Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene

Faustoviruses are the first giant viruses of amoebae isolated on Vermamoeba vermiformis. They are distantly related to African swine fever virus, the causative agent of lethal hemorrhagic fever in domestic pigs. Structural studies have shown the presence of a double protein layer encapsidating the double-stranded DNA genome of Faustovirus E12, the prototype strain. The major capsid protein (MCP) forming the external layer has been shown to be 645-amino acid-long. Unexpectedly, its encoding sequence has been found to be scattered along a 17 kbp-large genomic region. Using RNA-seq, we studied expression of Faustovirus E12 genes at nine time points over its entire replicative cycle. Paired-end 250 bp-long read sequencing on MiSeq instrument and double-round spliced alignment enabled the identification of 26 different splice-junctions. Reads corresponding to junctions represented 2% of mapped reads and mostly matched with the predicted MCP encoding sequences. Moreover, our study enabled describing a 1,939 bp-long transcript that corresponds to the MCP, delineating 13 exons. At least two types of introns coexist in the MCP gene: group I introns that can self-splice (n = 5) and spliceosome-like introns with non-canonical splice sites (n = 7). All splice-sites were non-canonical with five types of donor/acceptor splice-sites among which AA/TG was the most frequent association

Expression of genetic information in giant viruses

La découverte des virus géant a ouvert le champs a tout un pan de la virosphère. La diversité des tailles, formes, génomes et écosystèmes où ces virus sont isolés suggère la présence de mécanismes divers dans l'expression de leur information génétique au cours de leur cycle de réplication. Dans ce travail, nous avons utilisé la technologie de séquençage massif parallèle d’ARN afin d’élucider quelques-uns de ces mécanismes dans trois familles de virus géants infectant les amibes.Dans un premier travail, l’étude du transcriptome de Faustovirus E12 a permis de mettre en évidence la présence d’un épissage complexe du gène codant pour la protéine de capside majeure et d’ainsi corriger sa structure. Il a également montré l’autonomie relatif de ce virus vis-à-vis de son hôte au cours de l’infection. Le deuxième projet s’est intéressé à la cinétique d’expression des gènes de Marseillevirus T19 au cours de son cycle de réplication sur Acanthamoeba castellanii Neff. Nos résultats ont montré une régulation temporelle de l’expression génique de ce virus indépendante d’un motif de promoteur prédit, suggérant l’interventions de mécanismes plus complexes. Ils ont par ailleurs montré une altération de l’expression génique de l’amibe au profit du virus au cours de l’infection.Chez Pandoravirus, la régulation de la transcription des gènes viraux semble moins délétère pour l’hôte suggérant une cohabitation plus ancienne entre ce virus et les Acanthamoebae et son adaptation au protiste. Les résultats de cette thèse montrent la diversité des mécanismes de transcription des virus géants infectant les amibes et de leurs interactions avec leurs hôtes.The discovery of giant revealed a whole new part of the virosphere. The diversity of the sizes, shapes, genomes, and ecosystems in which giant viruses are isolated suggests the presence of diverse mechanisms for the expression of their genomic information during their replication cycle.In this work, we used massive parallel RNA sequencing technology or RNA-seq to elucidate some of these mechanisms in three families of giant viruses infecting amoebas. In the first project, the study of the Faustovirus E12 transcriptome highlighted the presence of a complex splicing of the gene coding for the major capsid protein and thus allowed us to correct its structure. The results also showed the relative autonomy of this virus from its host during infection.In a second work, we sought to determine the kinetics of expression of Marseillevirus T19 genes during its replication cycle on Acanthamoeba castellanii Neff. Our results showed a temporal regulation of the gene expression of Marseillevirus independent from the promoter motif suggesting the presence of a complex regulation mechanism and highlighted an alteration in the amoeba's gene expression in favor of the virus during the infection.In pandoraviruse, the regulation of transcription of viral genes seems less deleterious for the host suggesting an older cohabitation between pandoraviruses and Acanthamoeba and an adaptation of this virus to its protist.These results show the diversity of the transcription mechanisms of giant amoeba viruses and their interactions with their hosts

Vermamoeba vermiformis CDC-19 draft genome sequence reveals considerable gene trafficking including with candidate phyla radiation and giant viruses

International audienc

Analysis of a Marseillevirus Transcriptome Reveals Temporal Gene Expression Profile and Host Transcriptional Shift

Marseilleviruses comprise a family of large double-stranded DNA viruses belonging to the proposed order ``Megavirales.'' These viruses have a circular genome of similar to 370 kbp, coding hundreds of genes. Over a half of their genes are associated with AT-rich putative promoter motifs, which have been demonstrated to be important for gene regulation. However, the transcriptional profile of Marseilleviruses is currently unknown. Here we used RNA sequencing technology to get a general transcriptional profile of Marseilleviruses. Eight million 75-bp-long nucleotide sequences were robustly mapped to all 457 genes initially predicted for Marseillevirus isolate T19, the prototype strain of the family, and we were able to assemble 359 viral contigs using a genome-guided approach with stringent parameters. These reads were differentially mapped to the genes according to the replicative cycle time point from which they were obtained. Cluster analysis indicated the existence of three main temporal categories of gene expression, early, intermediate and late, which were validated by quantitative reverse transcription polymerase chain reaction assays targeting several genes. Genes belonging to different functional groups exhibited distinct expression levels throughout the infection cycle. We observed that the previously predicted promoter motif, AAATATTT, as well as new predicted motifs, were not specifically related to any of the temporal or functional classes of genes, suggesting that other components are involved in temporally regulating virus transcription. Moreover, the host transcription machinery is heavily altered, and many genes are down regulated, including those related to translation process. This study provides an overview of the transcriptional landscape of Marseilleviruses

Proteomics and Lipidomics Investigations to Decipher the Behavior of Willaertia magna C2c Maky According to Different Culture Modes

International audienceWillaertia magna C2c Maky is a free-living amoeba that has demonstrated its ability to inhibit the intracellular multiplication of some Legionella pneumophila strains, which are pathogenic bacteria inhabiting the aquatic environment. The Amoeba, an industry involved in the treatment of microbiological risk in the water and plant protection sectors, has developed a natural biocide based on the property of W. magna to manage the proliferation of the pathogen in cooling towers. In axenic liquid medium, amoebas are usually cultivated in adhesion on culture flask. However, we implemented a liquid culture in suspension using bioreactors in order to produce large quantities of W. magna. In order to investigate the culture condition effects on W. magna, we conducted a study based on microscopic, proteomics and lipidomics analyzes. According to the culture condition, amoeba exhibited two different phenotypes. The differential proteomics study showed that amoebas seemed to promote the lipid metabolism pathway in suspension culture, whereas we observed an upregulation of the carbohydrate pathway in adherent culture. Furthermore, we observed an over-regulation of proteins related to the cytoskeleton for W. magna cells grown in adhesion. Regarding the lipid analysis, suspension and adhesion cell growth showed comparable lipid class compositions. However, the differential lipid analysis revealed differences that confirmed cell phenotype differences observed by microscopy and predicted by proteomics. Overall, this study provides us with a better insight into the biology and molecular processes of W. magna in different culture lifestyles