Analysis of Virion Structural Components Reveals Vestiges of the Ancestral Ichnovirus Genome

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts

Génome et facteurs de virulence d'un polydnavirus d'hyménoptère parasitoïde

The hymenoptera Cotesia congregata (Microgastrinae ; Braconidae) lays its eggs inside its host, a caterpillar of the lepidoptera Manduca sexta (Noctuidae ; Sphingidae) and introduces bracovirus (CcBV) particles, containing 30 double-stranded DNA circles. The viral genes carried by these circles encode for a range of proteins expressed in the tissues of the parasitized caterpillar. These viral proteins play an essential role for succesful parasitism. Viral gene expression leads to host physiology modifications such as alteration of immunity allowing the parasitoid larvae to develop. Additionnally, the parasitized host undergo a prewandering developmental arrest.The characterization of bracovirus genome has advanced and allowed the discovery of many gene families. The introduction of this thesis summarizes up-to-date knowledge about insect immunity and bracoviral genes potentially involved in host physiology control. During my thesis, sequencing and genome analyses demonstrated that the CcBV genome is composed of 30 double-stranded DNA circles coding for 9 multigenic families (Espagne et al 2004) comprising the protein tyrosine phosphatase (PTP) family (27 genes) (Provost et al 2004), the cystatin family (3 genes) (Espagne et al soumis) and the ankyrin motif encoding genes (6 genes) (Pennacchio et al en préparation). The detailed characterisation of the viral PTP gene family was performed. Field inversion gel electrophoresis (FIGE) allowed to physically localize PTP genes on the viral genome. Their expression was analysed on different host tissues by a multiplex RT PCR approach. Biochemical activity tests of 2 PTPs were performed by in vitro production using the baculovirus expression system.Globally, genes of the described families are expressed in the parasitized host and some proteins have the biochemical function predicted by their conserved domains, suggesting these proteins play a role in the host physiological modifications induced by parasitism.The characterization of viral genes expressed in the host is a key step toward further identification of the role of each protein in host physiology alteration.L'hyménoptère Cotesia congregata (Microgastrinae ; Braconidae) pond ses oeufs à l'intérieur de son hôte, la chenille du lépidoptère Manduca sexta (Noctuidae ; Sphingidae) et introduit des particules virales de bracovirus contenant 30 cercles d'ADN double brin. Les gènes viraux portés par ces cercles codent pour une série de protéines qui sont produites dans les tissus de la chenille parasitée. Ces protéines virales jouent un rôle indispensable à la réussite parasitaire. En effet, l'expression des gènes viraux entraîne de nombreuses altérations de la physiologie de l'hôte, notamment un contournement de l'immunité de la chenille qui permet le développement des larves du parasite. D'autre part, le développement de l'hôte est bloqué à un stade pré-pupal. Les travaux portant sur la caractérisation des génomes de bracovirus ont beaucoup progressé et plusieurs familles de gènes ont été découvertes. Une synthèse des connaissances actuelles sur l'immunité des insectes et les gènes de bracovirus potentiellement impliqués dans le contrôle de l'immunité et du développement des lépidoptères est présentée en introduction.Au cours de ma thèse, le séquençage et l'analyse du génome du bracovirus de Cotesia congregata ont été réalisés (Espagne et al 2004). L'existence de plusieurs familles multigéniques a été mise en évidence, notamment la famille des protéines tyrosines phosphatases (PTP) composée de 27 gènes (Provost et al 2004), la famille des cystatines composée de 3 gènes (Espagne et al soumis) et enfin celle des protéines à motif ankyrine composée de 6 gènes (Pennacchio et al en préparation). La caractérisation détaillée de la famille des PTP a été effectuée. La technique d'électrophorèse en champs inversés (FIGE) a permis la localisation physique de ces gènes sur l'ensemble du génome viral, et leur expression a été analysée dans une série de tissus de l'hôte parasité grâce à une méthode de PCR multiplex. Enfin, des tests d'activité biochimique de PTP de bracovirus produites in vitro grâce à un système d'expression en baculovirus.Les gènes des familles décrites sont exprimés dans l'hôte parasité et les protéines possèdent, en général, la fonction biochimique prédite grâce aux domaines conservés qu'elles contiennent. Ceci suggère que ces protéines virales jouent un rôle actif dans les modifications de la physiologie de l'hôte induite par le parasitisme. La caractérisation des gènes viraux exprimés dans l'hôte est une étape indispensable vers l'identification du rôle individuel de chaque protéine dans le contrôle de la physiologie des chenilles parasitées

Génome et facteurs de virulence d'un polydnavirus d'hyménoptère parasitoïde

The hymenoptera Cotesia congregata lays its eggs into its host, a caterpillar of the lepidoptera Manduca sexta and introduces bracovirus (CcBV) particles. The proteins encoded by viral genes expressed in the parasitized host play an essential role for succesful parasitism, leading to modifications of host physiology such as alteration of immunity and prewandering developmental arrest. The introduction of this thesis summarizes up-to-date knowledge about insect immunity and bracoviral genes potentially involved in host physiology control. Sequencing and genome analyses demonstrated that the CcBV genome is composed of 30 double-stranded DNA circles coding for 9 multigenic families comprising protein tyrosine phosphatases, cystatins and ankyrin motif containing proteins. The characterisation of the viral genes expressed in the host is a key step toward further identification of the role of each protein in host physiology alteration. Field inversion gel electrophoresis (FIGE) allowed to physically localize PTP genes on the viral genome. Their expression was analysed on different host tissues by a multiplex RT PCR method. Next, biochemical activity tests of 2 PTPs were performed. Globally, genes of the described families are expressed in the parasitized host and some proteins have the biochemical function predicted by their conserved domains, suggesting these proteins play a role in the host physiological modifications induces by parasitism.L'hyménoptère Cotesia congregata pond dans un hôte, la chenille du lépidoptère Manduca sexta et introduit des particules virales de bracovirus (CcBV). Les protéines codées par les gènes viraux exprimées dans l'hôte parasité jouent un rôle indispensable à la réussite parasitaire, entraînant des modifications de la physiologie de l'hôte, notamment une altération de l'immunité et un arrêt du développement au stade pré-pupal. Une synthèse des connaissances actuelles sur l'immunité des insectes et les gènes de bracovirus potentiellement impliqués dans le contro le de la physiologie des hôtes est présentée en introduction. Le séquençage et l'analyse du génome de CcBV ont montré que ce génome est composé de 30 cercles d'ADN double-brin porteurs de 9 familles multigéniques : la famille des protéines tyrosines phosphatases (PTP), des cystatines, des protéines ankyrines... La caractérisation des gènes viraux exprimés dans l'hôte est une étape indispensable vers l'identification du rôle individuel de chaque protéine dans le contrôle de la physiologie de l'hôte. La technique d'électrophorèse en champs inversés (FIGE) a permis la localisation physique sur l'ensemble du génome viral des gènes de PTP. Leur expression a été analysée dans différents tissus de l'hôte parasité par une méthode de PCR multiplex. Puis des tests d'activité biochimique de 2 PTP ont été menés. Les gènes des familles décrites sont exprimés dans l'hôte parasité et certaines protéines possèdent la fonction biochimique prédite par les domaines conservés, suggérant que ces protéines jouent un rôle dans la modification de la physiologie de l'hôte induite par le parasitisme.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Bracoviruses Contain a Large Multigene Family Coding for Protein Tyrosine Phosphatases

The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The virions produced in the wasp ovaries are injected into host lepidopteran larvae, where virus genes are expressed, allowing successful development of the parasite by inducing host immune suppression and developmental arrest. Bracovirus-bearing wasps have a common phylogenetic origin, and contemporary bracoviruses are hypothesized to have been inherited by chromosomal transmission from a virus that originally integrated into the genome of the common ancestor wasp living 73.7 ± 10 million years ago. However, so far no conserved genes have been described among different braconid wasp subfamilies. Here we show that a gene family is present in bracoviruses of different braconid wasp subfamilies (Cotesia congregata, Microgastrinae, and Toxoneuron nigriceps, Cardiochilinae) which likely corresponds to an ancient component of the bracovirus genome that might have been present in the ancestral virus. The genes encode proteins belonging to the protein tyrosine phosphatase family, known to play a key role in the control of signal transduction pathways. Bracovirus protein tyrosine phosphatase genes were shown to be expressed in different tissues of parasitized hosts, and two protein tyrosine phosphatases were produced with recombinant baculoviruses and tested for their biochemical activity. One protein tyrosine phosphatase is a functional phosphatase. These results strengthen the hypothesis that protein tyrosine phosphatases are involved in virally induced alterations of host physiology during parasitism

Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses

International audienceThe Polydnaviridae is a family of double-stranded DNA viruses that are symbionts of parasitoid wasps. The family is currently divided into two genera, the Ichnovirus (IV) and Bracovirus (BV), which are associated with wasps in the families Ichneumonidae and Braconidae, respectively. IVs and BVs have similar immunosuppressive and developmental effects on parasitized hosts but their encapsidated genomes largely encode different genes. To assess whether IV and BV infection has similar or disparate effects on the transcriptome of shared hosts, we characterized the effects of Hyposoter didymator Ichnovirus (HdIV) and Microplitis demolitor Bracovirus (MdBV) on the fat body and hemocyte transcriptome of Spodoptera frugiperda (Lepidoptera: Noctuidae). Our results indicated that HdIV and MdBV infection alters the abundance of a relatively low proportion of S. frugiperda transcripts at 24 h post-infection. A majority of the transcripts affected by infection also differed between MdBV and HdIV. However, we did identify some host transcripts that were similarly affected by both viruses. A majority of these genes were transcribed in the fat body and most belonged to functional classes with roles in immunity, detoxification, or cell structure. Particularly prominent in this suite of transcripts were genes encoding for predicted motor-related and collagen IV-like proteins. Overall, our data suggest that the broadly similar effects that HdIV and MdBV have on host growth and immunity are not due to these viruses inducing profound changes in host gene expression. Given though that IVs and BVs encode few shared genes, the host transcripts that are similarly affected by HdIV and MdBV could indicate convergence by each virus to target a few processes at the level of transcription that are important for successful parasitism of hosts by H. didymator and M. demolitor

Homozygous Deletion in the Coding Sequence of the c-mer Gene in RCS Rats Unravels General Mechanisms of Physiological Cell Adhesion and Apoptosis

International audienceThe RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy؊ and RCS rdy؉ cDNAs were sequenced. The RCS rdy؊ cDNAs carried a significant deletion in the 5 part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties