The Nature and Chromosomal Landscape of Endogenous Retroviruses (ERVs) Integrated in the Sheep Nuclear Genome

Round 1

Reviewer 1 Report

The authors use whole-genome sequencing to identify endogenous retroviruses (ERVs) in the genome of two Iraqi sheep breeds. They find that ERVs comprise 0.55% of analyzed sequence reads. Using in situ hybridization, they find variable distribution of ERV sequences across sheep chromosomes. Further, the authors assemble the complete genome of endogenous Jaagsiekte sheep retrovirus (enJSRV) in these sheep breeds and find a close phylogenetic relationship to previously assembled enJSRV genomes worldwide.

Major points

•    ERVs account for 3.20% of the Bos taurus genome (Garcia-Etxebarria and Jugo, 2010), the Alpaca genome has 5-6% ERVs (https://www.repeatmasker.org/species/vicPac.html). The authors estimate a genomic proportion of 0.55% in the Iraqi sheep breed. How do the authors account for this discrepancy? How does that compare to the repeat / ERV content estimated for other sheep species (e.g. Li et al, Nature Communication 2020, PMID: 32499537)?
•    The enJSRV retrovirus genome has been extensively characterized in previous studies (e.g., Arnaud et al 2007). The motivation or novelty of the authors’ analysis needs to be explained in more detail, so readers can appreciate this work better. Is this the first attempt at genome-wide detection of ERVs in this specific sheep species, or has similar analysis been performed previously? It would increase the scope of this study to extend the author's analysis to publicly available datasets (e.g. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA624020). Do the authors results differ from the abundance or location of ERV sequences in closely related, sequenced sheep species?

Minor points

•    Line 36: suggest ‘descended’ in place of ‘ascended’.
•    Lines 52-55: sentence is not intelligible.
•    Line 61: typo. ‘Method’ should be ‘methods’.
•    Figures 1-5: scale bars do not have units.
•    Figure 6: the units of the horizonal are not given in the legend.
•    Lines 312-313: sentence is not intelligible.
•    Lines 338-340: this is a rather niche example of ERV domestication – there are numerous examples in mammals that are of greater relevance to sheep, including examples of retrovirus restriction factors derived from ERVs. In fact, Cumer et al. (2018, Heredity) have described a mutation in an enJSRV copy that protects sheep from exogenous infection, so the author's speculation in line 341 should be replaced by a description of these findings.

Author Response

Dear Reviewer,

Please kindly see the attachment

 

kind regards,

Authors

Author Response File: Author Response.docx

Reviewer 2 Report

Review

The nature and chromosomal landscape of endogenous retroviruses (ERVs) integrated in the sheep nuclear genome

By Sarbast Ihsan Mustafa, Trude Schwarzacher and JS Heslop-Harrison

For publication in DNA

 

Whereas the endogenous retroviruses (ERVs) of humans, pigs, mice, cats, and koalas are well characterized, nearly nothing is known about the ERVs in sheeps. Therefore, this manuscript is highly welcome.

High-throughput sequencing (HTS) as used in these investigations is an optimal method to characterize endogenous retroviruses in a species. Using different strategies (graph-based read clustering, frequency analysis of short motifs (k-mers) alignment to reference assemblies and fluorescent in situ hybridization) the authors identified three classes of sheep ERVs.

They studied sheep ERVs in the genome of five sheeps from the Iraqi Kurdistan region, their genomic DNA was sequenced.

Three classes of ERV, including ERV1, ERV2 and ERV3,  were identified. A total genomic proportion of all classes of ERV about 0.55% from all analyzed whole genome sequencing raw reads was estimated. Some 0.02% of the sheep reads were assembled with ancestral reads.

When amplified PCR products representing selected ERVs were labelled and hybridized to male sheep metaphase chromosomes, signals varied from dispersed over all or most chromosomes, to localized at centromeres or interstitial positions. This was illustrated with remarkable figures.

In the second part of the manuscript, the complete consensus endogenous betaretroviruses enJSRV (endogenous Jaagsiekte sheep retrovirus) genome of three Hamdani and two Karadi sheep breeds were assembled. Each genome was 7941bp long, 71-124 copies were found (0.0087-0.0118% genomic proportion). A phylogenetic study indicated the sequences from the Kurdistan sheeps were placed among sequences of viruses sampled from geographically different locations. The authors should give some additional information of the closest relatives, enJSRVs from Mongolia and USA and indicated the most striking differences.

Of great interest is the localization of many ERVs in the centromers. A similar pericentromeric locations of the human endogenous retrovirus – K (HERV-K) was reported.

Of interest is the low copy number of sheep ERV. The authors should discuss in more detail whether the low genomic proportions in sheeps compared with the high proportion of 8% in humans.

In pigs the copy number of porcine endogenous retroviruses (PERVs) depend on the breed, on the age, on the method used for detection and on the organ used for detection because PERVs are still active in the living animals (Denner J., How Active Are Porcine Endogenous Retroviruses (PERVs)? Viruses. 2016 Aug 3;8(8):215). How is the situation in sheeps? Concerning the method, sequencing always underestimated the copy number in the case of PERVs as well as in the case of HERV-K. The authors should analyze this.

The authors should – when discussing the biological significance of sheep ERVs – discuss that the enJSRV mRNA is abundant in organs of the reproductive tract, in trophectoderm cells and particularly in trophoblast giant binucleate cells (BNC) and multinucleated syncytia which are required for implantation and formation of placentosomes and that inhibition of Env expression by antisense oligonucleotide inhibits giant BNC differentiation and results in loss of pregnancy.

Author Response

Dear Reviewer,

Please kindly see the attachment

 

kind regards,

Authors

Author Response File: Author Response.docx