Virus-encoded histone doublets are essential and form nucleosome-like structures.

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.

Genome chromatinization in giant double-stranded DNA viruses.

Giant viruses have extravagantly large double-stranded (ds)DNA genomes that are packaged into exceedingly complex virions. In two recent papers, Liu et al. and Valencia-Sánchez, Abini-Agbomson et al. show that some giant viruses encode unique histone doublets, which form nucleosomes remarkably similar to those found across the eukaryotic domain of life.

Particle Morphology of Medusavirus Inside and Outside the Cells Reveals a New Maturation Process of Giant Viruses.

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoebae infected with the medusavirus showed four types of particles, i.e., pseudo-DNA-empty (p-Empty), DNA-empty (Empty), semi-DNA-full (s-Full), and DNA-full (Full). Time-dependent changes in the four types of particles and their intracellular localization suggested a new maturation process for the medusavirus. Viral capsids and viral DNAs are produced independently in the cytoplasm and nucleus, respectively, and only the empty particles located near the host nucleus can incorporate the viral DNA into the capsid. Therefore, all four types of particles were found outside the cells. The cryo-EM of these particles showed that the intact virus structure, covered with three different types of spikes, was preserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the Empty and p-Empty particles. The results suggest that these open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses. IMPORTANCE Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture. Time-course analysis and intracellular localization of the medusavirus in the infected cells suggested a new maturation process via the four types of particles. Like the previously reported pandoravirus, the viral DNA of medusavirus is replicated in the host's nucleus. However, viral capsids are produced independently in the host cytoplasm, and only empty capsids near the nucleus can take up viral DNA. As a result, many immature particles were released from the host cell along with the mature particles. The capsid structure is well conserved among the four types of particles, except for the open membrane structures in the empty particles, suggesting that they are used to exchange scaffold proteins for viral DNAs. These findings indicate that medusavirus has a unique maturation process.

Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organisms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible function of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Chromosome-level genome of Pedinomonas minor (Chlorophyta) unveils adaptations to abiotic stress in a rapidly fluctuating environment.

The Pedinophyceae (Viridiplantae) comprise a class of small uniflagellate algae with a pivotal position in the phylogeny of the Chlorophyta as the sister group of the 'core chlorophytes'. We present a chromosome-level genome assembly of the freshwater type species of the class, Pedinomonas minor. We sequenced the genome using Pacbio, Illumina and Hi-C technologies, performed comparative analyses of genome and gene family evolution, and analyzed the transcriptome under various abiotic stresses. Although the genome is relatively small (55 Mb), it shares many traits with core chlorophytes including number of introns and protein-coding genes, messenger RNA (mRNA) lengths, and abundance of transposable elements. Pedinomonas minor is only bounded by the plasma membrane, thriving in temporary habitats that frequently dry out. Gene family innovations and expansions and transcriptomic responses to abiotic stresses have shed light on adaptations of P. minor to its fluctuating environment. Horizontal gene transfers from bacteria and fungi have possibly contributed to the evolution of some of these traits. We identified a putative endogenization site of a nucleocytoplasmic large DNA virus and hypothesized that endogenous viral elements donated foreign genes to the host genome, their spread enhanced by transposable elements, located at gene boundaries in several of the expanded gene families.

Diversification of giant and large eukaryotic dsDNA viruses predated the origin of modern eukaryotes.

Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ∼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it.

Structure-Based Deep Mining Reveals First-Time Annotations for 46 Percent of the Dark Annotation Space of the 9,671-Member Superproteome of the Nucleocytoplasmic Large DNA Viruses.

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover.IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesvirus surface glycoprotein gB in cytoplasmic viruses. This study provided the first prediction of an endonuclease in 10 of the 20 viruses examined; the first report in a virus of a phenolic acid decarboxylase, proteasomal subunit, or cysteine knot (defensin) protein; and the first report of a prokaryotic-type ribosomal protein in a eukaryotic virus.

Characterization of Mollivirus kamchatka, the First Modern Representative of the Proposed Molliviridae Family of Giant Viruses.

Microbes trapped in permanently frozen paleosoils (permafrost) are the focus of increasing research in the context of global warming. Our previous investigations led to the discovery and reactivation of two Acanthamoeba-infecting giant viruses, Mollivirus sibericum and Pithovirus sibericum, from a 30,000-year old permafrost layer. While several modern pithovirus strains have since been isolated, no contemporary mollivirus relative was found. We now describe Mollivirus kamchatka, a close relative to M. sibericum, isolated from surface soil sampled on the bank of the Kronotsky River in Kamchatka, Russian Federation. This discovery confirms that molliviruses have not gone extinct and are at least present in a distant subarctic continental location. This modern isolate exhibits a nucleocytoplasmic replication cycle identical to that of M. sibericum Its spherical particle (0.6 µm in diameter) encloses a 648-kb GC-rich double-stranded DNA genome coding for 480 proteins, of which 61% are unique to these two molliviruses. The 461 homologous proteins are highly conserved (92% identical residues, on average), despite the presumed stasis of M. sibericum for the last 30,000 years. Selection pressure analyses show that most of these proteins contribute to virus fitness. The comparison of these first two molliviruses clarify their evolutionary relationship with the pandoraviruses, supporting their provisional classification in a distinct family, the Molliviridae, pending the eventual discovery of intermediary missing links better demonstrating their common ancestry.IMPORTANCE Virology has long been viewed through the prism of human, cattle, or plant diseases, leading to a largely incomplete picture of the viral world. The serendipitous discovery of the first giant virus visible under a light microscope (i.e., >0.3 µm in diameter), mimivirus, opened a new era of environmental virology, now incorporating protozoan-infecting viruses. Planet-wide isolation studies and metagenome analyses have shown the presence of giant viruses in most terrestrial and aquatic environments, including upper Pleistocene frozen soils. Those systematic surveys have led authors to propose several new distinct families, including the Mimiviridae, Marseilleviridae, Faustoviridae, Pandoraviridae, and Pithoviridae We now propose to introduce one additional family, the Molliviridae, following the description of M. kamchatka, the first modern relative of M. sibericum, previously isolated from 30,000-year-old arctic permafrost.

The African Swine Fever Virus Transcriptome.

African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5' ends of transcription units, adding quasitemplated AU- and AUAU-5' extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.

Isolation of Yasminevirus, the First Member of Klosneuvirinae Isolated in Coculture with Vermamoeba vermiformis, Demonstrates an Extended Arsenal of Translational Apparatus Components.

The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis This giant virus, called Yasminevirus, has a 2.1-Mb linear double-stranded DNA genome encoding 1,541 candidate proteins, with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery, with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids (aa), 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic, with ∼34% of genes having their closest homologues in other viruses, followed by ∼13.2% in Eukaryota, ∼7.2% in Bacteria, and less than 1% in Archaea Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses.IMPORTANCE Yasminevirus is an icosahedral double-stranded DNA virus isolated from sewage water by amoeba coculture. Here its structure and replicative cycle in the amoeba Vermamoeba vermiformis are described and genomic and evolutionary studies are reported. This virus belongs to the Klosneuvirinae group of giant viruses, representing the second isolated and cultivated giant virus in this group, and is the first isolated using a coculture procedure. Extended translational machinery pointed to Yasminevirus among the quasiautonomous giant viruses with the most complete translational apparatus of the known virosphere.

Medusavirus, a Novel Large DNA Virus Discovered from Hot Spring Water.

Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260 nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the host nucleus, whereas the progeny virions are assembled in the cytoplasm. Furthermore, the medusavirus genome harbored genes for all five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. In contrast, the host amoeba encoded many medusavirus homologs, including the major capsid protein. These facts strongly suggested that amoebae are indeed the most promising natural hosts of medusavirus, and that lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their coevolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and by evolutionarily long lasting virus-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that medusavirus represents a new family, MedusaviridaeIMPORTANCE We have isolated a new nucleocytoplasmic large DNA virus (NCLDV) from hot spring water in Japan, named medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes, including that encoding DNA polymerase, and its genome surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many medusavirus homologs in its genome, including the major capsid protein, suggesting that the amoeba is the genuine natural host from ancient times of this newly described virus and that lateral gene transfers have repeatedly occurred between the virus and amoeba. These results suggest that medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and virus-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that medusavirus represents a new viral family, Medusaviridae.

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton.

Phytoplankton community structure is shaped by both bottom-up factors, such as nutrient availability, and top-down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.

First genetic characterization of sturgeon mimiviruses in Ukraine.

A group of pathogenic nucleocytoplasmic large DNA viruses (NCLDVs) related to the Mimiviridae family infect farmed sturgeons across Europe, causing mild-to-severe losses. One of these viruses, Acipenser iridovirus-European (AcIV-E), was identified in six sturgeon species. During the 2018-2019 period, nine sick Siberian (A. baerii) and Russian (A. gueldenstaedtii) sturgeons were sampled in Ukrainian farms and tested for the presence of AcIV-E using real-time PCR. The presence of AcIV-E was confirmed in some samples. High-resolution melting (HRM) assay and Sanger sequencing demonstrated the presence in three farms of two alleles of the major capsid protein (MCP) gene, called var1 and var2. Five samples carried both var1 and var2 at varying ratios, and the sixth sample was infected with only var1. These results constitute the first detection of AcIV-E in Ukraine and the first detection of a sample carrying only var1. The full-length sequences of the MCP genes confirmed the existence of two genetic lineages of AcIV-E, tentatively named V1 and V2, each displaying multiple substitutions in the MCP gene. Some of the MCP sequences showed a genetic relationship to both V1 and V2 lineages, depending on the fragment examined. Most likely, these sequences resulted from recombination events.

Morphologic and Genomic Analyses of New Isolates Reveal a Second Lineage of Cedratviruses.

Giant viruses have been isolated and characterized in different environments, expanding our knowledge about the biology of these unique microorganisms. In the last 2 years, a new group was discovered, the cedratviruses, currently composed of only two isolates and members of a putative new family, "Pithoviridae," along with previously known pithoviruses. Here we report the isolation and biological and genomic characterization of two novel cedratviruses isolated from samples collected in France and Brazil. Both viruses were isolated using Acanthamoeba castellanii as a host cell and exhibit ovoid particles with corks at either extremity of the particle. Curiously, the Brazilian cedratvirus is ∼20% smaller and presents a shorter genome of 460,038 bp, coding for fewer proteins than other cedratviruses. In addition, it has a completely asyntenic genome and presents a lower amino acid identity of orthologous genes (∼73%). Pangenome analysis comprising the four cedratviruses revealed an increase in the pangenome concomitant with a decrease in the core genome with the addition of the two novel viruses. Finally, phylogenetic analyses clustered the Brazilian virus in a separate branch within the group of cedratviruses, while the French isolate is closer to the previously reported Cedratvirus lausannensis Taking all together, we propose the existence of a second lineage of this emerging viral genus and provide new insights into the biodiversity and ubiquity of these giant viruses.IMPORTANCE Various giant viruses have been described in recent years, revealing a unique part of the virosphere. A new group among the giant viruses has recently been described, the cedratviruses, which is currently composed of only two isolates. In this paper, we describe two novel cedratviruses isolated from French and Brazilian samples. Biological and genomic analyses showed viruses with different particle sizes, genome lengths, and architecture, revealing the existence of a second lineage of this new group of giant viruses. Our results provide new insights into the biodiversity of cedratviruses and highlight the importance of ongoing efforts to prospect for and characterize new giant viruses.

Prasinovirus Attack of Ostreococcus Is Furtive by Day but Savage by Night.

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analyzed the life cycle of Ostreococcus tauri virus 5 (OtV5), a lytic virus, using transcriptome sequencing (RNA-Seq) from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 h, but during the night, viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the dark appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4% of the host's genes. Some host cells underwent lysis progressively during the night, but a larger proportion were lysed the following morning. Our data suggest that the life cycles of algal viruses mirror the diurnal rhythms of their hosts.IMPORTANCE Prasinoviruses are common in marine environments, and although several complete genomes of these viruses and their hosts have been characterized, little is known about their life cycles. Here we analyze in detail the transcriptional changes occurring over a 27-h-long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime, when the most energy (from light) is available, but during the night viral transcription activates, accompanied by expression of a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

A Proteomic Atlas of the African Swine Fever Virus Particle.

African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information.IMPORTANCE African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.

Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses.

African swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus.IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members.

Molecular systematics of sturgeon nucleocytoplasmic large DNA viruses.

Namao virus (NV) is a sturgeon nucleocytoplasmic large DNA virus (sNCLDV) that can cause a lethal disease of the integumentary system in lake sturgeon Acipenser fulvescens. As a group, the sNCLDV have not been assigned to any currently recognized taxonomic family of viruses. In this study, a data set of NV DNA sequences was generated and assembled as two non-overlapping contigs of 306,448 bp and then used to conduct a comprehensive systematics analysis using Bayesian inference of phylogeny for NV, other sNCLDV and representative members of six families of the NCLDV superfamily. The phylogeny of NV was reconstructed using protein homologues encoded by nine nucleocytoplasmic virus orthologous genes (NCVOGs): NCVOG0022 - mcp, NCVOG0038 - DNA polymerase B elongation subunit, NCVOG0076 - VV A18-type helicase, NCVOG0249 - VV A32-type ATPase, NCVOG0262 - AL2 VLTF3-like transcription factor, NCVOG0271 - RNA polymerase II subunit II, NCVOG0274 - RNA polymerase II subunit I, NCVOG0276 - ribonucleotide reductase small subunit and NCVOG1117 - mRNA capping enzyme. The accuracy of our phylogenetic method was evaluated using a combination of Bayesian statistical analysis and congruence analysis. Stable tree topologies were obtained with data sets differing in target molecule(s), sequence length and taxa. Congruent topologies were obtained in phylogenies constructed using individual protein data sets. The major capsid protein phylogeny inferred that ten representative sNCLDV form a monophyletic group comprised of four lineages within a polyphyletic Mimi-Phycodnaviridae group of taxa. Overall, the analyses revealed that Namao virus is a member of the Mimiviridae family with strong and consistent support for a clade containing NV and CroV as sister taxa.

The Autonomous Glycosylation of Large DNA Viruses.

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production.