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Bernard La Scola - One of the best experts on this subject based on the ideXlab platform.
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Isolation and genomic characterization of a new Mimivirus of lineage B from a Brazilian river
Archives of Virology, 2020Co-Authors: Ludmila Karen Dos Santos Silva, Bernard La Scola, Anthony Levasseur, Ana Claudia Santos Pereira Andrade, Rodrigo Araujo Lima Rodrigues, Hiroyuki Hikida, Julien Andreani, Jonatas Santos AbrahaoAbstract:Since its discovery, the first identified giant virus associated with amoebae, Acanthamoeba polyphaga Mimivirus (APMV), has been rigorously studied to understand the structural and genomic complexity of this virus. In this work, we report the isolation and genomic characterization of a new Mimivirus of lineage B, named “Borely moumouvirus”. This new virus exhibits a structure and replicative cycle similar to those of other members of the family Mimiviridae . The genome of the new isolate is a linear double-strand DNA molecule of ~1.0 Mb, containing over 900 open reading frames. Genome annotation highlighted different translation system components encoded in the DNA of Borely moumouvirus, including aminoacyl-tRNA synthetases, translation factors, and tRNA molecules, in a distribution similar to that in other lineage B Mimiviruses. Pan-genome analysis indicated an increase in the genetic arsenal of this group of viruses, showing that the family Mimiviridae is still expanding. Furthermore, phylogenetic analysis has shown that Borely moumouvirus is closely related to moumouvirus australiensis. This is the first Mimivirus lineage B isolated from Brazilian territory to be characterized. Further prospecting studies are necessary for us to better understand the diversity of these viruses so a better classification system can be established.
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Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System
Frontiers in Microbiology, 2019Co-Authors: Said Mougari, Jonatas Abrahao, Graziele Oliveira, Jacques Bou Khalil, Bernard La ScolaAbstract:MIMIVIRE is a defense system described in lineage A Mimivirus (Mimiviridae family) that mediates resistance against Zamilon virophage. It is composed of putative helicase and nuclease associated with a gene of unknown function called R349, which contains four 15 bp repeats homologous to the virophage sequence. In a previous study, the silencing of such genes restored virophage susceptibility. Moreover, the CRISPR Cas-4 like activity of the nuclease has recently been characterized. In this study, a recently isolated Mimivirus of lineage A with R349 gene lacking 3 of 4 repeats was demonstrated to be susceptible to Zamilon. To reinforce the importance of the R349 gene in the MIMIVIRE system, we developed and presented, for the first time to our knowledge, a protocol for Mimivirus genomic editing. By knocking out R349 gene in a Mimivirus lineage A, we observed the replication of Zamilon, indicating that this gene is critical in the resistance against this specific group of virophages.
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Table_1_Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System.XLSX
2019Co-Authors: Said Mougari, Jonatas Abrahao, Graziele P. Oliveira, Jacques Bou Y. Khalil, Bernard La ScolaAbstract:MIMIVIRE is a defense system described in lineage A Mimivirus (Mimiviridae family) that mediates resistance against Zamilon virophage. It is composed of putative helicase and nuclease associated with a gene of unknown function called R349, which contains four 15 bp repeats homologous to the virophage sequence. In a previous study, the silencing of such genes restored virophage susceptibility. Moreover, the CRISPR Cas-4 like activity of the nuclease has recently been characterized. In this study, a recently isolated Mimivirus of lineage A with R349 gene lacking 3 of 4 repeats was demonstrated to be susceptible to Zamilon. To reinforce the importance of the R349 gene in the MIMIVIRE system, we developed and presented, for the first time to our knowledge, a protocol for Mimivirus genomic editing. By knocking out R349 gene in a Mimivirus lineage A, we observed the replication of Zamilon, indicating that this gene is critical in the resistance against this specific group of virophages.
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Image_1_Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System.TIF
2019Co-Authors: Said Mougari, Jonatas Abrahao, Graziele P. Oliveira, Jacques Bou Y. Khalil, Bernard La ScolaAbstract:MIMIVIRE is a defense system described in lineage A Mimivirus (Mimiviridae family) that mediates resistance against Zamilon virophage. It is composed of putative helicase and nuclease associated with a gene of unknown function called R349, which contains four 15 bp repeats homologous to the virophage sequence. In a previous study, the silencing of such genes restored virophage susceptibility. Moreover, the CRISPR Cas-4 like activity of the nuclease has recently been characterized. In this study, a recently isolated Mimivirus of lineage A with R349 gene lacking 3 of 4 repeats was demonstrated to be susceptible to Zamilon. To reinforce the importance of the R349 gene in the MIMIVIRE system, we developed and presented, for the first time to our knowledge, a protocol for Mimivirus genomic editing. By knocking out R349 gene in a Mimivirus lineage A, we observed the replication of Zamilon, indicating that this gene is critical in the resistance against this specific group of virophages.
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Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.
Journal of Virology, 2018Co-Authors: Meriem Bekliz, Philippe Decloquement, Said Azza, Didier Raoult, Herve Seligmann, Bernard La ScolaAbstract:The Acanthamoeba polyphaga Mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type Mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced Mimivirus, which were defined as up-or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of Mimivirus proteins and, thus, that Mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of Mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a Mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 Mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on Mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by Mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on Mimivirus translation-related factors will open the way to understanding the functions of these translational genes.
Didier Raoult - One of the best experts on this subject based on the ideXlab platform.
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Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.
Journal of Virology, 2018Co-Authors: Meriem Bekliz, Philippe Decloquement, Said Azza, Didier Raoult, Herve Seligmann, Bernard La ScolaAbstract:The Acanthamoeba polyphaga Mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type Mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced Mimivirus, which were defined as up-or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of Mimivirus proteins and, thus, that Mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of Mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a Mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 Mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on Mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by Mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on Mimivirus translation-related factors will open the way to understanding the functions of these translational genes.
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Mimivirus: leading the way in the discovery of giant viruses of amoebae
Nature Reviews Microbiology, 2017Co-Authors: Philippe Colson, Gustavo Caetano-anollés, Bernard La Scola, Anthony Levasseur, Didier RaoultAbstract:The accidental discovery of the giant virus of amoeba -Acanthamoeba polyphaga Mimivirus (APMV; more commonly known as Mimivirus) -in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, Mimiviruses are infected by virophages, defend against them through the Mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including Mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans.
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Mimivirus: leading the way in the discovery of giant viruses of amoebae
Nature Reviews Microbiology, 2017Co-Authors: Philippe Colson, Gustavo Caetano-anollés, Bernard La Scola, Anthony Levasseur, Didier RaoultAbstract:The accidental discovery of the giant virus of amoeba — Acanthamoeba polyphaga Mimivirus (APMV; more commonly known as Mimivirus) — in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, Mimiviruses are infected by virophages, defend against them through the Mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including Mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans. Acanthamoeba polyphaga Mimivirus (APMV) and subsequently discovered giant viruses of amoebae challenge the previous definition of viruses and their classification. The replication cycle, structure, genomic make-up and plasticity of giant viruses differ from those of traditional viruses. They extend the definition of viruses into a broader range of biological entities, some of which are very simple and others of which have a complexity that is comparable to that of other microorganisms. Giant viruses of amoebae have virus particles as large as some microorganisms that are visible by light microscopy and that have a stunning level of complexity. Their genomes are mosaics and contain large repertoires of genes, some of which are hallmarks of cellular organisms, although the majority of which have unknown functions. Mimiviruses are associated with a specific mobilome and are parasitized by viruses that they can defend against. Several hypotheses on the ancient origin and evolutionary relationship between cellular organisms and giant viruses of amoebae have been proposed, and these topics continue to be debated. The detection of giant viruses of amoebae in humans and the study of their potential pathogenicity are emerging fields. The discovery of the giant amoebal virus Mimivirus, in 2003, opened up a new area of virology. Extended studies, including those of Mimiviruses, have since revealed that these viruses have genetic, proteomic and structural features that are more complex than those of conventional viruses.
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Mimivirus leading the way in the discovery of giant viruses of amoebae
Nature Reviews Microbiology, 2017Co-Authors: Philippe Colson, Bernard La Scola, Anthony Levasseur, Gustavo Caetanoanolles, Didier RaoultAbstract:The discovery of the giant amoebal virus Mimivirus, in 2003, opened up a new area of virology. Extended studies, including those of Mimiviruses, have since revealed that these viruses have genetic, proteomic and structural features that are more complex than those of conventional viruses. The accidental discovery of the giant virus of amoeba — Acanthamoeba polyphaga Mimivirus (APMV; more commonly known as Mimivirus) — in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, Mimiviruses are infected by virophages, defend against them through the Mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including Mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans.
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Recombinant expression of Mimivirus L725 ORFan gene product
Acta Virologica, 2017Co-Authors: Haitham Sobhy, Guillaume Gotthard, Eric Chabriere, Didier Raoult, P. ColsonAbstract:Mimivirus was the first discovered amoebal giant virus. The Mimivirus virions are covered by a dense layer of approximately 130 nm-long fibers, the length and shape of which diverge from those of other viruses. Here, we aimed at expressing the L725 protein to further confirm and study its role as a fiber-associated protein. We report Escherichia coli expression of the L725 protein, which is encoded by a Mimivirus ORFan, was previously identified by proteomics in purified viral fibers and demonstrated to be a fiber-associated protein by RNA-silencing experiments. The expressed protein was recognized by anti-Mimivirus fiber or anti-Mimivirus L725 polyclonal antibodies. This study is the only expression, to our knowledge, of a product from a Mimiviral ORFan gene.
Jean-michel Claverie - One of the best experts on this subject based on the ideXlab platform.
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genomics of megavirus and the elusive fourth domain of life
Communicative & Integrative Biology, 2012Co-Authors: Matthieu Legendre, Chantal Abergel, Defne Arslan, Jean-michel ClaverieAbstract:We recently described Megavirus chilensis, a giant virus isolated off the coast of Chile, also replicating in fresh water acanthamoeba. Its 1,259,197-bp genome encodes 1,120 proteins and is the largest known viral genome. Megavirus and its closest relative Mimivirus only share 594 orthologous genes, themselves sharing only 50% of identical residues in average. Despite this divergence, comparable to the maximal divergence exhibited by bacteria within the same division (e.g., gamma proteobacteria), Megavirus retained all of the genomic features unique to Mimivirus, in particular its genes encoding key-elements of the translation apparatus, a trademark of cellular organisms. Besides homologs to the four aminoacyl-tRNA synthetases (aaRS) encoded by Mimivirus, Megavirus added three additional ones, raising the total of known virus-encoded aaRS to seven: IleRS, TrpRS, AsnRS, ArgRS, CysRS, MetRS, TyrRS. This finding strongly suggests that large DNA viruses derived from an ancestral cellular genome by reductive evolution. The nature of this cellular ancestor remains hotly debated.
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distant Mimivirus relative with a larger genome highlights the fundamental features of megaviridae
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Defne Arslan, Chantal Abergel, Matthieu Legendre, Virginie Seltzer, Jean-michel ClaverieAbstract:Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein).
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breaking the 1000 gene barrier for Mimivirus using ultra deep genome and transcriptome sequencing
Virology Journal, 2011Co-Authors: Matthieu Legendre, Chantal Abergel, Sebastien Santini, Alain Rico, Jean-michel ClaverieAbstract:Background: Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes. Findings: We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. Conclusions: This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.
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Mimivirus and its Virophage
Annual Review of Genetics, 2009Co-Authors: Jean-michel Claverie, Chantal AbergelAbstract:Mimivirus, a virus infecting amoebae of the acanthamoeba genus, is the prototype member of the Mimiviridae, the latest addition to the family of the nucleocytoplasmic large DNA viruses, already including the Poxviridae, the Iridoviridae, the Asfarviridae, and the Phycodnaviridae. Because of the size of its particle—a fiber-covered icosahedral protein capsid 0.75 μm in diameter—Mimivirus was initially mistaken for a parasitic bacterium. Its 1.2-Mb genome sequence encodes more than 900 proteins, many of them associated with functions never before encountered in a virus, such as four aminoacyl-tRNA synthetases. These findings revived the debate about the origin of DNA viruses and their possible role in the emergence of the eukaryotic nucleus. The recent isolation of a new type of satellite virus, called a virophage, associated with a second strain of Mimivirus, confirmed its unique position within the virus world. Post-genomic studies are now in progress, slowly shedding some light on the physiology of the m...
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dissecting the unique nucleotide specificity of Mimivirus nucleoside diphosphate kinase
Journal of Virology, 2009Co-Authors: Sandra Jeudy, Audrey Lartigue, Jean-michel Claverie, Chantal AbergelAbstract:The analysis of the Acanthamoeba polyphaga Mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the Mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga Mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the Mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 A to 1.5 A. Our results suggest that the Mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.
Chantal Abergel - One of the best experts on this subject based on the ideXlab platform.
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genomics of megavirus and the elusive fourth domain of life
Communicative & Integrative Biology, 2012Co-Authors: Matthieu Legendre, Chantal Abergel, Defne Arslan, Jean-michel ClaverieAbstract:We recently described Megavirus chilensis, a giant virus isolated off the coast of Chile, also replicating in fresh water acanthamoeba. Its 1,259,197-bp genome encodes 1,120 proteins and is the largest known viral genome. Megavirus and its closest relative Mimivirus only share 594 orthologous genes, themselves sharing only 50% of identical residues in average. Despite this divergence, comparable to the maximal divergence exhibited by bacteria within the same division (e.g., gamma proteobacteria), Megavirus retained all of the genomic features unique to Mimivirus, in particular its genes encoding key-elements of the translation apparatus, a trademark of cellular organisms. Besides homologs to the four aminoacyl-tRNA synthetases (aaRS) encoded by Mimivirus, Megavirus added three additional ones, raising the total of known virus-encoded aaRS to seven: IleRS, TrpRS, AsnRS, ArgRS, CysRS, MetRS, TyrRS. This finding strongly suggests that large DNA viruses derived from an ancestral cellular genome by reductive evolution. The nature of this cellular ancestor remains hotly debated.
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distant Mimivirus relative with a larger genome highlights the fundamental features of megaviridae
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Defne Arslan, Chantal Abergel, Matthieu Legendre, Virginie Seltzer, Jean-michel ClaverieAbstract:Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein).
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Preliminary crystallographic analysis of a possible transcription factor encoded by the Mimivirus L544 gene.
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2011Co-Authors: Alexandre Ciaccafava, Audrey Lartigue, Sandra Jeudy, Pascal Mansuelle, Chantal AbergelAbstract:Mimivirus is the prototype of a new family (the Mimiviridae) of nucleocytoplasmic large DNA viruses (NCLDVs), which already include the Poxviridae, Iridoviridae, Phycodnaviridae and Asfarviridae. Mimivirus specifically replicates in cells from the genus Acanthamoeba. Proteomic analysis of purified Mimivirus particles revealed the presence of many subunits of the DNA-directed RNA polymerase II complex. A fully functional pre-transcriptional complex appears to be loaded in the virions, allowing Mimivirus to initiate transcription within the host cytoplasm immediately upon infection independently of the host nuclear apparatus. To fully understand this process, a systematic study of Mimivirus proteins that are predicted (by bioinformatics) or suspected (by proteomic analysis) to be involved in transcription was initiated by cloning and expressing them in Escherichia coli in order to determine their three-dimensional structures. Here, preliminary crystallographic analysis of the recombinant L544 protein is reported. The crystals belonged to the orthorhombic space group C2221 with one monomer per asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal present in a selenomethionine-substituted protein crystal.
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Preliminary crystallographic analysis of a possible transcription factor encoded by the Mimivirus L544 gene.
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2011Co-Authors: Alexandre Ciaccafava, Audrey Lartigue, Sandra Jeudy, Pascal Mansuelle, Chantal AbergelAbstract:Mimivirus is the prototype of a new family (the Mimiviridae) of nucleocytoplasmic large DNA viruses (NCLDVs), which already include the Poxviridae, Iridoviridae, Phycodnaviridae and Asfarviridae. Mimivirus specifically replicates in cells from the genus Acanthamoeba. Proteomic analysis of purified Mimivirus particles revealed the presence of many subunits of the DNA-directed RNA polymerase II complex. A fully functional pre-transcriptional complex appears to be loaded in the virions, allowing Mimivirus to initiate transcription within the host cytoplasm immediately upon infection independently of the host nuclear apparatus. To fully understand this process, a systematic study of Mimivirus proteins that are predicted (by bioinformatics) or suspected (by proteomic analysis) to be involved in transcription was initiated by cloning and expressing them in Escherichia coli in order to determine their three-dimensional structures. Here, preliminary crystallographic analysis of the recombinant L544 protein is reported. The crystals belonged to the orthorhombic space group C2221 with one monomer per asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal present in a selenomethionine-substituted protein crystal.
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breaking the 1000 gene barrier for Mimivirus using ultra deep genome and transcriptome sequencing
Virology Journal, 2011Co-Authors: Matthieu Legendre, Chantal Abergel, Sebastien Santini, Alain Rico, Jean-michel ClaverieAbstract:Background: Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes. Findings: We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. Conclusions: This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.
Herve Seligmann - One of the best experts on this subject based on the ideXlab platform.
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Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.
Journal of Virology, 2018Co-Authors: Meriem Bekliz, Philippe Decloquement, Said Azza, Didier Raoult, Herve Seligmann, Bernard La ScolaAbstract:The Acanthamoeba polyphaga Mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type Mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced Mimivirus, which were defined as up-or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of Mimivirus proteins and, thus, that Mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of Mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a Mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 Mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on Mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by Mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on Mimivirus translation-related factors will open the way to understanding the functions of these translational genes.
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Stem-Loop RNA Hairpins in Giant Viruses: Invading rRNA-Like Repeats and a Template Free RNA
Frontiers in Microbiology, 2018Co-Authors: Herve SeligmannAbstract:We examine the hypothesis that de novo template-free RNAs still form spontaneously, as they did at the origins of life, invade modern genomes, contribute new genetic material. Previously, analyses of RNA secondary structures suggested that some RNAs resembling ancestral (t) RNAs formed recently de novo, other parasitic sequences cluster with rRNAs. Here positive control analyses of additional RNA secondary structures confirm ancestral and de novo statuses of RNA grouped according to secondary structure. Viroids with branched stems resemble de novo RNAs, rod-shaped viroids resemble rRNA secondary structures, independently of GC contents. 5' UTR leading regions of West Nile and Dengue flavivirid viruses resemble de novo and rRNA structures, respectively. An RNA homologous with Megavirus, Dengue and West Nile genomes, copperhead snake microsatellites and levant cotton repeats, not templated by Mimivirus' genome, persists throughout Mimivirus' infection. Its secondary structure clusters with candidate de novo RNAs. The saltatory phyletic distribution and secondary structure of Mimivirus' peculiar RNA suggest occasional template-free polymerization of this sequence, rather than noncanonical transcriptions (swinger polymerization, posttranscriptional editing).