies, genome sizes, and hundreds of unique genes. This diversity spans from Yaravirus, which forms 80-nm icosahedral particles and carries a 45-kbp dsDNA genome encoding 74 predicted proteins (1), to Pandoravirus, which produces 1-µm ovoid particles and harbors a 2.5-Mbp genome encoding over 1,500 genes (2, 3). Despite this diversity, these viruses are unified by only ten core genes, most of which are not universally
conserved among all members (4). How, then, do these viruses maintain a coherent replication strategy? Virtually all members of *Nucleocytoviricota* (including Poxviridae and the giant viruses (5, 6)) form viral factories (VFs): cytoplasmic organelle-like compartments that spatially coordinate genome replication and/or virion assembly (7). Importantly, while some viruses have acquired nuclear genome replication, cytoplasmic VFs are still visualized by electron microscopy (8, 9). However, the molecular mechanisms underlying VF biogenesis remain poorly understood (10). To fill this gap, mimivirus was used as a primary model since it displays a biphasic nature when imaged by electron microscopy and a highly synchronized biogenesis and maturation (10). Moreover, mimivirus is a well-studied member of the family Mimiviridae, which is ubiquitous in the environment (11, 12). Mimivirus is a fully cytoplasmic giant virus for which virions are internalized into the host cell by phagocytosis (13). The acidic compartment of the phagosome, in combination with oxidative stress through Fenton reactions, triggers the opening of the stargate structure located at an apex of the icosahedral capsid (14-16). Once opened, the fusion of the membrane of the phagosome with the virion's internal membrane leads to the release of the core in the cytoplasm (13, 17, 18). The core contains the genome of the virus and the machinery needed to establish infection (19). The core consequently develops to establish a viral factory, a highly dynamic and complex organelle-like structure putatively formed by over 300 proteins (20). VF not only protects the viral DNA from cellular insults (21) but also segregates DNA replication and transcription from translation (20).

Biomolecular condensates, formed through phase separation, are increasingly recognized as key structures in viral replication, host manipulation, and morphogenesis (22, 23). Phase separation is the process by which biomolecules demix into membrane-less compartments (22-25). These structures typically contain scaffold proteins, which drive condensate formation, and client proteins, which are selectively recruited (22-25). VFs formed by phase separation have been described in several RNA and DNA viruses, including members of the Herpesviridae, Adenoviridae, Reoviridae, and the order Mononegavirales (reviewed in (22)).

Here, we hypothesize that phase separation provides a unifying mechanism for assembling viral replication centers across the genetically diverse *Nucleocytoviricota*, despite minimal sequence conservation. To test this, we leveraged newly developed genetic tools for giant viruses to investigate the nature of VFs across the phylum (26-28).

## Results

### Universal liquid-like properties of amoeba-infecting *Nucleocytoviricota* viral factories.

To gather information associated with the fine ultrastructure of VFs, we first fluorescently labelled proteins (described later in the manuscript) enriched in a previous proteome of mimivirus (order:
119 *Imitervirales* (Fig. 1A)) VFs (20) and identified two sub-compartments recapitulating the
120 ultrastructure visualized by electron microscopy (Fig. 1B-C). Multiple inner layers can be seen in
121 a single VF early during infection, while a single and continuous outer layer englobes all inner
122 layers (Fig. 1B-C and Fig. S1A). The number of inner layers per VF linearly increases with the
123 multiplicity of infection, suggesting that each genome unit induces the formation of individual inner
124 layers (Fig. S1B).

125 Live-cell imaging of mimivirus-infected cells revealed that upon contact, two VFs juxtaposed and
126 never separated during the remaining recording time (Fig. S1C), suggesting that the outer layer of
127 two independent VFs fused as observed by fluorescence and electron microscopy (Fig. 1B-C).
128 Importantly, imaging mimivirus-infected cells early during infection to record fusion events is
129 challenging for two main reasons. 1) The high mobility of the amoeba precludes the use of higher
130 magnification since the cell quickly exits the area being recorded. 2) It is extremely infrequent to
131 find individual mimivirus viral factories: independent inner layers without a shared outer layer
132 within the same cell. Indeed, once viral factories are sufficiently big to be visualized, the inner
133 layers of the viral factory almost always share the same outer layer. Recovery of fluorescence
134 after photobleaching (FRAP) was observed when an area of the outer layer of the VF was
135 bleached (Fig. 1D). Fluorescence gain was accompanied by a partial decrease in fluorescence in
136 the non-bleached area, but this decrease was incomplete, indicating some diffusion along with a
137 fraction of non-mobile molecules (Fig. 1D). Fixed VFs did not show any recovery, consistent with
138 crosslinking (Fig. S1D). Thus, mimivirus VFs exhibit some liquid-like properties but do not behave
139 as a classical condensate formed by liquid-liquid phase separation.

140 To compare with other viruses from the phylum, we labeled noumeavirus (29) (order:
141 *Pimascovirales* (Fig. 1A)) VFs using cells expressing the C-terminal GFP-tagged *mel_H2B-H2A*
142 (28). Noumeavirus is a good model for this experiment since: 1) it contains a single detectable
143 layer; 2) multiple VFs can be easily observed early in infection, and 3) noumeavirus infection
144 rapidly disrupts the movement of the amoeba (29). Noumeavirus VFs were observed as dynamic
145 organelles subjected to deformation, and the encounter of two VFs yielded coalescence (Fig. 1E).
146 To specifically address the liquid-like properties of VFs on the class *Pokkesviricetes*, we
147 fluorescently tagged PN-99 with GFP and performed FRAP experiments on pacmanvirus lostcity
148 (30) (order: *Asfuvirales* (Fig. 1A)) VFs (Fig. 1F). Fast recovery was observed upon
149 photobleaching and diffusion from the non-bleached side of the VF led to a homogenous
150 fluorescent intensity. Thus, both noumeavirus and pacmanvirus show classical behavior of
151 condensates formed by liquid-liquid phase separation.

152 To facilitate comparisons, we investigated whether these membrane-less organelles would
153 dissolve by treating infected cells with 10% 1,6-hexanediol (23) or hypotonic stress (31).
154 Mimivirus VF only partially dissolved during the treatment, further reinforcing non-classical liquid-
155 like properties of the factories (Fig. S2A-C). On the other hand, treatment with 10% 1,6-hexanediol efficiently dissolved the VFs of noumeavirus (Marseilleviridae), pithovirus (Pithoviridae), manivirus and the mammalian-infecting vaccinia virus (Poxviridae) (Fig. S2A-C). All VFs were also partially dissolved upon hypotonic stress (Fig. S2A), reinforcing some liquid-like phase separation behavior of these organelles.
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160 Additionally, a subgroup of members of the phylum transfer their genome to their host nucleus,
161 where it replicates. Mollivirus is one of those viruses (32). Regardless, mollivirus major capsid
162 protein (virion protein) accumulates in a cytoplasmic, uncharacterized sub-compartment (likely
163 the viral factories) in infected cells before loading into the particles (21). This localization could
164 also be disrupted by 1,6-hexanediol treatment (Fig. S2D). Importantly, both 1,6-hexanediol and
165 hypotonic shock treatment have limitations worth mentioning: 1,6-hexanediol has several known
166 off-target effects, including components of the cytoskeleton (33) and the inhibition of kinases and
167 phosphatases (34), all of which could affect VFs independently of liquid-like phase separation.
168 Hypotonic stress induces swelling of the cells, effectively reducing the concentration of proteins
169 within them. Regardless, viral factories are big in size (reaching up-to 20 µm diameter) and
170 occupy half of the cell cytoplasm volume. This likely diminishes the impact of the cell swelling
171 effect, potentially explaining the partial dissolution of viral factories with this treatment.
172 Nevertheless, the combination of these experiments with the live imaging (Supplementary Table
173 1) strongly suggests that the VFs of mimivirus, noumeavirus, manivirus and probably all
174 amoeba-infecting members of the phylum Nucleocytoviricota display characteristics of
175 biomolecular condensates ranging from prototypical compartments made by liquid-liquid phase
176 separation (like noumeavirus or manivirus) to condensates with some liquid-like properties
177 (like mimivirus).
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A two-scaffold system creates biphasic condensates in mimivirus.

179 Having identified a new hallmark of the amoeba-infecting *Nucleocytoviricota*, we aimed to
180 uncover the common components of these organelles between the extreme viral diversity of the
181 phylum. Phase separation is driven by the multivalency of proteins termed scaffold proteins (35).
182 Scaffolding proteins are abundant in condensates and tend to contain intrinsically disordered
183 regions (IDRs) as compacted means to achieve multivalency (35). To identify VF scaffold
184 proteins, we performed immunoprecipitations using three previously identified VF proteins as bait
185 (R562, R505 and R336/R337) (20). Two prey proteins (R561 and R252) containing predicted
186 IDRs (Fig. S3A), were consistently enriched indicating interaction with all three client proteins
187 (Dataset S1).
188 Expression of R561 (termed Outer Layer Scaffold 1 (OLS1)) in *Acanthamoeba castellanii*
189 demonstrated its ability to undergo phase separation in the amoeba cytoplasm (Fig. S3B). In
190 contrast, R252 (termed Inner Layer Scaffold 1 (ILS1)) exhibited a diffuse cytoplasmic localization
191 (Fig. S3B). Upon mimivirus infection of cells expressing both fluorescently tagged proteins, OLS1
192 and ILS1 re-localized to the viral factories, with OLS1 associating with the outer layer and ILS1
193 with the inner layer (Fig. S3B). Control cells expressing only GFP or RFP did not display similar
194 re-localizations (Fig. S3C). Notably, VF client proteins, such as R336/R337, localized not only to
195 the VF outer layer but also to biomolecular condensates formed by the overexpressed OLS1,
196 strongly supporting its role as a scaffolding protein forming the VF outer layer (Fig. S3D).
197 Furthermore, when OLS1 and ILS1 were co-expressed, ILS1 acted as a client protein and was
198 recruited to OLS1 biomolecular condensate (Fig. S3E).

199 Considering that ILS1 binds DNA (36), we reasoned that its recruitment to the VF outer layer
200 would allow contact with viral DNA, thereby enabling phase separation. To test this hypothesis,
201 recombinantly expressed ILS1 (Fig. S4A) was analyzed for phase separation in the presence of
202 DNA (Fig. 2A and S4B). Phase separation was observed only in the presence of DNA (Fig. 2A).
203 Moreover, DNA concentration significantly influenced the nature of phase separation (Fig. S4C),
204 being at lower DNA concentrations (2.5-20 µg/mL), networks and droplets predominated,
205 whereas higher DNA concentrations (>20 µg/mL) prompted the formation of large gels. ILS1
206 concentration did not affect the nature of phase separation (Fig. S4D). Both linear and circular
207 DNA equally triggered phase separation, but mimivirus genomic DNA induced larger gel
208 formation at lower DNA concentrations (Fig. S4E). Similar results were obtained with the
209 recombinant mCherry fused ILS1 (Fig. S4F). RNA did not trigger ILS1 phase separation (Fig.
210 S4B).

211 Recombinant OLS1 (Fig. S4A) made phase separation with classical liquid-liquid phase
212 separation morphology, independently of other macromolecules (Fig. 2B). Phase separation
213 depended on OLS1 concentrations (Fig. S5A) and salt concentrations (Fig. S5B). Similar to *in
214 cellula* conditions, *in vitro* OLS1 biomolecular condensate recruited ILS1 as a client protein (Fig.
215 S5C). Finally, adding DNA to the mixture of both proteins triggered the segregation into two
216 phases, independently of the order of component incorporation (Fig. 2C). Altogether, OLS1, ILS1
217 and DNA are sufficient to trigger VF-like biphasic phase separation *in vitro*.

218 To test if these proteins were responsible for the VFs formation, we attempted gene knockout of
219 both genes in host cells expressing a codon-optimized version of each protein for trans-
220 complementation (28). Recombinant OLS1 knockout viruses were obtained (Fig. S6A) with a 100-
221 fold reduction in burst size compared to wild-type viruses (Fig. 2D). Moreover, a lower viral DNA
222 accumulation was observed during infection (Fig. 2E), consistent with a delay in VF growth, as
shown by DAPI staining of infected cells (Fig. 2F-G). Depletion of OLS1 also reduced fusion events of VFs upon superinfection, as shown by the presence of multiple independent VFs in the same cells (Fig. 2F and 2H). Visualization of mutant infectious cycle by electron microscopy demonstrated that VFs of *ols1* KO viruses lack any visible outer layer, confirming that OLS1 is the scaffold protein for this sub-compartment of the VF (Fig. 2I and S6B). Moreover, endogenous tagging of client proteins of the outer layer of the VF (like R336/R337 or R322) displayed a cytoplasmic localization upon deletion of *ols1* (Fig. 2J-K and S6C-D), indicating that the role of the outer layer of the VF of mimivirus is to concentrate important components for VF functions. All phenotypes were fully restored by trans-complementation (Fig. 2D-K). On the other hand, we were unable to obtain clonal *ils1* KO viruses. Using the trans-complementing line, we demonstrated that *ils1* is likely an essential gene (Fig. S6E).

## A conserved molecular grammar predicts scaffold protein throughout the phylum.

The molecular grammar of an IDR refers to compositional bias and sequence patterns in its primary structure (24, 25). This grammar allows condensation and specific recruitment of molecules, including client proteins (37-42). We thus reasoned that due to the plethora of client proteins recruited to the VFs, despite major changes in the primary sequence of their scaffold proteins, the molecular grammar of the condensate would not easily change. Concordantly, host-expressed mimivirus (*Imitervirales*) OLS1-GFP and ILS1-RFP re-localized to noumeavirus (*Pimascovirales*) VF upon infection (Fig. S7A-B).

Thus, to identify scaffold proteins in all *Nucleocytoviricota*, we built a pipeline to analyze the genomes of these viruses. First, we predicted the “IDRome” encoded in representative genomes of isolated viruses and extended the analysis to metagenomes from the giant virus database (4), the permafrost (43), and Egovirales (44) (Supplementary Dataset S2). We also capitalized on methods previously described to study the biochemistry of nucleolar phase separation, relying on Nardini and CIDER (42) and adjusted 2 parameters for faster (Fig. S8A) and more precise (Fig. S8B) discrimination of particular proteins. Since the inner layer of the VF is only present in members of the *Mimiviridae*, we excluded ILS1 from the following analysis and focused bioinformatic computations on OLS1. Using 98 features (36 from Nardini and 62 from CIDER), we created a preliminary machine learning classifier that identified 4 candidate scaffold proteins in noumeavirus (Fig. S7C).

To experimentally challenge these predictions, the four genes were codon-optimized and expressed in the amoeba. Two showed a localization coherent with biomolecular condensates (Fig. 3A) and re-localized to the noumeavirus VF upon infection (Fig. 3B). Moreover, both proteins also re-localized to the mimivirus VFs upon infection, confirming a shared molecular grammar for phase separation for these 2 viruses' VFs (Fig. S7D). In contrast, the other two
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protein candidates did not spontaneously form biomolecular condensates in the amoeba cytoplasm (Fig. S7E).

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Using the newly confirmed noumeavirus scaffold proteins (NMV_095 and NMV_238) and detected homologs, we re-trained the predictive machine learning classifier (Fig. S7F). Features for the classifier were selected by comparing the feature values of the three scaffold proteins' IDRs and homologs to the rest of the combined IDRome of mimivirus and noumeavirus (Fig. S8C-D). Final predictions of scaffold proteins in all orders of *Nucleocytoviricota* were then generated with this optimized classifier (Dataset S2).

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Clear segregation of resulting candidate IDRs can be observed in the unsupervised UMAP representation of the *Nucleocytoviricota* IDRome features (Fig. 3C and S9A). As expected, scaffold IDRs have a low Anchor2 score (45) and are not predicted to become structured upon changes in the environment (i.e. binding to an interacting protein) (Fig. S9B). The most discriminative features in our classification, and thus, the features associated with the molecular grammar of the VFs, were related to charge segregation (Fig. 3E-F and S9A and C). Within the negative charges, glutamate seems more important, as confirmed scaffold IDRs and candidates have 15% of this residue (+/- 6), while the rest of the IDRs have 6% (+/- 8). Scaffold IDRs and candidates have also a relatively higher E/D ratio (0.25 +/- 0.26 vs 0 +/- 0.45) and a higher K/R ratio (0.48 +/- 0.36 vs 0.07 +/- 0.53).

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Putative scaffold proteins could be identified to be encoded by members of all *Nucleocytoviricota* (15 families and unclassified viruses) (Fig. 3C-D and F). In all these orders, between 4.4% (*Algalvirales*) and 5.4% (*Chitovirales*) of all predicted IDRs were classified as candidate scaffold proteins for phase separation and VFs generation. The only exception was the *Egovirales*, scoring 8.7% of positive proteins in their respective IDRomes. In *Pimascovirales*, homologous proteins were identified as scaffolds in pithovirus sibericum and cedratvirus kamchatka (pv_12 and ck125, 39% identity/63% similarity), highlighting the consistency of the method. Similarly, orthopoxvirus' H5 protein was identified as the only candidate in the vaccinia and monkeypox viruses (93% identity). On the other hand, in *Pandoravirales* (which present a nuclear DNA replication (46)), 8 candidate proteins were identified. Regardless, all candidate VF scaffold IDRs predicted in all *Nucleocytoviricota* are distributed on the UMAP without order segregation, further reinforcing the shared molecular grammar for the cytoplasmic VFs (Fig. 3C).

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To challenge scaffold protein predictions across the *Nucleocytoviricota*, we expressed recombinantly vaccinia virus (order: *Chitovirales*) H5 (as previously described (47)) and noumeavirus (order: *Pimascovirales*) NMV_238 (Fig. S10A). Similar to OLS1, both H5 and NMV_238 formed biomolecular condensates in the absence of other macromolecules (Fig. 3G-H). Phase separation depended on protein concentration (Fig. S10B-C) and salt concentrations
293 (Fig. S10D-E). Interestingly, in contrast to OLS1, which does not bind dsDNA, both H5 and
294 NMV_238 were capable of binding and concentrating DNA, as shown by DAPI staining (Fig. 3G-
295 H). Moreover, condensation was slightly favored in the presence of dsDNA (Fig. S10B-E).
296 Overall, both scaffold proteins display the capacity to phase separate independently of other
297 macromolecules upon diminishing ionic strength (similar to OLS1), but display DNA binding and
298 seclusion similarly to ILS1.

299 While H5 shows a propensity to form biomolecular condensates, the recombinant protein did not
300 show the expected liquid-liquid phase separation behavior (however, H5 is heavily
301 phosphorylated during the infectious cycle (48), potentially affecting phase separation). To further
302 address the validity of scaffold predictions in the *Pokkesviricetes*, we expressed the three
303 candidates identified as potential scaffold proteins from manivirus lostcity (order: *Asfuvirales*)
304 in *A. castellanii*. PN-99-GFP showed the classical pattern of phase separation similar to mimivirus
305 OLS1 (Fig. 3I). PN_47-GFP displayed similar characteristics but also strongly interacted with the
306 contractile vacuole (Fig. 3I). Finally, PN-12-GFP showed a nuclear localization (Fig. 3I).
307 Moreover, upon infection with manivirus, both PN-99-GFP and PN_47-GFP re-localized to the
308 VFs while PN-12-GFP remained nuclear (Fig. 3J). Interestingly, homologues of PN-99-GFP and
309 PN_47-GFP were consistently identified as scaffolds in all manivirus isolates, while PN-12-
310 GFP was only identified in manivirus lostcity and A23 (Dataset S2). Taking this data together,
311 both PN-99-GFP and PN_47-GFP are scaffold proteins encoded by manivirus.

312 Importantly, while candidate scaffold proteins were found in all orders of *Nucleocytoviricota*
313 irrespective of their host, the experimental validation of this grammar was mostly centered on
314 amoeba-infecting viruses. Thus, further analysis on other host-pathogen models will be
315 necessary to confirm its universality.

316 Functional sub-compartmentalization of viral factories

317 While a previous study reported proteins tentatively localized at the VFs of mimivirus (20), it did
318 not differentiate proteins associated with each sub-compartments of the VFs and displayed high
319 rates of false positives (Fig. S11A-B). To fill this gap, we performed immunoprecipitations of the
320 two scaffold proteins and identified co-purified proteins by mass spectrometry (MS)-based
321 proteomics (Fig. 4A, S11C and Dataset S3). Several proteins enriched with OLS1 and/or ILS1
322 were endogenously tagged to confirm their localization (Fig. 4B and S11B). None of the false-
323 positive examples identified in the previous proteome study were detected in these
324 immunoprecipitations (Dataset S3). Moreover, enrichment of proteins localized to the outer layer
325 of the VF co-purified with OLS1, while inner layer proteins or virion proteins were co-
326 immunoprecipitated majorly with ILS1 (Fig. 4C). Importantly, while this study shows a low rate of
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false positive identifications, other proteins not detected in the immunoprecipitations are still
localized at the VF (Fig. 4C).

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Several host ribosomal proteins were enriched in the immunoprecipitations of OLS1-GFP (but not with ILS1-RFP), suggesting some proximity between the outer layer of the VF and the ribosomes (Fig. 5A and Dataset S3). Regardless, VFs are thought to segregate replication and transcription from translation (20). To test the current consensus, we generated recombinant viruses or amoebae encoding tagged versions of several proteins involved in the central dogma (Fig. 5A-D and Fig. S12A). As previously suggested (20), host ribosomes (Fig. 5C and Fig. S12A) and the viral translation-associated protein SUI1 (Fig. 5D) were excluded from the VFs. On the other hand, the virally encoded eIF4e localized both at the cytoplasm of the host and the outer layer of the VF (Fig. 5D). In eukaryotes, besides its classical cytoplasmic function in translation initiation, eIF4E also localizes at the nucleus, where it participates in the export of a subset of mRNA (49). If such export function is conserved in the virally encoded eIF4E, it remains to be explored, but it would explain the dual localization of the protein.

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Proteins associated with replication and transcription are incorporated into the VFs but sub-
compartmentalized. While replication proteins localized to the outer layer of the VF, transcription
proteins accumulated at the inner layer (Fig. 4A and Fig. 5A-B). Pulse-labeling of mRNAs using
5-ethynyluridine (EU) for 15 minutes strongly suggests that mRNA production site is located at
the inner layer of the VF (Fig. 5E), colocalizing with the RNA polymerase (Fig. 5B). On the other
hand, we were unable to efficiently label DNA by 5-Ethynyl-2-deoxyuridine (EdU) in wild-type
viruses (Fig. S12B). We reasoned that due to the high AT richness of mimivirus genome (50), de
novo synthesis of dTMP would be particularly efficient in infected cells, allowing thymidine to
outcompete EdU for its incorporation into the DNA (Fig. S12C). Thus, we generated knockout
recombinant viruses on the virally encoded thymidylate synthase (TS), which concordantly
blocked viral de novo synthesis of dTMP (Fig. S12D-E). EdU labeling significantly improved in
these viruses, allowing the visualization of DNA replication with pulses of EdU labelling as short
as 5 minutes (Fig. 5F and Fig. S12F). Similarly to what was observed in the vaccinia virus (51),
pulse labeling of DNA replication in late VF resulted in a lower fluorescence intensity than
labelling in early VF (Fig. 5F-G), showing that DNA replication decreases during the late stages of
infection. 5-minute labelling with EdU showed an enrichment of newly synthesized DNA at the
periphery of the inner layer of the VF (Fig. 5F). Concordantly, DNA replication occurs at the
interface between the inner layer and the outer layer, where DNA and the DNA polymerase get in
contact. Finally, mRNA processing (including capping and Poly(A) synthesis) localized at the
inner layer of the VF, indicating that maturation of pre-mRNA occurs at the same sub-
compartment as transcription (Fig. 5H). Overall, a significant sub-compartmentalization of
functions is observed at mimivirus VFs.
363

364 Discussion

365

366 The molecular grammar of an IDR in a scaffold protein determines the nature of the interaction to achieve phase separation and the selective recruitment of client proteins (37-42, 52). Here, we demonstrate that amoeba-infecting members of the *Nucleocytoviricota* phylum share a common molecular grammar governing the formation of their VFs. This observation likely extrapolates to non-amoeba infecting viruses but further experiments would need to be performed with different host-pathogen models in order to confirm this prediction. Such compact means of functional maintenance allow its inheritance with little gene conservation, solving a long-standing paradigm of how such a diverse phylum can conserve a replication strategy. Importantly, since the common ancestor of all *Nucleocytoviricota* likely already possessed a VF with this molecular grammar, and as the origin of *Nucleocytoviricota* is thought to predate the origin of eukaryotes (and thus, the origin of organelles) (53), this organelle-like structure might represent a primordial solution for compartmentalization. Such conservation of the grammar is likely driven by the complex number of client proteins recruited to the VFs, which would need to change their biochemical/biophysical properties simultaneously if the molecular grammar of the VF suddenly changes. Such a scenario is parsimoniously unlikely. Regardless, it is unclear whether all scaffold protein IDRs share a common origin and diversification occurred by shuffling protein fragments (54) or if the same molecular grammar emerged in multiple IDRs on different occasions by convergent evolution (55). Which advantages the virus gains by modifying the scaffold proteins that form their VFs remains to be addressed but might be associated with emergent traits of different VFs (including the appearance of an inner layer in mimivirus allowing sub-compartmentalization of replication and transcription, or the endoplasmic reticulum wrapping in poxviruses (56)).

387

388 Such grammar allowed us to identify previously neglected but well-characterized scaffold proteins in other members of the phylum. In *Poxviridae*, H5 was predicted as the sole candidate for a scaffold protein for phase separation. H5 is an essential protein for the infectious cycle of the vaccinia virus (57) and was coined as a hub protein due to its importance in DNA replication, transcription, and virion morphogenesis (47). All those phenotypes correlate with a scaffolding function for phase separation. Moreover, H5 binds DNA (47) (a function modulated by phosphorylation (48)) and localizes as puncta upon heterologous expression in mammalian cells (indicating spontaneous phase separation of the protein) (57). Interestingly, when vaccinia virus uncoating occurs, early viral proteins associated with DNA replication localize to cytoplasmic puncta, including H5 (47, 58). We propose that these puncta (known as prereplication foci) are likely formed by phase separation using H5 as a scaffold protein. Upon maturation of the prereplication foci, VF gets surrounded by the ER membranes (56). In addition, H5 strongly accumulates inside the VF (59, 60), and the VFs dissolve in the presence of 1,6-hexanediol.
Moreover, VFs of the vaccinia virus have previously been shown to coalesce when present in the same cell (61). These data support the idea that phase separation is a major driver of the VF formation regardless of the ER wrapping. It has been proposed that H5 would be a key component for VF enlargement and wrapping by the ER (58). Overall, previously published data on H5 strongly support its role as an unrecognized scaffold protein for phase separation. In *Marseilleviridae*, at least two scaffold proteins localize to the VFs with differential transcriptional expression patterns (62). This allows us to hypothesize that NMV_095 might initiate VF formation while NMV_238 would allow its expansion and maturation. Further experiments will be needed to corroborate this hypothesis. In *Pandoravirales*, multiple IDRs containing a similar molecular grammar to VF scaffold proteins were identified. Regardless, these viruses transfer their DNA into the nucleus of their host. In evolutionary terms, it is parsimonious to assume that a fully cytoplasmic infectious cycle style of the majority of the members of the phylum originated prior to the nuclear one (29), as the origin of *Nucleocytoviricota* predated the origin of eukaryotes (and thus, the nucleus) (53). Thus, during the transition from cytoplasmic to nuclear viruses, the transfer of the DNA into the nucleus would generate a VF, which is no longer needed for replication and transcription but would still retain the functions associated with virion morphogenesis. Nonetheless, only imaging data (electron microscopy (8, 9) and immunofluorescence (21)) support the presence of these structures and further experiments would be needed to characterize such "Virion Factories" potentially assembled by nuclear giant viruses and their functions.

Mimivirus VFs are highly compartmentalized organelle-like structures (Fig. S13). Biogenesis of the VFs starts by utilizing the cores as nucleating points. Similar observations were previously made on vaccinia virus, indicating some conserved mechanisms of nucleation (60). Mimivirus VFs then develop into multilayered structures that contain at least two distinctive phases. The outer layer of the VF, formed by OLS1, acts as a selective barrier and recruits VF proteins. Importantly, while we hypothesize that the outer layer of mimivirus VFs is the phylogenetically conserved phase between different *Nucleocytoviricota*, OLS1 is dispensable in mimivirus. We theorize that the presence of the inner layer allows the protection of the genome of the virus in the absence of the outer layer and, despite losing the ability to selectively recruit proteins to the VF and considering the permissive conditions of the laboratory, the inner layer is sufficient to achieve successful infection. The DNA replication machinery localizes to the outer layer of the VF and maximizes DNA replication only at the interphase between outer layer and inner layer. This interphase is also the site of virion assembly, and the competition between these two processes may at least partially explain the decrease in DNA synthesis during the late stages of the infection cycle. The inner layer of the VF contains proteins associated with transcription, which are also packaged into the virions to establish a new cycle of infection (including the RNA polymerase,
transcription factor, RNA processing, etc.). Thus, the inner layer appears to be a compartment analogous to the internal content of the virion core, which is sufficient to restart RNA transcription of early genes upon infection of a new cell (13). Moreover, ILS1 has recently been proposed to work on mimivirus DNA condensation for its incorporation into the viral particle (36). Finally, translation occurs outside of the VF, a feature that differentiates mimivirus from vaccinia virus (63). Importantly, how mRNAs are exported from the VFs remains unknown. Nevertheless, since neither ILS1 nor OLS1 requires RNA for phase separation, a simple model can be envisioned where RNA is not retained by either the inner layer or outer layer of the VF. In such a case, diffusion would be sufficient to deliver mRNAs into the cytoplasm of the infected cell.

Overall, this discovery raises new questions, including how cytoplasmic viruses interact upon infection of the same host cell (can VFs from different viruses fuse?), and how biomolecular condensates accommodate the various functions required for VFs' multiple roles spatiotemporally. This work opens the door for developing generalist drugs to inhibit *Nucleocytiviricota* viral infections, including monkeypox and African swine fever viruses.

450

451

## Materials and Methods

453

### A. castellanii growth and virus production

The following viral strains have been used in this study: Acanthamoeba polyphaga mimivirus (50), noumeavirus (29), pithovirus sibericum (64), manivirus lost city (30), mollivirus sibericum (32), Modified Vaccinia virus Ankara (MVA) strain. See supplementary Materials and Methods. Viral purification was performed as shown in (65).

### Generation of DNA constructs and viral lines

See Supplementary Materials and Methods.

### Protein expression and purification and Phase separation assays

See Supplementary Materials and Methods.

### Immunofluorescence, fluorescence microscopy and electron microscopy imaging.

See Supplementary Materials and Methods.

### 1,6-hexanediol treatment, EU and EdU labelling

See Supplementary Materials and Methods.
467 **Immunoprecipitation and MS-based proteomic analyses**

468 Immunoprecipitation was performed as previously described (66). Proteins eluted from co-IP
469 experiments were either separated by SDS-PAGE or stacked as previously described (67).

470 Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (68)
471 (PXD054803). Peptides and proteins were identified by Mascot (version 2.8.3, Matrix Science).

472 The Proline software (69) (version 2.3) was used for the compilation, grouping and filtering of the
473 results. Statistical analysis was performed using the ProStaR software (70). See Supplementary
474 Materials and Methods for extended protocol.

475 **Prediction of the VF scaffold proteins**

476 A database was constituted with genomes of isolated viruses, predicted proteins from the Giant
477 virus database (4) (PRJEB47746), and *Egovirales* (44). Intrinsically disordered regions were
478 predicted using MobiDB-lite v3.10.0 (71). For the residue binary patterns, the python package
479 Nardini v1.1.1 was used. Compositional data and physical and chemical properties of IDRs were
480 predicted with localCIDER v0.1.21 (72). Nardini and CIDER features were normalized. All Z-
481 scores and normalized features were then saturated by the sigmoid function (73). Scaffold protein
482 homologues were identified by MMseqs2 v.12 (74) with an e-value cutoff of 1e-5 and further
483 confirmed by alignment with t-coffee v13.41.0 (75). HMM models were constructed with HMMER
484 v3.3.2 (76). Sequences were considered as homologs only for alignments with e-values <1e-10.

485 The final SVM classifier was built on a rbf kernel considering 11 features whose Spearman
486 correlation coefficient is under 0.55. These features included two Nardini features and 9 CIDER
487 features. MolPhase (77) and ParSe v2 (78) were also tested. See Supplementary Materials and
488 Methods for an extended method.

489
490 **Acknowledgments**

491 The authors also thank Matias Estaras Hermosel, Christian Betzel, Gamaleldin I. Harisa, Chunfu
492 Zheng, Anouk Willemse, Andrea Maria Guarino, Yves Gaudin, Juliana Cortines and Kekungu-u
493 Puro for the comments raised on the preprint version of the manuscript. We thank the members
494 of the PiCSL-FBI core facility (Nicolas Brouilly, Fabrice Richard and Aïcha Aouane), IBDM, AMU-
495 Marseille, and the IMM imaging platform (Artemis Kosta). NO and MB gratefully acknowledge the
496 Advanced Bioimaging Unit at the Institut Pasteur Montevideo & Universidad de la República.

497 **Funding:** European Research Council grant agreement No. 101160452, ERC-2024-STG (HB).
498 European Research Council grant agreement No 832601 ERC-2018-ADG (CA). Views and
499 opinions expressed are, however, those of the authors only and do not necessarily reflect those
500 of the European Union or the European Research Council (ERC). Neither the European Union
501 nor the granting authority can be held responsible for them. Agence Nationale de la Recherche
502 grant ProFI, ANR-10-INBS-08 (YC). Chemistry Biology Health (CBH) Graduate School of
503 University Grenoble Alpes grant ANR-17-EURE-0003 (YC)

504
505

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680 **Figures and Tables**

681

682 **Figure 1.** *Nucleocytoviricota* viral factories display liquid-like properties. (A) Taxonomy of viruses belonging to the *Nucleocytoviricota* phylum (Adapted from (6)). (B) Transmission electron microscopy imaging (and cartoon) of an ultrathin section of an infected *A. castellanii* cell with mimivirus VF formed in the cytoplasm. 6h post-infection at a MOI of 20. (C) *A. castellanii* cells expressing C-terminally tagged OLS1-GFP (R561, outer layer) and ILS1-RFP (R252, inner layer) infected with mimivirus. Line profiles (below) corresponding to the white dashed line show fluorescence patterns. 6h post infection at a MOI of 10. (D) Representative images of *Acanthamoeba* cells expressing OLS1-GFP were infected with mimivirus at a MOI of 5. Five hours post-infection, OLS1-GFP was photobleached and fluorescence recovery was imaged by confocal microscopy. FRAP data were normalized, corrected for background and represented in the recovery curve. Data represent the mean ± SD of 7 experiments. (E) Live-cell imaging of noumeavirus-infected *A. castellanii* expressing mel_H2B-H2A-GFP as a marker of the VF. Recording was performed 2h post infection. Cell boundaries are indicated by a dashed line. (F) Representative images of Acanthamoeba cells expressing PN99-GFP were infected with Pacmanvirus lost city at a MOI of 10. Three hours post-infection, PN99-GFP was photobleached and fluorescence recovery was imaged by confocal microscopy. FRAP data were normalized, corrected for background and represented in the recovery curve. Data represent the mean ± SD of 10 experiments.

700
Figure 2. Two scaffold proteins are involved in mimivirus viral factory assembly. (A) *In vitro* phase separation of ILS1 in the presence or absence of DNA. DAPI was used to confirm nucleic acid presence in the condensate. (B) *In vitro* phase separation of mCherry-OLS1 at 50mM NaCl. (C) *In vitro* phase separation of mCherry-OLS1 and ILS1 in the presence of 10 µg/mL DNA. DAPI was used to confirm nucleic acid presence in the condensate. A plot profile is shown demonstrating a biphasic interaction between the two phase-separated compartments. (D) Quantification of the number of viruses produced upon knock-out of *ols1* in mimivirus. Data correspond to the mean ± SD of 3 independent experiments. (E) DNA replication was analyzed by qPCR. Viral DNA is represented as a percentage of total DNA in the sample. Data correspond to the mean ± SD of 3 independent experiments. (F) Representative light fluorescence microscopy images of *A. castellanii* cells infected with wild-type mimivirus or *ols1* KO viruses 6h pi. DAPI: VFs. (G) Quantification of the size of VF generated, as shown in (F). At least 50 VF were recorded per condition during 3 independent experiments. The area of DAPI staining was measured using ImageJ. (H) Quantification of the number of VF either fused or separated in infected cells as shown in (F). At least 100 infected cells were recorded per condition. Data correspond to the mean ± SD of 3 independent experiments. (I) Transmission electron microscopy imaging (and representative cartoon) of the mimivirus replication cycle in *A. castellanii*. Images were acquired 4-6h pi (MOI=20). (J) Localization of client protein (R336/337) from the outer layer of the VF endogenously tagged with C-terminal 3xHA. 6 hpi. DAPI: VFs. (K) Quantification of the number of (I). At least 100 infected cells were quantified per condition. Data correspond to the mean ± SD of 3 independent experiments. ns (P > 0.05), \* (P ≤ 0.05), \*\* (P ≤ 0.01), \*\*\* (P ≤ 0.001) and \*\*\*\* (P ≤ 0.0001).

724
Figure 3. A common grammar allows the identification scaffold proteins across the entire *Nucleocytoviricota phylum*. (A) *A. castellanii* cells expressing C-terminally GFP-tagged NMV_095 or NMV238. (B) *A. castellanii* cells expressing C-terminally GFP-tagged NMV_095 or NMV238 were infected with noumeavirus. VFs were labelled using DAPI 2-3 h pi. (C) UMAP representation of the IDRs in representative genomes and metagenomics giant viruses, based on the 11 features selected for the classifier. Each point represents one predicted IDR. IDRs corresponding to negative predictions are shown in grey. Inset: Scaffold candidates were labeled according to their Order classification (or higher-level classification available). (D) and (E) Details of the most discriminant features on the UMAP. Pos.neg (positive from negative segregation) and pos.pos (positive to all other residues) are plotted. (F) Count of candidate proteins for VF phase separation predicted by the classifier in representatives of each *Nucleocytoviricota* family. (G) NMV_238 and (H) H5 form biomolecular condensates *in vitro* in the absence and presence of DNA. The solution contains 25 mM NaCl and 0.6 µg/mL DNA, where indicated. (I) and (J) *A. castellanii* cells expressing C-terminally tagged scaffold candidates of pacmanvirus lostcity were either non-infected (I) or infected with pacmanvirus (J). VFs were labelled using DAPI 6 h pi.

740

741

742
**Figure 4.** Viral factory proteins are sub-compartmentalized. (A) Immunoprecipitation experiments in *A. castellanii* cells expressing ILS1-RFP or OLS1-GFP and infected by mimivirus. Cells expressing RFP or GFP were utilized as controls. Crosslinking was performed prior to cell lysis. Immunoprecipitated proteins were analyzed through MS-based label-free quantitative proteomics (three replicates per condition). The volcano plots represent the -log10 (limma p-value) on the y axis plotted against the log2(Fold Change bait vs control) on x axis for each quantified protein (upper panel: OLS1-GFP versus GFP, bottom panel: ILS1-RFP versus RFP). Each dot represents a protein. Proteins with log2 (FoldChange) ≥ 1.6 (FoldChange > 3) and -log10(p-value) ≥ 2 (p-value = 0.01) compared to controls, were considered significant (Benjamini-Hochberg FDR < 2%) and are shown in black. Detailed data are presented in Dataset S3. (B) Immunofluorescence demonstrating localization of proteins enriched in A. Proteins were endogenously tagged with 3xHA at the C-terminal of each mimivirus gene, and infection was carried out for 6 hours before fixation. VFs were labelled using DAPI. (C) Immunofluorescence demonstrating localization of proteins not enriched in A but still displaying a VF localization. Proteins were endogenously tagged with 3xHA at the C-terminal, and infection was carried out for 6 hours before fixation. VFs were labelled using DAPI.
**Figure 5.** Central dogma functions are sub-compartmentalized at the mimivirus viral factories. (A) Localization of proteins associated with DNA replication endogenously tagged with C-terminal 3xHA. 6 hpi. (B) Localization of the RNA polymerase subunit 2 (rbp2), associated with transcription, endogenously tagged with C-terminal 3xHA. 6 hpi. (C) Localization of host ribosomal protein RPL22-RFP, associated with translation. Protein is expressed from a second copy plasmid encoding *rpl22-rfp*. 6 hpi. (D) Localization of viral proteins associated with translation endogenously tagged with C-terminal 3xHA. 6 hpi. (E) Detection of RNA synthesis by EU labelling. Viral infection was allowed to proceed for 4-6 hours, and labelling time is indicated. (F) Detection of DNA synthesis by EdU labelling. Viral infection by *thymidylate synthase* KO viruses was allowed to proceed for 4-6 hours, and labelling time is indicated. (G) Quantification of the corrected total VF fluorescence (CT(VF)F) of EdU labeling as shown in F. Twenty VFs were reordered during 3 independent experiments, and the intensity of EdU staining was measured using ImageJ. **** (P ≤ 0.0001). (H) Localization of proteins associated with mRNA maturation endogenously tagged with C-terminal 3xHA. 6 hpi. In all cases: Line profiles corresponds to the white dashed lines. DAPI: VF inner layer. R366/337-3xHA or OLS1-GFP: VF outer layer.
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /></figure>

Host: A. castellanii
Virus: Mimivirus

<figure><img src="image_5.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_6.png" /></figure>

ILS1 (R252)-RFP Merge

Host: A. castellanii
Virus: Mimivirus

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

Imitervirales

<figure><img src="image_8.png" /></figure>

Merge: OLS1-GFP and DIC

Host: A. castellanii
Virus: Mimivirus

<figure><img src="image_9.png" /><figcaption>E</figcaption></figure>

Pimascovirales

<figure><img src="image_10.png" /></figure>

mel_H2B-H2A-GFP

Host: A. castellanii
Virus: Numeavirus

<caption>F</caption>

Asfuvirales

<figure><img src="image_11.png" /></figure>

Merge: PN_99-GFP and DIC

Host: A. castellanii
Virus: Pacmanvirus

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_23.png" /></figure>

Host: *A. castellanii*

Virus: Mimivirus
A

<figure><img src="image_24.png" /></figure>

NMV_95-GFP DIC

<figure><img src="image_25.png" /></figure>

NMV_238-GFP DIC

Host: A.i cast@l.m

B

<figure><img src="image_26.png" /></figure>

NMV_95-GFP

DAPI

Merge

<figure><img src="image_27.png" /></figure>

NMV_238-GFP

DAPI

Merge

D

Paramecium_bursaria_Chlorella_virus_1- ②

African_swine_fever_virus- ①

Vaccinia_virus- ①

Medusavirus- ⑤

Acanthamoeba_polyphaga_mimivirus- ⑤

Pyramimonas_orientalis_virus- ①

Phaeocystis_globosa_virus- ①

Aureococcus_anophagefferens_virus- ④

Pandoravirus_neocaledonia- ⑧

Noumeavirus- ②

Pithovirus_sibericum- ①

Hydrivirus- ③

Orpheovirus_IHUMI-LCC2- ①

Invertebrate_iridescent_virus_6- ①

Yaravirus_brasiensis- ①

C

<figure><img src="image_28.png" /></figure>

E

<figure><img src="image_29.png" /></figure>

pos.neg

<figure><img src="image_30.png" /></figure>

F

<figure><img src="image_31.png" /></figure>

pos.pos

<figure><img src="image_32.png" /></figure>

G

<figure><img src="image_33.png" /></figure>

DAPI

DIC

Merge

NMV_238: 11.1 µM

I

<figure><img src="image_34.png" /></figure>

PLN_12-GFP

DIC

1 µm

J

<figure><img src="image_35.png" /></figure>

PLN_12-GFP

DAPI

Merge

1 µm

<figure><img src="image_36.png" /></figure>

DAPI

DIC

Merge

H5: 30 µM

<figure><img src="image_37.png" /></figure>

PLN_47-GFP

DIC

1 µm

<figure><img src="image_38.png" /></figure>

PLN_47-GFP

DAPI

Merge

1 µm

<figure><img src="image_39.png" /></figure>

PLN_99-GFP

DIC

1 µm

<figure><img src="image_40.png" /></figure>

PLN_99-GFP

DAPI

Merge

1 µm

Host: A.i cast@l.m

Virus: Pacman virus
<figure><img src="image_41.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_43.png" /></figure>

HA-tag
Host: *A. castellanii*
Virus: Mimivirus

<figure><img src="image_44.png" /><figcaption>B</figcaption></figure>

HA-tag
Host: *A. castellanii*
Virus: Mimivirus
<figure><img src="image_45.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /><figcaption>Translation</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>Translation</figcaption></figure>

<figure><img src="image_63.png" /></figure>

Host: *A. castellanii*
Virus: Mimivirus