Periodic Reporting for period 3 - VIREVOL (Cells and giant viruses: a win-win co-evolution)
Okres sprawozdawczy: 2022-10-01 do 2024-03-31
The VIREVOL project is a basic science project to better understand virus and cell co-evolution and their respective impact on each partner evolution.
One of the aims of the VIREVOL project was to elucidate the genome organization of giant DNA viruses in order to compare it with what is found in the cellular world and eventually get some insights on the role they may have played for the evolution of the cells. We addressed the genome organization on two families of viruses. We studied the Mimiviridae and the Marseilleviridae which are giant and large DNA viruses infecting unicellular eukaryotes from the Acanthamoeba genus. We used Cryo-EM in both cases to analyze this organization. Surprisingly, they use opposite strategies while it results in both cases to the efficient compaction of the genomes in their icosahedral capsids. We also determined the structure of the large polysaccharides decorating Mimivirus capsids. They are synthesized by an autonomous glycosylation machinery encoded by the virus and we demonstrated that the composition of these glycans varies depending on the lineages, offering an efficient toolbox to these viruses to outcompete other viruses and bacteria infecting their amoebal host. Finally, DNA methylation is an important epigenetic mark that contributes to various regulations in all cellular domains of life. Yet, virtually nothing was known about the methylation of giant viruses DNA. We reveal that DNA methylation is widespread in giant viruses and correspond to additional tools to again outcompete bacteria and viruses targeting the same host.
Overall, this provides new insights in the ecology of these viruses and the forces at work directing their evolution. Beyond the fundamental knowledge, understanding such evolutionary trends may have consequences in the way we handle environmental viruses as emerging threats for humanity.
One of the aims of the VIREVOL project was to elucidate the genome organization of giant DNA viruses in order to compare it with what is found in the cellular world and eventually get some insights on the role they may have played for the evolution of the cells. We addressed the genome organization on two families of viruses. We studied the Mimiviridae and the Marseilleviridae which are giant and large DNA viruses infecting unicellular eukaryotes from the Acanthamoeba genus. We used Cryo-EM in both cases to analyze this organization. Surprisingly, they use opposite strategies while it results in both cases to the efficient compaction of the genomes in their icosahedral capsids. We also determined the structure of the large polysaccharides decorating Mimivirus capsids. They are synthesized by an autonomous glycosylation machinery encoded by the virus and we demonstrated that the composition of these glycans varies depending on the lineages, offering an efficient toolbox to these viruses to outcompete other viruses and bacteria infecting their amoebal host. Finally, DNA methylation is an important epigenetic mark that contributes to various regulations in all cellular domains of life. Yet, virtually nothing was known about the methylation of giant viruses DNA. We reveal that DNA methylation is widespread in giant viruses and correspond to additional tools to again outcompete bacteria and viruses targeting the same host.
Overall, this provides new insights in the ecology of these viruses and the forces at work directing their evolution. Beyond the fundamental knowledge, understanding such evolutionary trends may have consequences in the way we handle environmental viruses as emerging threats for humanity.
One of the aims of the VIREVOL project was to elucidate the genome organization of giant viruses in order to compare it with what is found in the cellular world and eventually get some insights on the role they may have played for the evolution of the cells. We addressed the genome organization on two families of viruses. We studied the Mimiviridae and the Marseilleviridae which are giant and large DNA viruses infecting unicellular eukaryotes from the Acanthamoeba genus. Surprisingly, they use opposite genome organization while it results in both cases to the efficient compaction of the genomes in their icosahedral capsids. We used Cryo-EM in both cases to analyze this organization.
Mimivirus genomic fibre:
For Mimivirus, after capsid opening, we managed to purify the 30 nm rod-shaped structure expelled from the capsids and analyzed them using Cryo-EM and proteomics to determine its structure and composition. The 1.2Mb genome is encased into a ~30 nm wide helical and flexible protein shell where the dsDNA is compacted with 5 to 6 DNA strands lining the interior of the protein shell. The shell is surprisingly made of an enzyme also composing the glycosylated fibrils surrounding the capsids. Additional proteins, including the viral RNA can fit in the central channel, revealing a remarkable evolutionary strategy for packaging and protecting the viral genome, in a state ready for immediate transcription upon delivery in the host cytoplasm (doi :10.1101/2022.02.17.480895).
Marseilleviridae genome compaction:
While organization of genomic DNA into nucleosomes was considered a hallmark of eukaryotes, Marseilleviridae, encoding fused remote homologs of the four eukaryotic histones, are also able to form nucleosomes, resembling cell nucleosomes, to compact their genomes. Our results further expand the range of ‘organisms’ that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses. We also developed the genetics of this virus and were able to demonstrate that histone doublets are essential for infectivity (doi : 10.1016/j.cell.2021.06.032).
Another aim of the VIREVOL project was to understand the processes at work in giant virus co-evolution with their host.
Autonomous glycosylation machinery of Mimiviridae:
A specific study on members of the Mimiviridae family revealed they are making complex glycans using their virally encoded glycosylation machinery, thus expanding glycosylation to the viral world. For instance, Mimivirus makes two huge polysaccharides of 70kDa and 25 kDa, made of rare capsular sugars which are branched on the fibrils surrounding the capsids (doi: 10.1002/anie.202106671). The sugar composition also appears to be clade specific suggesting giant virus built a versatile, constantly evolving toolbox, most likely to cope with other viruses and bacteria competing for a same host (microLife, doi.org/10.1093/femsml/uqac002)
Epigenetics in giant viruses:
DNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Giant viruses are widespread dsDNA viruses with gene contents overlapping the cellular world that also encode DNA methyltransferases. Yet, virtually nothing is known about the methylation of their DNA. We used single-molecule real-time sequencing to study the complete methylome of a large spectrum of giant viruses. We show that DNA methylation is widespread, affecting 2/3 of the tested families and identified the corresponding viral methyltransferases and show that they are subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. Most methyltransferases are conserved, functional and under purifying selection, suggesting that they increase the viruses' fitness. Some virally encoded methyltransferases are also paired with restriction endonucleases forming Restriction-Modification systems. Our data suggest that giant viruses' methyltransferases are involved in diverse forms of virus-pathogens interactions during coinfections (doi:10.1038/s41467-020-16414-2 )
Mimivirus genomic fibre:
For Mimivirus, after capsid opening, we managed to purify the 30 nm rod-shaped structure expelled from the capsids and analyzed them using Cryo-EM and proteomics to determine its structure and composition. The 1.2Mb genome is encased into a ~30 nm wide helical and flexible protein shell where the dsDNA is compacted with 5 to 6 DNA strands lining the interior of the protein shell. The shell is surprisingly made of an enzyme also composing the glycosylated fibrils surrounding the capsids. Additional proteins, including the viral RNA can fit in the central channel, revealing a remarkable evolutionary strategy for packaging and protecting the viral genome, in a state ready for immediate transcription upon delivery in the host cytoplasm (doi :10.1101/2022.02.17.480895).
Marseilleviridae genome compaction:
While organization of genomic DNA into nucleosomes was considered a hallmark of eukaryotes, Marseilleviridae, encoding fused remote homologs of the four eukaryotic histones, are also able to form nucleosomes, resembling cell nucleosomes, to compact their genomes. Our results further expand the range of ‘organisms’ that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses. We also developed the genetics of this virus and were able to demonstrate that histone doublets are essential for infectivity (doi : 10.1016/j.cell.2021.06.032).
Another aim of the VIREVOL project was to understand the processes at work in giant virus co-evolution with their host.
Autonomous glycosylation machinery of Mimiviridae:
A specific study on members of the Mimiviridae family revealed they are making complex glycans using their virally encoded glycosylation machinery, thus expanding glycosylation to the viral world. For instance, Mimivirus makes two huge polysaccharides of 70kDa and 25 kDa, made of rare capsular sugars which are branched on the fibrils surrounding the capsids (doi: 10.1002/anie.202106671). The sugar composition also appears to be clade specific suggesting giant virus built a versatile, constantly evolving toolbox, most likely to cope with other viruses and bacteria competing for a same host (microLife, doi.org/10.1093/femsml/uqac002)
Epigenetics in giant viruses:
DNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Giant viruses are widespread dsDNA viruses with gene contents overlapping the cellular world that also encode DNA methyltransferases. Yet, virtually nothing is known about the methylation of their DNA. We used single-molecule real-time sequencing to study the complete methylome of a large spectrum of giant viruses. We show that DNA methylation is widespread, affecting 2/3 of the tested families and identified the corresponding viral methyltransferases and show that they are subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. Most methyltransferases are conserved, functional and under purifying selection, suggesting that they increase the viruses' fitness. Some virally encoded methyltransferases are also paired with restriction endonucleases forming Restriction-Modification systems. Our data suggest that giant viruses' methyltransferases are involved in diverse forms of virus-pathogens interactions during coinfections (doi:10.1038/s41467-020-16414-2 )
One important breakthrough for giant virus studies is the implementation of the genetic of the host and of the viruses.
Acanthamoeba Castellanii is a complex organism with 25 copies of a genome of ~50 Mb. To be able to perform the genetics of our viruses we first needed to develop the host genetics and check whether recombination could work, if we could manage to express Cas9 and optimize vectors to be transfected to perform CrispR/Cas9 editing.
First aim:
Demonstrate that Mollivirus did hijack the cellulose synthase from the host to build up their particles which had the consequence to alleviate the importance of the Major capsid protein and gave them the opportunity to make a transition from a icosahedral capsid to an ovoid one, which was the starting point of capsid and genome expansion.
For this we needed to knock out the 3 gene encoding for the cellulose synthase for the 25 copies of the genome. During this first period, we succeeded in implementing the CrispR-Cas9 system in the amoeba and demonstrated that despite its polyploidy we were able to perform gene knock out.
As a result, when infected by Mollivirus the knocked-out cells produced virions that were not infectious anymore.
Interestingly, this is not the case for Pandoravirus which means that it may not depend on the cell to make cellulose opening a new challenge to understand why and how Pandoraviruses manage to still produce cellulose.
We also managed to use CrispR/Cas9 genetics for giant viruses having a nuclear phase. Namely Pandoraviruses and Molliviruses.
We finally managed to develop genetics for exclusively cytoplasmic giant viruses, namely Mimiviridae and Pithoviridae.
Various follow up studies have been initiated that produce paradigm shifting results that we hope to finalize before the end of the project.
Acanthamoeba Castellanii is a complex organism with 25 copies of a genome of ~50 Mb. To be able to perform the genetics of our viruses we first needed to develop the host genetics and check whether recombination could work, if we could manage to express Cas9 and optimize vectors to be transfected to perform CrispR/Cas9 editing.
First aim:
Demonstrate that Mollivirus did hijack the cellulose synthase from the host to build up their particles which had the consequence to alleviate the importance of the Major capsid protein and gave them the opportunity to make a transition from a icosahedral capsid to an ovoid one, which was the starting point of capsid and genome expansion.
For this we needed to knock out the 3 gene encoding for the cellulose synthase for the 25 copies of the genome. During this first period, we succeeded in implementing the CrispR-Cas9 system in the amoeba and demonstrated that despite its polyploidy we were able to perform gene knock out.
As a result, when infected by Mollivirus the knocked-out cells produced virions that were not infectious anymore.
Interestingly, this is not the case for Pandoravirus which means that it may not depend on the cell to make cellulose opening a new challenge to understand why and how Pandoraviruses manage to still produce cellulose.
We also managed to use CrispR/Cas9 genetics for giant viruses having a nuclear phase. Namely Pandoraviruses and Molliviruses.
We finally managed to develop genetics for exclusively cytoplasmic giant viruses, namely Mimiviridae and Pithoviridae.
Various follow up studies have been initiated that produce paradigm shifting results that we hope to finalize before the end of the project.