Contrasted Patterns of Selection on MHC-Linked Microsatellites in Natural Populations of the Malagasy Plague Reservoir

Plague (Yersinia pestis infection) is a highly virulent rodent disease that persists in many natural ecosystems. The black rat (Rattus rattus) is the main host involved in the plague focus of the central highlands of Madagascar. Black rat populations from this area are highly resistant to plague, whereas those from areas in which the disease is absent (low altitude zones of Madagascar) are susceptible. Various lines of evidence suggest a role for the Major Histocompatibility Complex (MHC) in plague resistance. We therefore used the MHC region as a candidate for detecting signatures of plague-mediated selection in Malagasy black rats, by comparing population genetic structures for five MHC-linked microsatellites and neutral markers in two sampling designs. We first compared four pairs of populations, each pair including one population from the plague focus and one from the disease-free zone. Plague-mediated selection was expected to result in greater genetic differentiation between the two zones than expected under neutrality and this was observed for one MHC-class I-linked locus (D20Img2). For this marker as well as for four other MHC-linked loci, a geographic pattern of genetic structure was found at local scale within the plague focus. This pattern would be expected if plague selection pressures were spatially variable. Finally, another MHC-class I-linked locus (D20Rat21) showed evidences of balancing selection, but it seems more likely that this selection would be related to unknown pathogens more widely distributed in Madagascar than plague

Gamma- and delta-retroviral envelope glycoproteins (Envs) and their receptors : identification of new Env receptors associated to cell metabolism and identification of related endogenous Env with receptor-binding potentals

Couverture)Les rétrovirus sont des virus enveloppés à ARN simple brin omniprésents dans le monde animal et sources de nombreuses pathologies. Les rétrovirus de vertébrés comprennent sept genres dont les gamma et deltarétrovirus qui sont l’objet de ces travaux. Les rétrovirus dits endogènes (ERV), par opposition à leurs homologues infectieux exogènes, sont présents dans les cellules germinales et font partie intégrante du patrimoine génétique, avec transmission mendélienne. Au cours de l'évolution, les ERV ont fait l'objet de mutations, rendant défectives la plupart des copies dans les génomes de vertébrés, avec quelques exceptions notoires. De fait, certaines copies maintiennent de larges cadres de lecture suite à une pression de sélection positive.Rétrovirus exogènes et ERV partagent une organisation génétique similaire. Leurs glycoprotéines d’enveloppe (Env), dont une des propriétés est de lier un récepteur cellulaire, comprennent une composante de surface (SU) associée à une partie transmembranaire (TM). La SU des Env γ et -rétrovirales porte un module RBD (Receptor-Binding Domain) qui lie un récepteur appartenant à la famille SLC (Solute Carriers) des transporteurs de nutriments. Les SLC présents à la surface cellulaire conditionnent le métabolisme des cellules. Afin de pallier l'absence d'anticorps fiables reconnaissant les parties extracellulaires (exofaciales) des SLC, le laboratoire a dérivé des RBD solubles comme ligands des SLC, permettant de suivre leur expression à la surface cellulaire et ainsi, évaluer le métabolisme cellulaire.Parmi les ERV, certaines env partiellement ou entièrement conservées jouent un rôle physiologique essentiel dans les organismes qui les portent. Une hypothèse de mon laboratoire d’accueil est l’existence de RBD endogènes de mammifères capables de moduler le métabolisme cellulaire de leurs hôtes. Dans ce contexte, mes travaux sont articulés autour de deux axes : (i) identifier et produire de nouveaux RBD dérivés des ERV et (ii) identifier de nouveaux transporteurs de type SLC reconnus par des RBD issus de rétrovirus exogènes et ERV de mammifères. Nous avons identifié et caractérisé deux nouveaux RBD humains endogènes (HERV-41 et HERV-89), entrés et conservés chez les primates de l’Ancien Monde il y a environ 35 millions d’années. Nous avons caractérisé leurs séquences PBS (Primer Binding Site), amorces putatives de la réplication rétrovirale, comme étant complémentaires de l’ARNtLeu ou ARNtArg pour HERV-89, et de l'ARNtGlu pour HERV-41. Les séquences env les plus proches dans le génome humain présentent respectivement 38% et 69% d'identité, indiquant l'appartenance de HERV-89 à deux nouvelles familles d'Env. Nous avons pu produire le RBD soluble de HERV-89, montrer que son récepteur est distinct de l'Env HERV ayant la séquence la plus homologue, et étudier sa distribution tissulaire. Le RBD HERV-89 lie un récepteur sur de nombreuses cellules souches et lignées cellulaires établies et nous avons montré par immunohistochimie que le récepteur est exprimé de manière différentielle dans les tissus humains sains et tumoraux. Parallèlement, nous avons dérivé une banque d'expression de 170 SLC que nous avons utilisée pour le criblage à haut-débit de récepteurs des Env gamma et deltarétrovirales. Cette banque nous a permis d'identifier le récepteur, longtemps recherché, de l’Env du virus de la leucémie bovine (BLV). De plus, en utilisant la transfection d'une banque d’expression d’ADNc dans des cellules de hamster, nous avons aussi identifié le récepteur du virus endogène félin ERV-DC14/FeLV-D comme étant le transporteur de cuivre et de cisplatine CTR1/SLC31A1.L’identification du récepteur de BLV pourrait notamment aider dans la lutte contre la transmission du virus et les pathologies associées qui affectent environ 5% du bétail infecté. De plus, les BLV-RBD et DC14-RBD constituent respectivement de nouveaux marqueurs et modulateurs potentiels du métabolisme, dont celui du cuivreRetroviruses are enveloped, single-stranded RNA viruses, that are omnipresent in animals and the causal agents of a large array of pathologies. Vertebrate retroviruses are divided into seven genera, including the γ and -retroviral groups, which we study particularly. Endogenous retroviruses (ERV), as opposed to exogenous infectious viruses, are present in germline cells and as such are bona fide components of the host genome, with Mendelian transmission. Most ERV have been inactivated by purifying mutations during evolution, although a few copies have been subjected to positive selection pressure with conserved open reading frames (ORFs).Exogenous viruses and ERV that belong to gamma and deltaretroviruses share similar genetic organization and their envelope glycoproteins (Env) comprises a transmembrane (TM) and a surface (SU) component, which binds a specific receptor on the host cell membrane. The SU contains a receptor-binding domain (RBD), responsible for receptor recognition, while TM engages membrane fusion and harbors an immunosuppressive domain. Noticeably, some ERVs have maintained entire or partial ORFs in env, which have been shown, in certain cases, to have essential physiological functions.Another common feature of gamma and deltaretroviral Env is the nature of their receptors, which, when identified, all belong to the solute carrier family of nutrient transporters (SLCs). The laboratory derived soluble RBDs from complete Env that can bind cognate receptors and be used to monitor SLC receptor expression at the cell surface. This important property of RBDs overcomes the notorious lack of reliable anti-SLC exofacial antibodies and provides a new way to evaluate, or even modulate, cell metabolism.Our laboratory postulates that some endogenous RBD-coding genes have been positively selected in their hosts for properties linked to binding SLCs and modulating host cell metabolism. In this context, the aim of my work was to: (i) search for new natural endogenous RBDs and (ii) characterize SLC transporters recognized by RBDs derived from ERVs or exogenous infectious mammalian retroviruses.Here, we describe the identification of two novel human endogenous RBDs (HERV-41 and HERV-89), which each harbor a significant ORF. We estimated that both RBDs have been introduced into Old World primate genomes 35 MYA ago, after the separation with New World monkeys. HERV-89 and HERV-41 are included within retroviral elements that comprise potential primer binding sites (PBS) complementary to tRNALeu or tRNAArg, for HERV-89, and tRNAGlu, for HERV-41. The envs of HERV-89 and HERV-41 do not share more than 38% and 69% amino acid identity with the closest known HERVs, respectively, which indicates that they belong to two new Env families. We derived a soluble HERV-89 RBD and monitored its receptor cell and tissue distribution. Using the ligand by flow cytometry, we observed that a HERV-89 receptor is expressed in a large panel of established cell lines and stem cells. Immunohistochemistry on 94 healthy and tumor human tissue samples showed that HERV-89 receptor is largely distributed, with distinct expression patterns in healthy and tumor tissues. In parallel, we derived a 170 gene-containing SLC expression library for high throughput screening of SLC/ligand interactions. Using this partial human SLC library, we identified the long-sought receptor for bovine leukemia virus (BLV). Moreover, transfection of a cDNA library expression into hamster cells, led us to identify CTR1/SLC31A1, the copper and cisplatin transporter, as the receptor for the feline ERV-DC14/FeLV-D.As a ligand for the BLV receptor, BLV-RBD may be used to help controlling BLV transmission and prevent associated pathologies that affect 5% of infected cattle. Also, BLV-RBD and DC14-RBD can now be used as metabolic markers and modulators of their SLC cognate receptors, including copper metabolism, in the case of DC14-RBD

Results of population differentiation tests for the Madagascar dataset analyses.

<p>The <i>p</i>-values associated with each of the two tests (Fdist2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032814#pone.0032814-Beaumont1" target="_blank">[43]</a> and DetSel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032814#pone.0032814-Vitalis1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032814#pone.0032814-Vitalis2" target="_blank">[45]</a> analyses) for each of the four population pairs are reported for the two MHC-linked loci: Msat-Tnf and D20Img2. The supposedly neutral microsatellites for which a significant result was obtained in the analyses are also indicated, together with their <i>p</i>-values (in the case of Fdist2 analysis, all the presumed neutral loci revealing significant had F_ST higher than expected).</p

List of the populations analyzed in this study.

<p>Sample sizes and genetic diversity (expected heterozygosity: H_E) are shown. C.H.: central highlands; L.A.: lowland areas.</p

Location of the sampled sites.

<p>For the Madagascar dataset, each population pair, consisting of one population from the plague focus and one from the plague-free zone, is identified by a different symbol. The principal Malagasy plague focus is outlined by a black dotted line and the region of Betafo is outlined in grey.</p

Characteristics of the five MHC-linked microsatellites loci analyzed in this study.

<p>The five loci are ranked as on the chromosome, with D20Rat21 the closest to the telomere.</p><p>The neighboring genes indicated are based on the <i>R. norvegicus</i> genome sequence.</p

Partial Mantel tests carried out on the Betafo dataset for each of the five MHC-linked microsatellites.

<p>Partial Mantel tests carried out on the Betafo dataset for each of the five MHC-linked microsatellites.</p