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. 2010 Sep;84(18):9078-85.
doi: 10.1128/JVI.00950-10. Epub 2010 Jul 7.

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model

Sarah G Black  1 , Frederick Arnaud, Robert C Burghardt, M Carey Satterfield, Jo-Ann G W Fleming, Charles R Long, Carol Hanna, Lita Murphy, Roman Biek, Massimo Palmarini, Thomas E Spencer
Affiliations

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model

Sarah G Black et al. J Virol. 2010 Sep.

Abstract

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.

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Figures

FIG. 1.
FIG. 1.
Western blot analysis of viral particles isolated from the lumen of the ovine uterus. Viral particles were purified by ultracentrifugation of uterine flush samples over a sucrose cushion. Proteins were separated by 12% SDS-PAGE, and Western blot analysis was conducted using a rabbit antiserum toward the major capsid protein of JSRV. An immunoreactive protein of ∼25 kDa in size, which is the correct size of the mature enJSRVs capsid (CA), was detected in uterine flushings from all ewes (n = 16). Representative results from six ewes are presented. Cell lysates from NIH 3T3 cells were used as a negative control.
FIG. 2.
FIG. 2.
Transspecies embryo transfer model. Schematic illustrating experimental design of transspecies embryo transfer conducted to test the working hypothesis that enJSRV particles are released from the uterine endometrial epithelia and infect the conceptus trophectoderm.
FIG. 3.
FIG. 3.
Analysis of microdissected bovine conceptus trophectoderm for presence of enJSRV RNA and DNA. (A) RT-PCR analysis of bovine conceptus trophectoderm. RNA was extracted from microdissected bovine conceptus trophectoderm cells and analyzed by RT-PCR for LGALS15, IFNT, CSH1, HYAL2, and enJSRV mRNAs. Representative results are shown from 10 different bovine conceptuses. Negative controls included water as template and no reverse transcriptase (−RT) to assess genomic DNA contamination. (B) PCR analysis of bovine conceptus trophectoderm. DNA was extracted from microdissected bovine conceptus trophectoderm cells and analyzed by PCR for ovine and bovine MT-CO1 genes as well as enJSRV env. Representative results are shown from 11 bovine conceptuses. The negative control used water as a template, and the positive controls were bovine or ovine genomic DNA (gDNA).
FIG. 4.
FIG. 4.
Phylogenetic analysis of enJSRVs expressed in the uterus. Bayesian majority rule consensus trees of 200 partial env sequences from endometrium (red), uterine flush (blue), and conceptus (purple) of four ewes receiving bovine blastocysts. Sequences from 26 known endogenous betaretroviruses (enJSRVs) were also included (in black); enJSRV-6 was used to root the tree as it is the oldest enJSRV provirus integrated in the sheep genome containing the sequences analyzed in this figure. Branch width represents the level of posterior support, with most internal branches receiving 100% support. Scale bars represent the expected substitutions per site. For easier visual representation, deeper parts of the phylogeny (gray branches) are shown at a different scale.
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