A puzzling anomaly in the 4-mer composition of the giant pandoravirus genomes reveals a stringent new evolutionary selection process

International audienceThe Pandoraviridae is a rapidly growing family of giant viruses, all of which have been isolated using laboratory strains of Acanthamoeba. The genomes of ten distinct strains have been fully characterized, reaching up to 2.5 Mb in size. These double-stranded DNA genomes encode the largest of all known viral proteomes and are propagated in oblate virions that are among the largest ever-described (1.2 μm long and 0.5 μm wide). The evolutionary origin of these atypical viruses is the object of numerous speculations. Applying the Chaos Game Representation to the pandoravirus genome sequences, we discovered that the tetranucleotide (4-mer) "AGCT" is totally absent from the genomes of 2 strains (P. dulcis and P. quercus) and strongly underrepresented in others. Given the amazingly low probability of such an observation in the corresponding randomized sequences, we investigated its biological significance through a comprehensive study of the 4-mer compositions of all viral genomes. Our results indicate that "AGCT" was specifically eliminated during the evolution of the Pandoraviridae and that none of the previously proposed host-virus antagonistic relationships could explain this phenomenon. Unlike the three other families of giant viruses (Mimiviridae, Pithoviridae, Molliviridae) infecting the same Acanthamoeba host, the pandoraviruses exhibit a puzzling genomic anomaly suggesting a highly specific DNA editing in response to a new kind of strong evolutionary pressure.IMPORTANCE The recent years have seen the discovery of several families of giant DNA viruses all infecting the ubiquitous amoebozoa of the genus Acanthamoeba. With dsDNA genomes reaching 2.5 Mb in length packaged in oblate particles the size of a bacterium, the pandoraviruses are the most complex and largest viruses known as of today. In addition to their spectacular dimensions, the pandoraviruses encode the largest proportion of proteins without homolog in other organisms which are thought to result from a de novo gene creation process. While using comparative genomics to investigate the evolutionary forces responsible for the emergence of such an unusual giant virus family, we discovered a unique bias in the tetranucleotide composition of the pandoravirus genomes that can only result from an undescribed evolutionary process not encountered in any other microorganism

mRNA maturation in giant viruses: variation on a theme

International audienceGiant viruses from the Mimiviridae family replicate entirely in their host cytoplasm where their genes are transcribed by a viral transcription apparatus. mRNA polyadenylation uniquely occurs at hairpin-forming palindromic sequences terminating viral transcripts. Here we show that a conserved gene cluster both encode the enzyme responsible for the hairpin cleavage and the viral polyA polymerases (vPAP). Unexpectedly, the vPAPs are homodimeric and uniquely self-processive. The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication. A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails. These findings elucidate how the arm wrestling between hosts and their viruses to access the translation machinery is taking place in Mimiviridae

Mimivirus and the emerging concept of "giant" virus

The recently discovered Acanthamoeba polyphaga Mimivirus is the largest known DNA virus. Its particle size (>400 nm), genome length (1.2 million bp) and large gene repertoire (911 protein coding genes) blur the established boundaries between viruses and parasitic cellular organisms. In addition, the analysis of its genome sequence identified new types of genes not expected to be seen in a virus, such as aminoacyl-tRNA synthetases and other central components of the translation machinery. In this article, we examine how the finding of a giant virus for the first time overlapping with the world of cellular organisms in terms of size and genome complexity might durably influence the way we look at microbial biodiversity, and force us to fundamentally revise our classification of life forms. We propose to introduce the word "girus" to recognize the intermediate status of these giant DNA viruses, the genome complexity of which make them closer to small parasitic prokaryotes than to regular viruses.Comment: Submitted to Virus Researc

The Astounding World of Glycans from Giant Viruses

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

Complex membrane remodeling during virion assembly of the 30,000 years-old Mollivirus sibericum

International audienceCellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary 3D-electron microscopy techniques including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles, never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant and DNA is packed within virions, prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggests that the molecular mechanisms for cellular membrane remodeling and persistence are unique.ImportanceSince the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the World and appear to be unique in several aspects. They belong to at least four viral families and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae" which was isolated from a 30,000 years-old permafrost sample, and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna; (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled; (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding on how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly

Metagenomic survey of the microbiome of ancient Siberian permafrost and modern Kamchatkan cryosols

In the context of global warming, the melting of arctic permafrost raises the threat of a re-emergence of microorganisms some of which were shown to remain viable in ancient frozen soils for up to half a million years. In order to evaluate this risk, it is of interest to acquire a better knowledge of the composition of the microbial communities found in this understudied environment. Here we present a metagenomics analysis of 12 soil samples from Russian Arctic and subarctic pristine areas: Chukotka, Yakutia, and Kamchatka, including 9 permafrost samples collected at various depths. These large datasets (9.2 1011 total bp) were assembled (525,313 contigs > 5kb), their encoded protein contents predicted, then used to perform taxonomical assignments of bacterial, archaeal, and eukaryotic organisms, as well as DNA viruses. The various samples exhibited variable DNA contents and highly diverse taxonomic profiles showing no obvious relationship with their locations, depths or deposit ages. Bacteria represented the largely dominant DNA fraction (95%) in all samples, followed by archaea (3.2%), surprisingly little eukaryotes (0.5%), and viruses (0.4%). Although no common taxonomic pattern was identified, the samples shared unexpected high frequencies of β-lactamase genes, almost 0.9 copy/bacterial genome. In addition of known environmental threats, the particularly intense warming of the Arctic might thus enhance the spread of bacterial antibiotic resistances, today's major challenge in public health. β-lactamases were also observed at high frequency in other types of soils, suggesting their general role in the regulation of bacterial populations

Identification of an L-Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection

Comparative Genomics of Multidrug Resistance in Acinetobacter baumannii

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island—the largest identified to date—in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance

Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes

With genomes of up to 2.7 Mb propagated in μm-long oblong particles and initially predicted to encode more than 2000 proteins, members of the Pandoraviridae family display the most extreme features of the known viral world. The mere existence of such giant viruses raises fundamental questions about their origin and the processes governing their evolution. A previous analysis of six newly available isolates, independently confirmed by a study including three others, established that the Pandoraviridae pan-genome is open, meaning that each new strain exhibits protein-coding genes not previously identified in other family members. With an average increment of about 60 proteins, the gene repertoire shows no sign of reaching a limit and remains largely coding for proteins without recognizable homologs in other viruses or cells (ORFans). To explain these results, we proposed that most new protein-coding genes were created de novo, from pre-existing non-coding regions of the G+C rich pandoravirus genomes. The comparison of the gene content of a new isolate, pandoravirus celtis, closely related (96% identical genome) to the previously described p. quercus is now used to test this hypothesis by studying genomic changes in a microevolution range. Our results confirm that the differences between these two similar gene contents mostly consist of protein-coding genes without known homologs, with statistical signatures close to that of intergenic regions. These newborn proteins are under slight negative selection, perhaps to maintain stable folds and prevent protein aggregation pending the eventual emergence of fitness-increasing functions. Our study also unraveled several insertion events mediated by a transposase of the hAT family, 3 copies of which are found in p. celtis and are presumably active. Members of the Pandoraviridae are presently the first viruses known to encode this type of transposase