Recombinant origin and interspecies transmission of a HERV-K(HML-2)-related primate retrovirus with a novel RNA transport element
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Evaluation Summary:
This work is a bioinformatic analysis of HML-2-like proviruses found in the genomes of Rhesus Macaques, which convincingly argues that an HML-2 provirus underwent an ancient recombination event with a HERV-K (HML-8) related virus. The authors also provide data to suggest that the recombinant retrovirus may have acquired a distinct mechanism for the regulation of expression of spliced and unspliced transcripts. This paper should be of broad interest to virologists as it uses molecular 'fossil-like' evidence contained in the genomes of modern pirates to document the generation of what could be considered a previously undescribed retrovirus species, through recombination.
(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 name with the authors.)
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Abstract
HERV-K(HML-2), the youngest clade of human endogenous retroviruses (HERVs), includes many intact or nearly intact proviruses, but no replication competent HML-2 proviruses have been identified in humans. HML-2-related proviruses are present in other primates, including rhesus macaques, but the extent and timing of HML-2 activity in macaques remains unclear. We have identified 145 HML-2-like proviruses in rhesus macaques, including a clade of young, rhesus-specific insertions. Age estimates, intact open reading frames, and insertional polymorphism of these insertions are consistent with recent or ongoing infectious activity in macaques. 106 of the proviruses form a clade characterized by an ~750 bp sequence between env and the 3′ long terminal repeat (LTR), derived from an ancient recombination with a HERV-K(HML-8)-related virus. This clade is found in Old World monkeys (OWM), but not great apes, suggesting it originated after the ape/OWM split. We identified similar proviruses in white-cheeked gibbons; the gibbon insertions cluster within the OWM recombinant clade, suggesting interspecies transmission from OWM to gibbons. The LTRs of the youngest proviruses have deletions in U3, which disrupt the Rec Response Element (RcRE), required for nuclear export of unspliced viral RNA. We show that the HML-8-derived region functions as a Rec-independent constitutive transport element (CTE), indicating the ancestral Rec–RcRE export system was replaced by a CTE mechanism.
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Author response:
Reviewer #1 (Public Review):
Weaknesses:
With the exception of the PCR analysis and the reporter assays, the manuscript does not contain any experiments or attempts to analyze current expression from any of the identified proviruses. No long-read RNASeq or other RNA analysis on cytoplasmic RNA was performed, nor any experiments to show that proteins are indeed expressed.
We agree that an investigation of RNA and protein expression from these proviruses would be very interesting, and we hope to do such work in the future to test whether this clade is still actively infecting any primate species. However, we believe that such an investigation is out of the scope of this manuscript, which is focused on the past evolutionary history of these viruses. However, it is worth noting that we do show evidence for proviral …
Author response:
Reviewer #1 (Public Review):
Weaknesses:
With the exception of the PCR analysis and the reporter assays, the manuscript does not contain any experiments or attempts to analyze current expression from any of the identified proviruses. No long-read RNASeq or other RNA analysis on cytoplasmic RNA was performed, nor any experiments to show that proteins are indeed expressed.
We agree that an investigation of RNA and protein expression from these proviruses would be very interesting, and we hope to do such work in the future to test whether this clade is still actively infecting any primate species. However, we believe that such an investigation is out of the scope of this manuscript, which is focused on the past evolutionary history of these viruses. However, it is worth noting that we do show evidence for proviral expression at the RNA level in Fig. 6 supplement 1, showing alignment of publically available rhesus macaque iPSC RNAseq data to the SERV-K1 provirus, including both spliced and full length viral RNA. Interestingly, there appear to be reads derived from multiple proviruses, as some reads originate from proviruses with large internal deletions, while others derive from full length proviruses.
The findings of a potential CTE are interesting, but the sequences that were appended to the reporter construct are much longer than previously identified CTEs. No data were presented to indicate whether this sequence show similarity to previously identified CTEs and no experiments to show whether this sequence functionally interacts with Nxf1, the protein shown to interact with previously identified bona fide CTEs. Also, since nucleo-cytoplasmic export was not directly analyzed, it remains possible that the sequences that were inserted into the reporter contained splice sites that would allow the RNA to be spliced "downstream" of the GFP gene, allowing the export of a "spliced" GFP mRNA.
While it is true that the HML8-derived sequences we have tested are much longer than the canonical MPMV CTE and many other known CTEs, there are other reports of elements with CTE-like activity that are much longer and more complex than the MPMV CTE, including one, the MLV PTE, which is ~1400 nt long, even longer than the HML8-derived sequence we have identified. We have compared the MER11 sequence to known CTEs from MPMV, IAP, MusD, MLV, and RSV, as well as the woodchuck hepatitis virus WPRE, which is not a canonical CTE but has been shown to promote nuclear export of RNA; none of these sequences showed any clear sequence similarity to our sequences of interest. We have added a section discussing these questions in some detail (l. 535-547).
Although the question of what pathway or pathways these elements co-opt is obviously of great interest, we believe it is outside the scope of this manuscript. It is worth noting that a number of cis-acting RNA transport elements do not bind NXF1, either indirectly recruiting NXF1 (IAP RTE), using CRM1 (MLV, WPRE, foamy viruses), or have an unknown mechanism (MusD). We agree that there are potential pitfalls of the reporter system used, and thus have added experiments to directly test the CTE activity of these elements, detailed above.
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Evaluation Summary:
This work is a bioinformatic analysis of HML-2-like proviruses found in the genomes of Rhesus Macaques, which convincingly argues that an HML-2 provirus underwent an ancient recombination event with a HERV-K (HML-8) related virus. The authors also provide data to suggest that the recombinant retrovirus may have acquired a distinct mechanism for the regulation of expression of spliced and unspliced transcripts. This paper should be of broad interest to virologists as it uses molecular 'fossil-like' evidence contained in the genomes of modern pirates to document the generation of what could be considered a previously undescribed retrovirus species, through recombination.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
This work is a bioinformatic analysis of HML-2-like proviruses found in the genomes of Rhesus Macaques, which convincingly argues that an HML-2 provirus underwent an ancient recombination event with a HERV-K (HML-8) related virus. The authors also provide data to suggest that the recombinant retrovirus may have acquired a distinct mechanism for the regulation of expression of spliced and unspliced transcripts. This paper should be of broad interest to virologists as it uses molecular 'fossil-like' evidence contained in the genomes of modern pirates to document the generation of what could be considered a previously undescribed retrovirus species, through recombination.
(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 name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Williams et al. present a mainly bioinformatic analysis of HERV-K(HML-2) related retroviruses in Old World Monkey species. HML-2 viruses represent the youngest clade of human retroviruses and have also been shown in other primates. This manuscript is focused on identifying and characterizing HML-2-like proviruses in rhesus macaques. A majority of the identified proviruses (106/145) contain sequences that the authors determine were derived from ancient recombination with a HERV-K(HML-8)-related virus (likely after the OWM/great ape split). They further identify similar sequences in white-cheeked gibbons, which they conclude suggest interspecies transmission. Several of the viruses have deletions that disrupt the Rec-Response-Element (RcRE), which interacts with the viral Rec protein.This …
Reviewer #1 (Public Review):
In this manuscript, Williams et al. present a mainly bioinformatic analysis of HERV-K(HML-2) related retroviruses in Old World Monkey species. HML-2 viruses represent the youngest clade of human retroviruses and have also been shown in other primates. This manuscript is focused on identifying and characterizing HML-2-like proviruses in rhesus macaques. A majority of the identified proviruses (106/145) contain sequences that the authors determine were derived from ancient recombination with a HERV-K(HML-8)-related virus (likely after the OWM/great ape split). They further identify similar sequences in white-cheeked gibbons, which they conclude suggest interspecies transmission. Several of the viruses have deletions that disrupt the Rec-Response-Element (RcRE), which interacts with the viral Rec protein.This has previously been shown to be essential to overcome the host cell restrictions to nucleo-cytoplasmic export of unspliced/partially spliced mRNA. Export of this kind of RNA is essential for viral structural protein expression and genome packaging. The authors suggest that sequences derived from HML-8 contain a Constitutive Transport Element (CTE) that replaced the Rec-RcRE export system in the recombinant proviruses.
Strengths:
This manuscript presents a comprehensive bioinformatic analysis of HML-2 like HERV-K proviruses present in rhesus macaques. Several of the authors are well-recognized experts in this kind of analysis and have published extensively in this area. They present convincing evidence (based on analysis of age, intact Open Reading Frames (ORFs), as well as insertional polymorphism) for recent and maybe even ongoing infectious activity of these viruses in rhesus macaques. The identification of the ancient recombination with HML-8 sequences is interesting and allows the authors to reasonably suggest that this event happened after the split of old world monkeys and great apes. The analysis led to the discovery of LTR deletions in recombinant proviruses. These were verified using PCR on genomic DNA and since the deletions included parts of the RcRE, this would indeed suggest that Rec/RcRE function is absent. This was also supported by the lack of function in conjunction with Rec, using a previously described reporter system, where GFP expression suggests the export of unspliced mRNA. However, when sequences from the 3' LTR (derived from HML-8) in the recombinant proviruses were tested in this reporter, GFP protein expression was observed in the absence of Rec. Based on this, the authors suggest that these sequences contain a Constitutive Transport Element (CTE). This is a potentially very interesting finding.Weaknesses:
With the exception of the PCR analysis and the reporter assays, the manuscript does not contain any experiments or attempts to analyze current expression from any of the identified proviruses. No long-read RNASeq or other RNA analysis on cytoplasmic RNA was performed, nor any experiments to show that proteins are indeed expressed. The findings of a potential CTE are interesting, but the sequences that were appended to the reporter construct are much longer than previously identified CTEs. No data were presented to indicate whether this sequence show similarity to previously identified CTEs and no experiments to show whether this sequence functionally interacts with Nxf1, the protein shown to interact with previously identified bona fide CTEs. Also, since nucleo-cytoplasmic export was not directly analyzed, it remains possible that the sequences that were inserted into the reporter contained splice sites that would allow the RNA to be spliced "downstream" of the GFP gene, allowing the export of a "spliced" GFP mRNA.
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Reviewer #2 (Public Review):
The authors use molecular 'fossil-like' evidence from genome sequence to document the generation of what could be considered a previously undescribed retrovirus species, through recombination. The authors show, convincingly, that the recombinant retrovirus has acquired a distinct mechanism for the regulation of expression of spliced and unspliced transcripts, employing a novel constitutive transport element rather than a Rev-RRE-like mechanism from one of the parental viruses. The authors document the evolutionary history of the recombinant virus in certain OWM species and demonstrate that it has been transmitted among Asian primate species within the past few million years.
The key strengths of the paper are its definitiveness - the data are clear, robust, and support the conclusions
In my opinion, there …
Reviewer #2 (Public Review):
The authors use molecular 'fossil-like' evidence from genome sequence to document the generation of what could be considered a previously undescribed retrovirus species, through recombination. The authors show, convincingly, that the recombinant retrovirus has acquired a distinct mechanism for the regulation of expression of spliced and unspliced transcripts, employing a novel constitutive transport element rather than a Rev-RRE-like mechanism from one of the parental viruses. The authors document the evolutionary history of the recombinant virus in certain OWM species and demonstrate that it has been transmitted among Asian primate species within the past few million years.
The key strengths of the paper are its definitiveness - the data are clear, robust, and support the conclusions
In my opinion, there are no major weaknesses - one could quibble about whether additional analyses of recent transmission in other Asian primates might add to the story, (its unclear to me how extensive the search for SERV-K/MER11 was in these species). Did the authors look for and not find recent entry deposited proviruses in, say, pig-tailed macaques or other asian monkeys? I empahasise that this is a minor criticism and doesn't detract from, or in any way undermine the study.
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