Analyses biochimiques de la recombinaison homologue méiotique chez schizosaccharomyces pombe

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010Les cassures double-brin de l'acide désoxyribonucléique (ADN) sont parmi les lésions les plus cytotoxiques car une seule lésion non réparée est létale chez la levure. Dans le but de réparer efficacement ces dommages, la cellule dispose de différents mécanismes tels que le Non Homologous End Joining (NHEJ). Toutefois ces mécanismes peuvent causer des mutations et, lorsqu' il est possible, la cellule privilégie un système qui permet une réparation très fiable: la recombinaison homologue. Ce processus peut aussi être utilisé lors de la méiose pour créer de la diversité génétique. La méiose est un mécanisme complexe et une mauvaise régulation peut mener à des problèmes tels que l' aneuploïdie. La recombinaison homologue méiotique se divise en quatre étapes majeures: (i) l' initiation qui crée des cassures double-brin par un complexe muItiprotéique incluant Rec12; (ii) la résection de l'ADN ou les protéines majeures sont Rad32/Rad50/Nbsl; (iii) l'invasion d'un duplex homologue avec les protéines RadSl et Dmcl, et pour terminer (iv) la résolution des jonctions de Holliday. Lors de mes études de doctorat, je me suis concentré sur la caractérisation biochimique des principales protéines des trois premières étapes (initiation, résection et particulièrement l'invasion) de la recombinaison méiotique chez Schizosaccharomyces pombe permettant la réparation des cassures double-brin. Mes travaux avaient pour but de caractériser les protéines Dmcl et le complexe Hop2/Mndl chez S.pombe. Nous avons déterminé que Dmcl avait comme Rad 51 la capacité de former un filament hélical sur l'ADN simple brin, ansi que de catalyser des réactions d'échange de brin. D'autre part j'ai mis en évidence le rôle que joue le complexe Hop2/Mndl dans la stimulation de Dmc 1, ainsi que les différences entre les protéines de S.pombe et de la SOurIS

Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model

It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity

Distinct expression profiles of TGF-β1 signaling mediators in pathogenic SIVmac and non-pathogenic SIVagm infections

BACKGROUND: The generalized T-cell activation characterizing HIV-1 and SIVmac infections in humans and macaques (MACs) is not found in the non-pathogenic SIVagm infection in African green monkeys (AGMs). We have previously shown that TGF-β1, Foxp3 and IL-10 are induced very early after SIVagm infection. In SIVmac-infected MACs, plasma TGF-β1 induction persists during primary infection [1]. We raised the hypothesis that MACs are unable to respond to TGF-β1 and thus cannot resorb virus-driven inflammation. We therefore compared the very early expression dynamics of pro- and anti-inflammatory markers as well as of factors involved in the TGF-β1 signaling pathway in SIV-infected AGMs and MACs. METHODS: Levels of transcripts encoding for pro- and anti-inflammatory markers (tnf-α, ifn-γ, il-10, t-bet, gata-3) as well as for TGF-β1 signaling mediators (smad3, smad4, smad7) were followed by real time PCR in a prospective study enrolling 6 AGMs and 6 MACs. RESULTS: During primary SIVmac infection, up-regulations of tnf-α, ifn-γ and t-bet responses (days 1–16 p.i.) were stronger whereas il-10 response was delayed (4(th )week p.i.) compared to SIVagm infection. Up-regulation of smad7 (days 3–8 p.i.), a cellular mediator inhibiting the TGF-β1 signaling cascade, characterized SIV-infected MACs. In AGMs, we found increases of gata-3 but not t-bet, a longer lasting up-regulation of smad4 (days 1–21 p.i), a mediator enhancing TGF-β1 signaling, and no smad7 up-regulations. CONCLUSION: Our data suggest that the inability to resorb virus-driven inflammation and activation during the pathogenic HIV-1/SIVmac infections is associated with an unresponsiveness to TGF-β1

Plasmacytoid Dendritic Cell Infection and Sensing Capacity during Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infection.

International audienceHuman immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques (MAC) lead to chronic inflammation and AIDS. Natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM), are protected against SIV-induced chronic inflammation and AIDS. Here, we report that AGM plasmacytoid dendritic cells (pDC) express extremely low levels of CD4, unlike MAC and human pDC. Despite this, AGM pDC efficiently sensed SIVagm, but not heterologous HIV/SIV isolates, indicating a virus-host adaptation. Moreover, both AGM and SM pDC were found to be, in contrast to MAC pDC, predominantly negative for CCR5. Despite such limited CD4 and CCR5 expression, lymphoid tissue pDC were infected to a degree similar to that seen with CD4(+) T cells in both MAC and AGM. Altogether, our finding of efficient pDC infection by SIV in vivo identifies pDC as a potential viral reservoir in lymphoid tissues. We discovered low expression of CD4 on AGM pDC, which did not preclude efficient sensing of host-adapted viruses. Therefore, pDC infection and efficient sensing are not prerequisites for chronic inflammation. The high level of pDC infection by SIVagm suggests that if CCR5 paucity on immune cells is important for nonpathogenesis of natural hosts, it is possibly not due to its role as a coreceptor. The ability of certain key immune cell subsets to resist infection might contribute to the asymptomatic nature of simian immunodeficiency virus (SIV) infection in its natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM). This relative resistance to infection has been correlated with reduced expression of CD4 and/or CCR5. We show that plasmacytoid dendritic cells (pDC) of natural hosts display reduced CD4 and/or CCR5 expression, unlike macaque pDC. Surprisingly, this did not protect AGM pDC, as infection levels were similar to those found in MAC pDC. Furthermore, we show that AGM pDC did not consistently produce type I interferon (IFN-I) upon heterologous SIVmac/HIV type 1 (HIV-1) encounter, while they sensed autologous SIVagm isolates. Pseudotyping SIVmac/HIV-1 overcame this deficiency, suggesting that reduced uptake of heterologous viral strains underlays this lack of sensing. The distinct IFN-I responses depending on host species and HIV/SIV isolates reveal the host/virus species specificity of pDC sensing

Immune activation in HIV infection: what can the natural hosts of simian immunodeficiency virus teach us?

International audienceThe review summarizes studies in natural hosts, with a particular focus on the control of immune activation and new insights into viral reservoirs. We discuss why these findings are relevant for HIV research today. AIDS resistance in natural hosts is characterized by a rapid control of inflammatory processes in response to simian immunodeficiency virus infection despite persistent viremia. Although CD4 T cells are dramatically depleted in the intestine in primary infection, interleukin 17-producing T helper cells (Th17) are preserved and natural hosts lack microbial translocation. Thus, viral replication in the gut is not sufficient to explain mucosal damage, but additional factors are necessary. Natural hosts also display a lower infection rate of stem-cell memory, central memory and follicular helper T cells. The follicles are characterized by a lack of viral trapping and the viral replication in secondary lymphoid organs is rapidly controlled. Hence, the healthy status of natural hosts is associated with preserved lymphoid environments. Understanding the underlying mechanisms of preservation of Th17 and of the low contribution of stem-cell memory, central memory and follicular helper T cells to viral reservoirs could benefit the search for preventive and curative approaches of HIV. Altogether, the complementarity of the model helps to identify strategies aiming at restoring full capacity of the immune system and decreasing the size of the viral reservoirs

African Non Human Primates Infected by SIV - Why Dont they Get Sick? Lessons from Studies on the Early Phase of Non-Pathogenic SIV Infection

International audienceAfrican non human primates are natural hosts of SIV. The infection is generally non-pathogenic despite high steady-state levels of plasma viral RNA that in HIV-1 and SIVmac infections are associated with progression towards AIDS. The viral loads in the gut also are as high as in pathogenic HIV-1/SIVmac infections; but replication levels are lower in peripheral lymph nodes of chronically infected African green monkeys. There is a transient loss of CD4(+) T cells in the blood in SIVagm and SIVsm infections and an early dramatic and more persistent decrease in the gut. Although SIV in natural hosts is thus cytopathic, the continuous viral replication is not associated with immunopathology. T CD4(+) cells in blood, lymph nodes and gut manifest no or little increase of cell-death by apoptosis. The lymph node and gut architecture is not disrupted. The most striking difference between non-pathogenic SIV and pathogenic HIV-1/SIVmac infections is the lack of chronic T cell activation. Several studies are currently in progress to determine which factors are involved in the maintenance of the low activation level in the non-pathogenic SIV infections. There are two ways in which this could be achieved: (i) a lack of immune activation induction or (ii) an active downregulation of the immune activation. The arguments in favor of each of these two possible ways of immune activation control will be discussed in view of the most recent data in the literature. A particular focus is put on data on the innate immune system and the timing of induction of immunosuppressive mediators during the early phase of SIV infection

Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes

Non-human primates in HIV research: Achievements, limits and alternatives.

International audienceAn ideal model for HIV-1 research is still unavailable. However, infection of non-human primates (NHP), such as macaques, with Simian Immunodeficiency Virus (SIV) recapitulates most virological, immunological and clinical hallmarks of HIV infection in humans. It has become the most suitable model to study the mechanisms of transmission and physiopathology of HIV/AIDS. On the other hand, natural hosts of SIV, such as African green monkeys and sooty mangabeys that when infected do not progress to AIDS, represent an excellent model to elucidate the mechanisms involved in the capacity of controlling inflammation and disease progression. The use of NHP-SIV models has indeed enriched our knowledge in the fields of: i) viral transmission and viral reservoirs, ii) early immune responses, iii) host cell-virus interactions in tissues, iv) AIDS pathogenesis, v) virulence factors, vi) prevention and vii) drug development. The possibility to control many variables during experimental SIV infection, together with the resemblance between SIV and HIV infections, make the NHP model the most appropriate, so far, for HIV/AIDS research. Nonetheless, some limitations in using these models have to be considered. Alternative models for HIV/AIDS research, such as humanized mice and recombinant forms of HIV-SIV viruses (SHIV) for NHP infection, have been developed. The improvement of SHIV viruses that mimic even better the natural history of HIV infection and of humanized mice that develop a greater variety of human immune cell lineages, is ongoing. None of these models is perfect, but they allow contributing to the progress in managing or preventing HIV infection