Frederick Arnaud

Revealing the History of Sheep Domestication Using Retrovirus Integrations

Sheep retroviruses can be used to map the selective preferences of early farmers and trace livestock movements across Europe. Not Just Dinner on Legs Several thousand years ago, human beings realized the virtues of domesticating wild animals as easy meat. Soon other possibilities became apparent, and as revealed in a series of papers in this issue, early pastoralists became selective about breeding for wool, leather, milk, and muscle power. In two papers, Gibbs et al. report on the bovine genome sequence (p. 522; see the cover, the Perspective by Lewin, and the Policy Forum by Roberts) and trace the diversity and genetic history of cattle (p. 528), while Chessa et al. (p. 532) survey the occurrence of endogenous retroviruses in sheep and map their distribution to historical waves of human selection and dispersal across Europe. Finally, Ludwig et al. (p. 485) note the origins of variation in the coat-color of horses and suggest that it is most likely to have been selected for by humans in need of good-looking transport. The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their “retrotype” and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as “primitive” on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.

Interplay between Ovine Bone Marrow Stromal Cell Antigen 2/Tetherin and Endogenous Retroviruses

ABSTRACT Endogenous betaretroviruses (enJSRVs) of sheep are expressed abundantly in the female reproductive tract and play a crucial role in conceptus development and placental morphogenesis. Interestingly, the colonization of the sheep genome by enJSRVs is likely still ongoing. During early pregnancy, enJSRV expression correlates with the production of tau interferon (IFNT), a type I IFN, by the developing conceptus. IFNT is the pregnancy recognition signal in ruminants and possesses potent antiviral activity. In this study, we show that IFNT induces the expression of bone marrow stromal cell antigen 2 (BST2) (also termed CD317/tetherin) both in vitro and in vivo. The BST2 gene is duplicated in ruminants. Transfection assays found that ovine BST2 proteins (oBST2A and oBST2B) block release of viral particles produced by intact enJSRV loci and of related exogenous and pathogenic jaagsiekte sheep retrovirus (JSRV). Ovine BST2A appears to restrict enJSRVs more efficiently than oBST2B. In vivo, the expression of BST2A/B and enJSRVs in the endometrium increases after day 12 and remains high between days 14 and 20 of pregnancy. In situ hybridization analyses found that oBST2A is expressed mainly in the endometrial stromal cells but not in the luminal and glandular epithelial cells, in which enJSRVs are highly expressed. In conclusion, enJSRVs may have coevolved in the presence of oBST2A/B by being expressed in different cellular compartments of the same organ. Viral expression in cells unable to express BST2 may be one of the mechanisms used by retroviruses to escape restriction.

Mechanisms of Late Restriction Induced by an Endogenous Retrovirus

ABSTRACT The host has developed during evolution a variety of “restriction factors” to fight retroviral infections. We investigated the mechanisms of a unique viral block acting at late stages of the retrovirus replication cycle. The sheep genome is colonized by several copies of endogenous retroviruses, known as enJSRVs, which are highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV). enJS56A1, one of the enJSRV proviruses, can act as a restriction factor by blocking viral particles release of the exogenous JSRV. We show that in the absence of enJS56A1 expression, the JSRV Gag (the retroviral internal structural polyprotein) targets initially the pericentriolar region, in a dynein and microtubule-dependent fashion, and then colocalizes with the recycling endosomes. Indeed, by inhibiting the endocytosis and trafficking of recycling endosomes we hampered JSRV exit from the cell. Using a variety of approaches, we show that enJS56A1 and JSRV Gag interact soon after synthesis and before pericentriolar/recycling endosome targeting of the latter. The transdominant enJS56A1 induces intracellular Gag accumulation in microaggregates that colocalize with the aggresome marker GFP-250 but develop into bona fide aggresomes only when the proteasomal machinery is inhibited. The data argue that dominant-negative proteins can modify the overall structure of Gag multimers/viral particles hampering the interaction of the latter with the cellular trafficking machinery.

Coevolution of endogenous betaretroviruses of sheep and their host.

Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. Jaagsiekte sheep retrovirus (JSRV) is a pathogenic exogenous retrovirus and the causative agent of ovine pulmonary adenocarcinoma. The sheep genome contains at least 27 copies of endogenous retroviruses (enJSRVs) highly related to JSRV. enJSRVs have played several roles in the evolution of the domestic sheep as they are able to block the JSRV replication cycle and play a critical role in sheep conceptus development and placental morphogenesis. Available data strongly suggest that some dominant negative enJSRV proviruses (i.e. able to block JSRV replication) have been positively selected during evolution. Interestingly, viruses escaping the transdominant enJSRV loci have recently emerged (less than 200 years ago). Thus, endogenization of these retroviruses may still be occurring today. Therefore, sheep provide an exciting and unique system to study retrovirus-host coevolution. (Part of a multi-author review).

The Transdominant Endogenous Retrovirus enJS56A1 Associates with and Blocks Intracellular Trafficking of Jaagsiekte Sheep Retrovirus Gag

ABSTRACT The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.

Friendly viruses the special relationship between endogenous retroviruses and their host

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and have coevolved with their hosts for millions of years. Some ERVs play a critical role in placental development, contribute to genome plasticity, and protect the host against infection of related pathogenic and exogenous retroviruses, thus some ERVs have been positively selected and maintained in the host genome. The sheep genome contains 27 endogenous retroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. enJSRVs are able to protect their host against JSRV infection by blocking different steps of the viral replication cycle. In addition, enJSRVs are absolutely required for sheep placental development. Thus, enJSRVs‐JSRV provides a unique and interesting model to study the symbiotic relationship and interplay between host ERVs and evolution. This review will provide some examples of the biological functions of ERVs. In particular, the role of ERVs in reproductive biology and in protecting the host against pathogenic retrovirus infections will be emphasized using enJSRVs/JSRV and the sheep as a model.

The Signal Peptide of a Simple Retrovirus Envelope Functions as a Posttranscriptional Regulator of Viral Gene Expression

ABSTRACT Retroviruses use different strategies to regulate transcription and translation and exploit the cellular machinery involved in these processes. This study shows that the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus (JSRV) plays a major role in posttranscriptional viral gene expression. Expression of the JSRV Env in trans increases viral particle production by mechanisms dependent on (i) its leader sequence, (ii) an intact signal peptide cleavage site, (iii) a cis-acting RNA-responsive element located in the viral genome, (iv) Crm1, and (v) B23. The signal peptide of the JSRV Env (JSE-SP) is 80 amino acid residues in length and contains putative nuclear localization and export signals, in addition to an arginine-rich RNA binding motif. JSE-SP localizes both in the endoplasmic reticulum and in the nucleus, where it colocalizes with nucleolar markers. JSE-SP is a multifunctional protein, as it moderately enhances nuclear export of unspliced viral mRNA and considerably increases viral particle release by favoring a posttranslational step of the replication cycle.

Drosophila melanogaster as a Model Organism for Bluetongue Virus Replication and Tropism

ABSTRACT Bluetongue virus (BTV) is the etiological agent of bluetongue (BT), a hemorrhagic disease of ruminants that can cause high levels of morbidity and mortality. BTV is an arbovirus transmitted between its ruminant hosts by Culicoides biting midges (Diptera: Ceratopogonidae). Recently, Europe has experienced some of the largest BT outbreaks ever recorded, including areas with no known history of the disease, leading to unprecedented economic and animal welfare issues. The current lack of genomic resources and genetic tools for Culicoides restricts any detailed study of the mechanisms involved in the virus-insect interactions. In contrast, the genome of the fruit fly (Drosophila melanogaster) has been successfully sequenced, and it is used extensively as a model of molecular pathways due to the existence of powerful genetic technology. In this study, D. melanogaster is investigated as a model for the replication and tropism of BTV. Using reverse genetics, a modified BTV-1 that expresses the fluorescent mCherry protein fused to the viral nonstructural protein NS3 (BTV-1/NS3mCherry) was generated. We demonstrate that BTV-1/NS3mCherry is not only replication competent as it retains many characteristics of the wild-type virus but also replicates efficiently in D. melanogaster after removal of the bacterial endosymbiont Wolbachia pipientis by antibiotic treatment. Furthermore, confocal microscopy shows that the tissue tropism of BTV-1/NS3mCherry in D. melanogaster resembles that described previously for BTV in Culicoides. Overall, the data presented in this study demonstrate the feasibility of using D. melanogaster as a genetic model to investigate BTV-insect interactions that cannot be otherwise addressed in vector species.

Viral Particles of Endogenous Betaretroviruses Are Released in the Sheep Uterus and Infect the Conceptus Trophectoderm in a Transspecies Embryo Transfer Model

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.

The 5' untranslated region and Gag product of Idefix, a long terminal repeat-retrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA.

ABSTRACT Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5′UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.