Jean-Marie Alempic’s research while affiliated with Aix-Marseille University and other places

The microbial sampling of submarine hydrothermal vents remains challenging, with even fewer studies focused on viruses. Here we report the first isolation of a eukaryotic virus from the Lost City hydrothermal field, by co-culture with the laboratory host Acanthamoeba castellanii. This virus, named pacmanvirus lostcity, is closely related to previously isolated pacmanviruses (strains A23 and S19), clustering in a divergent clade within the long-established family Asfarviridae. Its icosahedral particles are 200 nm in diameter, with an electron-dense core surrounded by an inner membrane. Its genome of 395 708 bp (33% G + C) is predicted to encode 473 proteins. However, besides these standard properties, pacmanvirus lostcity was found associated with a new type of selfish genetic element, 7 kb in length, whose architecture and gene content are reminiscent of those of transpovirons, hitherto specific to the family Mimiviridae. Like previously described transpovirons, this element propagates as an episome within its host virus particles and exhibits partial recombination with its genome. In addition, an unrelated 2 kb long episome was also associated with pacmanvirus lostcity. Together, the transpoviron and the 2 kb episome might participate to exchanges between pacmanviruses and other large DNA virus families. It remains to be elucidated if the presence of these mobile genetic elements is restricted to pacmanviruses or was simply overlooked in other members of the Asfarviridae.

The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae, suggesting this may be the major evolutionary force behind their giant genomes.

Pithoviridae are amoeba-infecting giant viruses possessing the largest viral particles known so far. Since the discovery of Pithovirus sibericum, recovered from a 30,000-y-old permafrost sample, other pithoviruses, and related cedratviruses, were isolated from various terrestrial and aquatic samples. Here we report the isolation and genome sequencing of two Pithoviridae from soil samples, in addition to three other recent isolates. Using the 12 available genome sequences, we conducted a thorough comparative genomics study of the Pithoviridae family to decipher the organization and evolution of their genomes. Our study reveals a non-uniform genome organization in two main regions: one concentrating core genes, and another gene duplications. We also found that Pithoviridae genomes are more conservative than other families of giant viruses, with a low and stable proportion (5% to 7%) of genes originating from horizontal transfers. Genome size variation within the family is mainly due to variations in gene duplication rates (from 14% to 28%) and massive invasion by inverted repeats. While these repeated elements are absent from cedratviruses, repeat-rich regions cover as much as a quarter of the pithoviruses genomes. These regions, identified using a dedicated pipeline, are hotspots of mutations, gene capture events and genomic rearrangements, that contribute to their evolution.

The Mimivirus 1.2Mb genome is organized into a nucleocapsid-like genomic fiber composed of a mixture of two GMC-oxidoreductase paralogs (1). Surprisingly, these proteins also constitute the glycosylated fibril layer that decorates the virion (2). The individual inactivation of each gene by homologous recombination-based knockout (KO) confirmed that they are not essential. The resulting genomic fiber corresponds to a 5- and a 6-start helix, similar to the wild type structures, and is composed of the remaining GMC-oxidoreductase. A model is proposed explaining the transition from a 6- to a 5-start helix and their coexistence. The two single mutants and the double mutant, for which both genes have been KO, showed equivalent fitness in laboratory conditions with no impact on the glycosylation of the layer of fibrils surrounding the capsids. Their major protein components can be easily exchanged, as highlighted by the double KO fibril layer composition. Accordingly, we show that the proteins composing the fibril layer can already be different between different members of the family (moumouviruses and megaviruses), echoing their differences in glycan composition (3, 4). These results obtained on two distinct essential processes (genome packaging and virion infectivity) illustrate the exceptional functional redundancy of the Mimiviridae , which may be the major evolutionary force behind their giant genomes. One-Sentence Summary: Functional redundancy warrants mimivirus genomic fiber and fibril layer formation. Subject Area: Biological sciences/Microbiology/Virology/Viral evolution; Biological sciences/Microbiology/Virology/Virus structures

Pithoviruses are amoeba-infecting giant viruses possessing the largest viral particles known so far. Since the discovery of Pithovirus sibericum, recovered from a 30,000-y-old permafrost sample, other pithoviruses, and related cedratviruses, were isolated from various terrestrial and aquatic samples. Here we report the isolation and genome sequencing of two strains of Pithoviridae from soil samples, in addition to three other recent isolates. Using the 12 available genome sequences, we conducted a thorough comparative genomics study of the Pithoviridae family to decipher the organization and evolution of their genomes. Our study reveals a non-uniform genome organization in two main regions: one concentrating core genes, and another gene duplications. We also found that Pithoviridae genomes are more conservative than other families of giant viruses, with a low and stable proportion (5% to 7%) of genes originating from horizontal transfers. Genome size variation within the family is mainly due to variations in gene duplication rates (from 14% to 28%) and massive invasion by miniature inverted-repeats transposable elements (MITEs). While these repeated elements are absent from cedratviruses, repeat-rich regions cover as much as a quarter of the pithoviruses genomes. These regions, identified using a dedicated pipeline, are hotspots of mutations, gene capture events and genomic rearrangements, that likely contribute to their evolution.

One quarter of the Northern hemisphere is underlain by permanently frozen ground, referred to as permafrost. Due to climate warming, irreversibly thawing permafrost is releasing organic matter frozen for up to a million years, most of which decomposes into carbon dioxide and methane, further enhancing the greenhouse effect. Part of this organic matter also consists of revived cellular microbes (prokaryotes, unicellular eukaryotes) as well as viruses that have remained dormant since prehistorical times. While the literature abounds on descriptions of the rich and diverse prokaryotic microbiomes found in permafrost, no additional report about “live” viruses have been published since the two original studies describing pithovirus (in 2014) and mollivirus (in 2015). This wrongly suggests that such occurrences are rare and that “zombie viruses” are not a public health threat. To restore an appreciation closer to reality, we report the preliminary characterizations of 13 new viruses isolated from seven different ancient Siberian permafrost samples, one from the Lena river and one from Kamchatka cryosol. As expected from the host specificity imposed by our protocol, these viruses belong to five different clades infecting Acanthamoeba spp. but not previously revived from permafrost: Pandoravirus, Cedratvirus, Megavirus, and Pacmanvirus, in addition to a new Pithovirus strain.

Giant viruses (GVs) are a hotspot of unresolved controversies since their discovery, including the definition of “Virus” and their origin. While increasing knowledge of genome diversity has accumulated, GV functional genomics was largely neglected. Here, we describe an experimental framework to genetically modify nuclear GVs and their host Acanthamoeba castellanii using CRISPR/Cas9, shedding light on the evolution from small icosahedral viruses to amphora-shaped GVs. Ablation of the icosahedral major capsid protein in the phylogenetically-related mollivirus highlights a transition in virion shape and size. We additionally demonstrate the existence of a reduced core essential genome in pandoravirus, reminiscent of their proposed smaller ancestors. This proposed genetic expansion led to increased genome robustness, indicating selective pressures for adaptation to uncertain environments. Overall, we introduce new tools for manipulation of the unexplored genome of nuclear GVs and provide experimental evidence suggesting that viral gigantism has aroused as an emerging trait. Until today, genetic tools have been lacking to enable manipulation of amoebal giant viruses (GVs) by CRISPR/Cas9 technology. Here, Bisio et al. apply S. pyogenes Cas9 together with pU6- driven guide RNAs to investigate the replication of pandoravirus, a GV replication in the nucleus. Using this tool, they provide evidence for stepwise evolution and genetic expansion of viral gigantism.

One quarter of the Northern hemisphere is underlain by permanently frozen ground, referred to as permafrost. Due to climate warming, irreversibly thawing permafrost is releasing organic matter frozen for up to a million years, most of which decomposes into carbon dioxide and methane, further enhancing the greenhouse effect. Part of this organic matter also consists of revived cellular microbes (prokaryotes, unicellular eukaryotes) as well as viruses that remained dormant since prehistorical times. While the literature abounds on descriptions of the rich and diverse prokaryotic microbiomes found in permafrost, no additional report about live viruses have been published since the two original studies describing pithovirus (in 2014) and mollivirus (in 2015). This wrongly suggests that such occurrences are rare and that zombie viruses are not a public health threat. To restore an appreciation closer to reality, we report the preliminary characterizations of 13 new viruses isolated from 7 different ancient Siberian permafrost samples, 1 from the Lena river and 1 from Kamchatka cryosol. As expected from the host specificity imposed by our protocol, these viruses belong to 5 different clades infecting Acanthamoeba spp. but not previously revived from permafrost: pandoravirus, cedratvirus, megavirus, and pacmanvirus, in addition to a new pithovirus strain.

Giant viruses (GVs) are a hotspot of unresolved controversies since their discovery, including the definition of Virus and the existence of a fourth domain of life1-3. While increasing knowledge of genome diversity has accumulated4, functional genomics was largely neglected. Here, we describe an experimental framework to genetically modify nuclear GVs and its host Acanthamoeba castellanii using CRISPR/Cas9, allowing us to uncover the evolution from small icosahedral viruses to amphora-shaped GVs. Ablation of the icosahedral major capsid protein in the evolutionary intermediate mollivirus highlights a stepwise transition in virion shape and size. We additionally demonstrate the existence of a reduced core essential genome in pandoravirus, reminiscent of their proposed smaller ancestors. Genetic expansion led to increased genome robustness, indicating selective pressures for adaptation to uncertain environments. Overall, we introduce new tools for manipulation of the unexplored genome of nuclear GVs and demonstrate that viral gigantism can arise as an emerging trait.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a ~30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.