Virophages and retrotransposons colonize the genomes of a heterotrophic flagellate
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Evaluation Summary:
Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, the study by Hackl et al. reveals a large diversity of complete and fragmented virophage genomes - termed EMALEs- scattered throughout the genomes of four strains of the marine protist Cafeteria. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards deciphering the biology of these viruses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These e ndogenous ma virus- l ike e lements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.
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Author Response:
Reviewer #1 (Public Review):
This study is well-written and well-presented. The conclusions are clear and robustly supported by the data. The figures provide useful visualizations for the major findings. Virophage are an important and underappreciated component of global viral diversity, and they likely play important roles in eukaryotic genome evolution; this work is therefore quite timely. Relatively few studies focus on virophage or giant viruses compared to other viral lineages, so studies like this are highly valuable.
Strengths of this work include the high quality of the reference genomes, which were constructed using both short-read and long-read sequencing, as well as the diverse locations and isolation times of the host genomes.
We thank the reviewer for his encouraging and constructive comments!
I …
Author Response:
Reviewer #1 (Public Review):
This study is well-written and well-presented. The conclusions are clear and robustly supported by the data. The figures provide useful visualizations for the major findings. Virophage are an important and underappreciated component of global viral diversity, and they likely play important roles in eukaryotic genome evolution; this work is therefore quite timely. Relatively few studies focus on virophage or giant viruses compared to other viral lineages, so studies like this are highly valuable.
Strengths of this work include the high quality of the reference genomes, which were constructed using both short-read and long-read sequencing, as well as the diverse locations and isolation times of the host genomes.
We thank the reviewer for his encouraging and constructive comments!
I found no major weaknesses in this study. One minor issue is that the details of how EMALEs were delineated and initially detected seem a bit unclear to me. Based on my reading I am curious if some divergent or degraded EMALEs could have been missed. This may be important for assessing the consequences of possible retrotransposition-mediated EMALE inactivation.
Thank you for pointing this out. We added two sections to Materials and Methods called “Detection and annotation of EMALEs” and “Detection of Ngaro retrotransposons” where we describe the procedure in detail.
Based on our approach of visually screening the entire genome assemblies for GC anomalies, combined with blast searches of Cafeteria genomes using as input manually annotated EMALEs as well as databases of all available virophage sequences, we are quite confident that we have not missed any obvious virophage genomes. We would only have missed putative virophage sequences if their GC-contents were similar to that of the host (~70% GC) and if these sequences bore no detectable similarity to known virophage genes/proteins.
In contrast, our sequencing and assembly strategy probably did not result in a complete account of all EMALEs in these host genomes, as is evident from the large number of partially assembled EMALEs. However, partial does not equal degraded, but simply means that contig assembly stopped somewhere within the EMALE, resulting in an artificially truncated sequence. We therefore do not think that our approach introduced any relevant bias towards addressing the question whether retrotransposon insertion may lead to EMALE inactivation.
These points are now included in the discussion.
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Evaluation Summary:
Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, the study by Hackl et al. reveals a large diversity of complete and fragmented virophage genomes - termed EMALEs- scattered throughout the genomes of four strains of the marine protist Cafeteria. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards deciphering the biology of these viruses.
(This preprint has been reviewed by …
Evaluation Summary:
Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, the study by Hackl et al. reveals a large diversity of complete and fragmented virophage genomes - termed EMALEs- scattered throughout the genomes of four strains of the marine protist Cafeteria. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards deciphering the biology of these viruses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This study is well-written and well-presented. The conclusions are clear and robustly supported by the data. The figures provide useful visualizations for the major findings. Virophage are an important and underappreciated component of global viral diversity, and they likely play important roles in eukaryotic genome evolution; this work is therefore quite timely. Relatively few studies focus on virophage or giant viruses compared to other viral lineages, so studies like this are highly valuable.
Strengths of this work include the high quality of the reference genomes, which were constructed using both short-read and long-read sequencing, as well as the diverse locations and isolation times of the host genomes.
I found no major weaknesses in this study. One minor issue is that the details of how EMALEs were …
Reviewer #1 (Public Review):
This study is well-written and well-presented. The conclusions are clear and robustly supported by the data. The figures provide useful visualizations for the major findings. Virophage are an important and underappreciated component of global viral diversity, and they likely play important roles in eukaryotic genome evolution; this work is therefore quite timely. Relatively few studies focus on virophage or giant viruses compared to other viral lineages, so studies like this are highly valuable.
Strengths of this work include the high quality of the reference genomes, which were constructed using both short-read and long-read sequencing, as well as the diverse locations and isolation times of the host genomes.
I found no major weaknesses in this study. One minor issue is that the details of how EMALEs were delineated and initially detected seem a bit unclear to me. Based on my reading I am curious if some divergent or degraded EMALEs could have been missed. This may be important for assessing the consequences of possible retrotransposition-mediated EMALE inactivation.
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Reviewer #2 (Public Review):
The manuscript reports the in-depth analysis of 4 Cafeteria Burkhardea strains genome sequences revealing several integration events of mavirus-related virophages (EMALE) which can either correspond to ancestral integration events (present in several strains at the same genomic location) or unique to a given strain as a result of recent integration. Given the protective effect that Mavirus virophage have on the host against CroV infection, they are likely functional mobile genetic elements that can reactivate upon giant virus infections thus providing adaptative anti giant viruses defense to the host. Some integrated virophage genomes are incomplete and thus not functional anymore.
The EMALEs can be clustered into 8 different groups each endowed with different sets of gene promoters and are thus proposed to …
Reviewer #2 (Public Review):
The manuscript reports the in-depth analysis of 4 Cafeteria Burkhardea strains genome sequences revealing several integration events of mavirus-related virophages (EMALE) which can either correspond to ancestral integration events (present in several strains at the same genomic location) or unique to a given strain as a result of recent integration. Given the protective effect that Mavirus virophage have on the host against CroV infection, they are likely functional mobile genetic elements that can reactivate upon giant virus infections thus providing adaptative anti giant viruses defense to the host. Some integrated virophage genomes are incomplete and thus not functional anymore.
The EMALEs can be clustered into 8 different groups each endowed with different sets of gene promoters and are thus proposed to be virophages infecting different giant viruses, all infecting Cafeteria Burkhardea. Half of them are AT-rich and the others present medium AT-richness. The isolated mavirus prototype falls into type 4 of AT-rich EMALE. Type 2 could be the result of recombination between EMALE 1 and a polinton-like virus. The truncated type 8 lacks morphogenesis genes.
Interestingly, the study also reveals nested parasitism with integration of tyrosine recombinase retrotransposons of the Ngaro super family that can occur both in the host genome and in the virophage, with an example of a virophage integrating inside a Ngaro retrotransposon. There are four types of Ngaro retrotransposons based on their nucleotide sequences that share the same coding potential but differed in terms of integration preferences. When integrating into the host genome, the ORF1 encoding a GAG-like protein appears to be missing and this ORF could be determinant for integration site specificity.
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Reviewer #3 (Public Review):
Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, this study reveals a large diversity of complete and fragmented virophages genomes - termed EMALEs-scattered throughout the genomes of four strains of Cafeteria (a marine protist) expanding the known diversity of the Lavidaviridae family from a fresh slant. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards …Reviewer #3 (Public Review):
Approaching the search of novel viruses while in an endogenized stage, rather than as free virions, this study reveals a large diversity of complete and fragmented virophages genomes - termed EMALEs-scattered throughout the genomes of four strains of Cafeteria (a marine protist) expanding the known diversity of the Lavidaviridae family from a fresh slant. Given that the activation of the integrated virophage mavirus during infection by the giant virus, CroV, has been shown to have a protective effect on the Cafeteria population, this study provides a tantalizing window into the traces of virophage-giant virus¬-protist interactions in the marine environment. Given the enormous diversity of virophages and giant viruses that have been found in metagenomes with no known hosts, this study is a step towards deciphering the biology of these viruses. Intriguingly, the authors show that endogenized virophages themselves are predisposed to being targets of NGARO transposons, pointing to another potential player in an already complex (virophage-giant virus-protist) biological system.Strengths
The article is well-written and presented, so it is easy to follow the bioinformatic process and evaluate its technical soundness. Namely, how the search for endogenized elements was conducted, then how the EMALEs/NGAROs were classified by GC/nucleotide identity/whole genome synteny and phylogenetically.
The authors mapped each complete genomic element its genetic content and genomic context in great detail. This allows clear comparisons to be made between the EMALE types.
All genomic sequences and analyses are available, and the depth of the analyses available in supplementary is encyclopedic in scope, making this work highly transparent and also readily exploitable by others in the field.
The conclusions are well-supported by the data and the authors keep their discussion close to their findings.Weaknesses
There are no identifiable weaknesses in the technical analysis. There were a few points in the descriptions where a few points of clarification would be welcome.
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