The evolutionary interplay between exogenous and endogenous sheep betaretroviruses

Retroviruses must integrate their genome into the host DNA as a necessary step of their replication cycle. Normally, retroviruses integrate into somatic cells and are transmitted, from infected to uninfected hosts, as “exogenous” retroviruses. On rare occasions, they can infect germ line cells and become part of the host genome as “endogenous” retroviruses (ERVs), which are transmitted vertically to the offspring and inherited as Mendelian genes. During evolution, most ERVs have accumulated mutations that rendered them defective and unable to produce infectious viral particles. Some ERVs, however, have maintained intact open reading frames for some of their genes, and have been co-opted by the host as they fulfil important biological functions. Sheep betaretroviruses represent a unique model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) co-exists with the highly related endogenous JSRVs (enJSRVs). The sheep genome harbours at least twenty-seven enJSRV loci and, most likely, the process of endogenization is still occurring. During evolution, one of these enJSRV loci, enJS56A1, has acquired a defective and transdominant Gag polyprotein that blocks the late replication steps of related retroviruses, by a mechanism known as JSRV late restriction (JLR). Interestingly, enJSRV-26, a provirus that integrated in the sheep germ line less than two hundred years ago, possesses the unique ability to escape JLR. In this thesis, the molecular basis of JLR escape was investigated. The main determinant of JLR escape was identified in the signal peptide of enJSRV-26 envelope protein (SP26). A single amino acid substitution in SP26 was found to be responsible for altering its intracellular localization as well as its function as a post-transcriptional regulator of viral gene expression. Interestingly, interference assays demonstrated that enJSRV-26 relies on the presence of the functional signal peptide of enJS56A1 envelope protein (SP56) in order to escape JLR. In addition, the ratio between enJSRV-26 and enJS56A1 Gag polyproteins was found to be critical to elude JLR. Finally, sequence analyses revealed that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus, reinforcing the hypothesis that this locus has provided an evolutionary advantage to the host. This study unveils critical aspects of JLR that were previously unknown, and provides new insights on the molecular mechanisms governing the interplay between endogenous and exogenous sheep betaretroviruses

Comparative fluorescence in situ hybridization (FISH) mapping of twenty-three endogenous Jaagsiekte sheep retrovirus (enJSRVs) in sheep (Ovis aries) and river buffalo (Bubalus bubalis) chromosomes

Endogenous retroviruses (ERVs) are the remnants of ancient infections of host germline cells, thus representing key tools to study host and viral evolution. Homologous ERV sequences often map at the same genomic locus of different species, indicating that retroviral integration occurred in the genomes of the common ancestors of those species. The genome of domestic sheep (Ovis aries) harbors at least twenty-seven copies of ERVs related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRVs), thus referred to as enJSRVs. Some of these loci are unequally distributed between breeds and individuals of the host species due to polymorphic insertions, thereby representing invaluable tools to trace the evolutionary dynamics of virus populations within their hosts. In this study, we extend the cytogenetic physical maps of sheep and river buffalo by performing fluorescent in situ hybridization (FISH) mapping of twenty-three genetically characterized enJSRVs. Additionally, we report the first comparative FISH mapping of enJSRVs in domestic sheep (2n = 54) and river buffalo (Bubalus bubalis, 2n = 50). Finally, we demonstrate that enJSRV loci are conserved in the homologous chromosomes and chromosome bands of both species. Altogether, our results support the hypothesis that enJSRVs were present in the genomes of both species before they differentiated within the Bovidae family

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell

“Ménage à Trois”: The Evolutionary Interplay between JSRV, enJSRVs and Domestic Sheep

Sheep betaretroviruses represent a fascinating model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with a variety of highly related endogenous JSRVs, referred to as enJSRVs. During evolution, some of them were co-opted by the host as they fulfilled important biological functions, including placental development and protection against related exogenous retroviruses. In particular, two enJSRV loci, enJS56A1 and enJSRV-20, were positively selected during sheep domestication due to their ability to interfere with the replication of related competent retroviruses. Interestingly, viruses escaping these transdominant enJSRVs have recently emerged, probably less than 200 years ago. Overall, these findings suggest that in sheep the process of endogenization is still ongoing and, therefore, the evolutionary interplay between endogenous and exogenous sheep betaretroviruses and their host has not yet reached an equilibrium

The evolutionary interplay between exogenous and endogenous sheep betaretroviruses

Sheep betaretroviruses represent an interesting model to study the ­complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with at least 27 highly related endogenous JSRVs (enJSRVs). During evolution, some enJSRVs were co-opted by the host as they fulfilled important biological functions, including protection against infections by related exogenous retroviruses as well as conceptus development and placental morphogenesis. In particular, recent studies demonstrate that transdominant enJSRVs (i.e., those that are able to block JSRV replication) were positively selected during sheep domestication. Interestingly, viruses escaping these loci have recently emerged (less than 200 years ago). Overall, these findings suggest that the process of endogenization is still ongoing in sheep and, therefore, the evolutionary interplay between endogenous and exogenous sheep betaretroviruses and their hosts has not reached equilibrium

<i>In vitro</i> growth properties of rescued WT and ΔNS4 viruses during interferon treatment.

<p>CPT-Tert cells were treated (solid line) or mock treated (dashed line) with 1000 AVU/ml of interferon (<i>Tau,</i> IFNT or Universal, UIFN) for 20 h prior and 2 h after being infected by BTV-8 (dark red, square), BTV8-ΔNS4 (red, triangle), BTV-1 (blue, square) and BTV1-ΔNS4 (light blue, triangle) viruses. Cells were infected at a MOI of 0.01. Supernatants were collected at 24, 48 and 72 h after infection, and then titrated on BSR cells by limiting dilution analysis and virus titers expressed as log₁₀ (TCID₅₀/ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed three times, each time in duplicate.</p

The NS4 of BTV-1 displays similar biological properties to the homologous BTV-8 protein.

<p>(A) Western blotting of cellular extracts (lysate) of CPT-Tert cells infected with BTV-8 wt or BTV-8ΔNS4 at a MOI of 0.01. Cells were analyzed 24 h post-infection and blots were incubated with antisera against NS1, VP7, VP6, NS4 and γ-tubulin as indicated. (B) Schematic diagram of the BTV-8/BTV-1 reassortants and mutants used in this study. Note that BTV1 and BTV8 segments/proteins are coloured in blue and red, respectively. * indicates a point mutation, while # indicates the introduction of a stop codon in the NS4 ORF. (C) CPT-Tert cells were treated with 1000 AVU/ml of Universal IFN for 20 h prior, and 2 h after, being infected by the recombinant viruses indicated in the panel using a MOI of 0.01. Cell monolayers were stained 72 h post-infection using crystal violet. Values indicated below each well correspond to the relative quantification of the disrupted monolayer using Image-Pro Plus (MediaCybernetics, Inc.). (D) CPT-Tert cells were treated (solid line) or mock treated (dashed line) with 100 AVU/ml of Universal interferon (UIFN) for 20 h prior and 2 h after being infected by the viruses indicated in the panel. Cells were infected at a MOI of 0.01. Supernatants were collected at 24, 48 and 72 h after infection, and then titrated on BSR cells by limiting dilution analysis and virus titers expressed as log₁₀ (TCID₅₀/ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed two times, each time in duplicate.</p

Generation of ΔNS4 Bluetongue viruses by reverse genetics.

<p>(A) BTV segment 9 open reading frames. VP6 amino acid residues are written in black, NS4 amino acid residues are written in grey. The nucleotides at positions 183 (T), 252 (G) and 381 (T) were mutated to C, A and A, respectively (bold). Note that whilst these mutations do not change any amino acid residues of VP6, they remove the initiation codon of NS4 (position182) and introduce two stop codons into the NS4 coding sequence at amino acid positions 24 and 67. (B) Transfected BSR cells with BTV transcripts generated <i>in vitro</i> (0.5×10¹¹ molecules per segment for BTV-1 and 1×10¹¹ molecules per segment for BTV-8). Cell monolayers were stained using crystal violet at 72 h post-transfection. As negative controls, ΔVP6 assays correspond to using a segment 9 containing a stop codon at position 79 in the VP6 gene. (C) Agarose gel (1.5%) of purified BTV genomic dsRNA. BSR cells infected at a MOI of 0.01 were collected at 72 h post infection and BTV dsRNA was purified as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a>. 2 µg of dsRNA was loaded in each lane. (D) Western blotting of cellular extracts (lysate) of BSR cells infected at a MOI of 0.01. Cells were analyzed 36 h post-infection and blots were incubated with antisera against VP7, NS4 and γ-tubulin as indicated. Note that the double NS4 band in the BTV-1 sample is not a feature observed consistently. (E) Electron microscopy of BSR cells infected by BTV1-ΔNS4. Note cells display all the major features of BTV-infected cells including NS1 tubules (T), viral inclusion bodies (VIB) and viral particles (arrows). Scale bar = 1 µm.</p

NS4 expression profile.

<p>Confocal microscopy of BFAE cells infected with BTV-1 at a MOI of 1.5. Cells were fixed before infection (0 h) and at 0 h30, 2 h, 4 h, 8 h, 16 h and 24 h post-infection and processed for immunofluorescence using antibodies against VP7, NS1, NS2, NS3 and NS4 with an Alexa Fluor 488 secondary antibody as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002477#s4" target="_blank">Materials and Methods</a>. Scale bars correspond to 21.16 µm for 0 h to 4 h post-infection panels, and 13.6 µm for 8 h to 24 h post-infection panels.</p