Bone marrow stromal antigen 2 (BST-2/tetherin) is a type II transmembrane protein that plays various roles, including protective and detrimental roles in the host. Cellular responses to BST-2 expression or the lack thereof, may be cell type and context-dependent and may vary with time. When protective, BST-2 functions as an antiviral factor, renowned for its ability to tether budding enveloped viruses to the membrane of infected cells. Tethering of budding virions prevents their release into the extracellular milieu limiting infection of naïve cells. The antiviral role of BST-2 has been predominantly studied using cultured cells. Insight into the role of BST-2 in inhibition of viral infection in vivo came from our study of the alphavirus Chikungunya virus (CHIKV) and the retrovirus mouse mammary tumor virus, (MMTV). BST-2 prevents the release of CHIKV and MMTV virions from infected cells and limits the replication of both viruses in mice. In the context of CHIKV infection, BST-2 protects the host in a tissue-type dependent manner. In lymphoid and most non-lymphoid tissues, expression of BST-2 limits CHIKV replication. In addition, BST-2 regulates CHIKV-induced inflammatory responses in mice, an indication that BST-2 may function to initiate and amplify innate immune responses. Host response to MMTV infection depends on the stage of the infection and disease sequela. Acute infection of immune cells with MMTV results in an initial increase in BST-2 expression followed by a sharp decline. In contrast, in MMTV-induced mammary tumors, BST-2 mRNA and protein are elevated, so is the viral load. This is an indication that the antiviral role of BST-2 is not operative once mammary tumors have developed. These data provided the initial evidence that BST-2 may promote breast cancer progression. Indeed, data from two mouse models of breast cancer show that expression of BST-2 is necessary for cell to cell and cell to extracellular matrix interactions. Thus, BST-2 expression in breast cancer cells enhances cancer cell adhesion, anchorage-independency, migration, and invasion, culminating in increased tumor mass, increased metastases, and reduced host survival. Structurally, BST-2 homodimerization is important for its cancer-promoting role as dimers of BST-2 regulate anchorage-independency, resistance to anoikis, and enhanced adhesion between cancer cells and components (proteins and cells) of the tumor microenvironment. How BST-2 is enriched in breast cancer cells was elusive until our in silico analyses of a large human breast cancer dataset that revealed the involvement of epigenetic regulation of BST-2 in breast tumors. In highly aggressive breast cancers, specific CpG sites in and at close proximity to the BST-2 promoter are hypomethylated. This is in sharp contrast to non-aggressive luminal cancers and normal breast epithelial cells. These data suggest that a progressive loss of methylation on the BST-2 gene may contribute to constitutive overexpression of BST-2 in tumors. Overall, these findings show that 1) BST-2 contributes to the emergence and progression of breast malignancies and may be used as a therapeutic target or as a biomarker for aggressive breast cancers; and, 2) BST-2 acts as a viral sensor to initiate antiviral inflammatory responses and could be exploited therapeutically to treat viral infections. We highlight the need for additional research on the antiviral and cancer-promoting roles of BST-2 to reconcile both functions for the purpose of therapeutics.

Fatores de restrição são as primeiras proteínas celulares envolvidas no combate a infecções virais, são considerados uma defesa intrínseca das células, constituindo-se em uma rápida resposta frente a invasão de patógenos. Essas moléculas são bastante diversas e são capazes de interferir em algum ponto do ciclo viral, atenuando ou bloqueando a evolução da infecção. Após a descoberta da existência desses fatores, alguns estudos têm direcionado o foco para as possíveis alterações genéticas que podem influenciar a estrutura dessas proteínas e, deste modo, interferir sobre suscetibilidade e progressão de doenças infecciosas, como a infecção pelo HIV/aids. O objetivo do presente estudo foi avaliar três SNPs de três diferentes fatores de restrição (o TRIM5α – rs10838525, a APOBEC3F – rs2076101 e o CUL5 – rs7117111) e observar suas frequências em diferentes grupos étnicos, bem como a associação desses fatores com a suscetibilidade ao HIV e a progressão a aids, em um grupo de soronegativos e soropositivos. Foram selecionados 345 indivíduos HIV+ atendidos no setor de Infectologia do Hospital Nossa Senhora da Conceição e 324 indivíduos HIV– doadores de sangue do Hospital de Clínicas de Porto Alegre. Os SNPs foram identificados através da técnica de PCR TaqManTM. O teste qui-quadrado foi utilizado para a análise das frequências e por regressão logística univariada foi avaliado o OR com 95% de IC entre os modelos dominantes e recessivos. Entre os SNPs estudados apenas o rs7117111 apresentou resultado estatisticamente significativo para o genótipo GG em relação a proteção ao HIV-1 (OR 0,661, IC 95% 0,449-0,974, P=0,036) e esse mesmo genótipo, também, parece estar relacionado aos progressores rápidos, pois apresentou uma tendência nessa relação quando ajustado pela etnia (OR ajustado 2,115, IC 95% 0,990- 4,520, P=0,053). Tais achados demonstram que alterações genéticas, especificamente no gene CUL5, podem influenciar a suscetibilidade ao HIV-1 e podem, também, interferir na progressão a aids. Esses resultados geram questionamentos de grande valia para um maior entendimento da influência genética do sistema de defesa intrínseco celular no curso da infecção. / Host restriction factors are the first cellular proteins engaged in antiviral response, they are considerate an intrinsic cell defense with the aim to be a rapid answer against the invasion of pathogens. This molecules have a vary diversity in structure and each one act in a distinct stages of viral life cycle, however always with the same objective to attenuate or block the infection. After the discovery of this restriction factors, some researches focus in looking for genetic variation that can be influence in structure protein and with this way interfere in HIV susceptibility or progress to AIDS. The aim of present work was evaluate tree SNPs of tree different restrictions factors (TRIM5α – rs10838525, APOBEC3F – rs2076101 and CUL5 – rs7117111) and detect yours frequencies in different ethics group, as well as, evaluate the SNPs`s capacity in influence the susceptibility to HIV and progress to AIDS in a seronegative and seropositive groups. For this research was selected 345 samples of HIV+ individuals from the Infectology sector of Nossa Senhora da Conceição hospital and 324 HIV- samples from blood donors of Clinics Hospital of Porto Alegre. Through PCR TaqManTM assay the SNPs was genotyping. The qui-square test was used to analyze the frequencies and by unvaried logistic regression was estimate the OR with 95% CI to dominant and recessive models. Between the tree SNPs chosen only rs7117111 was statistically significant the GG genotype with the HIV-1 protection (GG, OR: 0,661, 95% CI 0,449-0,974, P=0.036) and this same genotype seems to be to related with rapid progress to AIDS, because the result shows a tendency when adjusted for ethnicity in the recessive model (adjusted OR 2,115, IC 95% 0,990-4,520, P=0,053). This finds shows the genetics alterations, specify in the CUL5 gene, can alter the susceptibility to HIV-1 and can interfere in the progress to AIDS. Theses results are also important for the understanding of the genetic alterations in the host antiviral intrinsic mechanisms anti-HIV and can bring new insights for strategies against HIV pandemic.

Environ 8% du génome humain est constitué de rétrovirus endogènes (HERV). La famille de bétarétrovirus HERV-K(HML2), l'une des plus actives chez l'homme, est entrée il y a 45 millions d'années dans le génome des primates et s'est amplifiée efficacement depuis, et ce malgré l'existence de nombreuses protéines cellulaires, appelées facteurs de restriction, qui s'opposent à la réplication du virus dans la cellule hôte. La Tetherin/BST2, l'un d'entre eux, est une protéine membranaire capable de bloquer le relargage des virions dans le milieu extracellulaire et est active sur la plupart des virus enveloppés testés jusqu'à présent, en particulier HERV-K(HML2). Nous avons tout d'abord mis en évidence que l'enveloppe (Env) de la famille HML2 est un antagoniste de la Tetherin, propriété qui a pu contribuer au succès de l'amplification de la famille HERV¬K(HML2) dans les génomes. Plusieurs domaines de l'enveloppe coopèrent pour s'opposer à l'action du facteur de restriction : la SU (domaine d'interaction), ainsi que la partie transmembranaire, alors que la queue cytoplasmique n'est pas indispensable. Le mécanisme de cette inhibition n'a pas été encore complètement élucidé, mais l'on sait, comme pour la glycoprotéine d'Ebola, que l'Env HERV-K(HML2) n'induit ni relocalisation, ni dégradation de la Tetherin. Etant donné le grand polymorphisme insertionnel de la famille HERV-K(1-IML2), il est très probable que cette activité anti-Tetherin endogène soit variable entre les individus, ce qui pourrait avoir des conséquences dans les pathologies où les éléments HERV-K(HML2) sont spécifiquement induits. Parmi ces pathologies, les cancers de la peau, du sein et de la lignée germinale présentent une association particulièrement forte avec l'expression de l'Env HERV-K(HML2), que nous avons voulu mieux comprendre dans la suite de ces travaux de thèse. Nous avons dans un premier temps montré que l'expression de l'Env dans des cellules humaines non transformées de l'épithélium de sein (MCF10A), induit la transition vers un phénotype mésenchymateux (EMT, transition épithélio-mésenchymateuse), caractéristique de l'apparition de métastases dans les cancers. Cette transition est associée à une augmentation de la mobilité des cellules (mise en évidence dans des tests Transwell), à un changement de morphologie des cellules et à une modification du profil d'expression de quelques marqueurs moléculaires caractéristiques (E-cadherin, N-cadherin, vimentin, fibronectin). Grâce à une étude transcriptomique en cellules 293T, nous avons mis en évidence que l'expression de l'Env HERV-K induit fortement plusieurs facteurs de transcription : ETV4, ETVS, ainsi que EGR1, qui ont été identifiés comme des marqueurs du processus de tumorigénèse dans différents modèles. Nous avons également montré que l'Env HERV-K active la voie des MAP kinases via ERK 1/2 —dérégulée dans un grand nombre de cancers- en amont de la kinase Raf. Ces phénomènes d'induction de la transduction de signal requièrent la présence de la queue cytoplasmique de l'enveloppe. De façon remarquable, seule l'enveloppe du bétarétrovirus de mouton JSRV, oncogénique in vivo, est capable d'activer les mêmes voies de signalisation, ce qui renforce l'hypothèse d'une implication de l'Env HERV-K(HML2) dans la tumorigenèse. / Human endogenous retroviruses (HERV) represent about 8% of our genomic content. HERV-K(HML2) betaretroviral family is one of the most active in humans. Although it entered 45 million years ago in the primate genomes, its members have amplified quite recently despite the existence of restriction factors, which are host proteins blocking viral replication in cells. Tetherin/BST2 is one of them and acts by keeping the viral particles attached to the cell surface. It targets most enveloped viruses tested so far including HERV-K(HML2). We show that the envelope protein (Env) of HML2 family is an antagonist of Tetherin retriction, property that probably helped the endogenous retrovirus to efficiently amplify in the genomes. We mapped several domains required for antagonism : the surface subunit of Env (SU), which interacts with Tetherin, and the transmembrane. We also show that the cytoplasmic tail is dispensable for counteraction. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate the restriction factor. Due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. This could have functional consequences in pathologies where HERV-K(HML2) is specifically induced. Among them, melanomas, breast cancers and germ line tumors display a strong association with HML2 Env expression, that we wanted to better analyse. We first show that Env expression in a model of epithelial human breast cancer cells induces the so-called EMT (epithelial mesenchymal transition), critical for cancer progression and the process of metastasis. This includes enhanced migratory capacities (shown by transwell assays), changes in cell morphology and characteristic modifications in a set of molecular markers (e.g. E-cadherin, N-cadherin, vimentin, fibronectin). Microarray experiments performed in 293T cells revealed that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETVS and EGRI, which have been associated with cellular transformation. Importantly, we also show that HERV-K(HML2) Env activates the MAP kinase pathway via ERK 1/2, key player in numerous cancers. This induction occurs upstream of the kinase Raf and involves the cytoplasmic tail of HERV-K(HML2) Env. In addition, this phenomenon is very specific, being absent with every other Env tested, except for JSRV Env which is already known to have transforming properties in vivo.

Fatores de restrição são as primeiras proteínas celulares envolvidas no combate a infecções virais, são considerados uma defesa intrínseca das células, constituindo-se em uma rápida resposta frente a invasão de patógenos. Essas moléculas são bastante diversas e são capazes de interferir em algum ponto do ciclo viral, atenuando ou bloqueando a evolução da infecção. Após a descoberta da existência desses fatores, alguns estudos têm direcionado o foco para as possíveis alterações genéticas que podem influenciar a estrutura dessas proteínas e, deste modo, interferir sobre suscetibilidade e progressão de doenças infecciosas, como a infecção pelo HIV/aids. O objetivo do presente estudo foi avaliar três SNPs de três diferentes fatores de restrição (o TRIM5α – rs10838525, a APOBEC3F – rs2076101 e o CUL5 – rs7117111) e observar suas frequências em diferentes grupos étnicos, bem como a associação desses fatores com a suscetibilidade ao HIV e a progressão a aids, em um grupo de soronegativos e soropositivos. Foram selecionados 345 indivíduos HIV+ atendidos no setor de Infectologia do Hospital Nossa Senhora da Conceição e 324 indivíduos HIV– doadores de sangue do Hospital de Clínicas de Porto Alegre. Os SNPs foram identificados através da técnica de PCR TaqManTM. O teste qui-quadrado foi utilizado para a análise das frequências e por regressão logística univariada foi avaliado o OR com 95% de IC entre os modelos dominantes e recessivos. Entre os SNPs estudados apenas o rs7117111 apresentou resultado estatisticamente significativo para o genótipo GG em relação a proteção ao HIV-1 (OR 0,661, IC 95% 0,449-0,974, P=0,036) e esse mesmo genótipo, também, parece estar relacionado aos progressores rápidos, pois apresentou uma tendência nessa relação quando ajustado pela etnia (OR ajustado 2,115, IC 95% 0,990- 4,520, P=0,053). Tais achados demonstram que alterações genéticas, especificamente no gene CUL5, podem influenciar a suscetibilidade ao HIV-1 e podem, também, interferir na progressão a aids. Esses resultados geram questionamentos de grande valia para um maior entendimento da influência genética do sistema de defesa intrínseco celular no curso da infecção. / Host restriction factors are the first cellular proteins engaged in antiviral response, they are considerate an intrinsic cell defense with the aim to be a rapid answer against the invasion of pathogens. This molecules have a vary diversity in structure and each one act in a distinct stages of viral life cycle, however always with the same objective to attenuate or block the infection. After the discovery of this restriction factors, some researches focus in looking for genetic variation that can be influence in structure protein and with this way interfere in HIV susceptibility or progress to AIDS. The aim of present work was evaluate tree SNPs of tree different restrictions factors (TRIM5α – rs10838525, APOBEC3F – rs2076101 and CUL5 – rs7117111) and detect yours frequencies in different ethics group, as well as, evaluate the SNPs`s capacity in influence the susceptibility to HIV and progress to AIDS in a seronegative and seropositive groups. For this research was selected 345 samples of HIV+ individuals from the Infectology sector of Nossa Senhora da Conceição hospital and 324 HIV- samples from blood donors of Clinics Hospital of Porto Alegre. Through PCR TaqManTM assay the SNPs was genotyping. The qui-square test was used to analyze the frequencies and by unvaried logistic regression was estimate the OR with 95% CI to dominant and recessive models. Between the tree SNPs chosen only rs7117111 was statistically significant the GG genotype with the HIV-1 protection (GG, OR: 0,661, 95% CI 0,449-0,974, P=0.036) and this same genotype seems to be to related with rapid progress to AIDS, because the result shows a tendency when adjusted for ethnicity in the recessive model (adjusted OR 2,115, IC 95% 0,990-4,520, P=0,053). This finds shows the genetics alterations, specify in the CUL5 gene, can alter the susceptibility to HIV-1 and can interfere in the progress to AIDS. Theses results are also important for the understanding of the genetic alterations in the host antiviral intrinsic mechanisms anti-HIV and can bring new insights for strategies against HIV pandemic.

Fatores de restrição são as primeiras proteínas celulares envolvidas no combate a infecções virais, são considerados uma defesa intrínseca das células, constituindo-se em uma rápida resposta frente a invasão de patógenos. Essas moléculas são bastante diversas e são capazes de interferir em algum ponto do ciclo viral, atenuando ou bloqueando a evolução da infecção. Após a descoberta da existência desses fatores, alguns estudos têm direcionado o foco para as possíveis alterações genéticas que podem influenciar a estrutura dessas proteínas e, deste modo, interferir sobre suscetibilidade e progressão de doenças infecciosas, como a infecção pelo HIV/aids. O objetivo do presente estudo foi avaliar três SNPs de três diferentes fatores de restrição (o TRIM5α – rs10838525, a APOBEC3F – rs2076101 e o CUL5 – rs7117111) e observar suas frequências em diferentes grupos étnicos, bem como a associação desses fatores com a suscetibilidade ao HIV e a progressão a aids, em um grupo de soronegativos e soropositivos. Foram selecionados 345 indivíduos HIV+ atendidos no setor de Infectologia do Hospital Nossa Senhora da Conceição e 324 indivíduos HIV– doadores de sangue do Hospital de Clínicas de Porto Alegre. Os SNPs foram identificados através da técnica de PCR TaqManTM. O teste qui-quadrado foi utilizado para a análise das frequências e por regressão logística univariada foi avaliado o OR com 95% de IC entre os modelos dominantes e recessivos. Entre os SNPs estudados apenas o rs7117111 apresentou resultado estatisticamente significativo para o genótipo GG em relação a proteção ao HIV-1 (OR 0,661, IC 95% 0,449-0,974, P=0,036) e esse mesmo genótipo, também, parece estar relacionado aos progressores rápidos, pois apresentou uma tendência nessa relação quando ajustado pela etnia (OR ajustado 2,115, IC 95% 0,990- 4,520, P=0,053). Tais achados demonstram que alterações genéticas, especificamente no gene CUL5, podem influenciar a suscetibilidade ao HIV-1 e podem, também, interferir na progressão a aids. Esses resultados geram questionamentos de grande valia para um maior entendimento da influência genética do sistema de defesa intrínseco celular no curso da infecção. / Host restriction factors are the first cellular proteins engaged in antiviral response, they are considerate an intrinsic cell defense with the aim to be a rapid answer against the invasion of pathogens. This molecules have a vary diversity in structure and each one act in a distinct stages of viral life cycle, however always with the same objective to attenuate or block the infection. After the discovery of this restriction factors, some researches focus in looking for genetic variation that can be influence in structure protein and with this way interfere in HIV susceptibility or progress to AIDS. The aim of present work was evaluate tree SNPs of tree different restrictions factors (TRIM5α – rs10838525, APOBEC3F – rs2076101 and CUL5 – rs7117111) and detect yours frequencies in different ethics group, as well as, evaluate the SNPs`s capacity in influence the susceptibility to HIV and progress to AIDS in a seronegative and seropositive groups. For this research was selected 345 samples of HIV+ individuals from the Infectology sector of Nossa Senhora da Conceição hospital and 324 HIV- samples from blood donors of Clinics Hospital of Porto Alegre. Through PCR TaqManTM assay the SNPs was genotyping. The qui-square test was used to analyze the frequencies and by unvaried logistic regression was estimate the OR with 95% CI to dominant and recessive models. Between the tree SNPs chosen only rs7117111 was statistically significant the GG genotype with the HIV-1 protection (GG, OR: 0,661, 95% CI 0,449-0,974, P=0.036) and this same genotype seems to be to related with rapid progress to AIDS, because the result shows a tendency when adjusted for ethnicity in the recessive model (adjusted OR 2,115, IC 95% 0,990-4,520, P=0,053). This finds shows the genetics alterations, specify in the CUL5 gene, can alter the susceptibility to HIV-1 and can interfere in the progress to AIDS. Theses results are also important for the understanding of the genetic alterations in the host antiviral intrinsic mechanisms anti-HIV and can bring new insights for strategies against HIV pandemic.

HIV-2 differs from HIV-1 in that many infected people experience normal survival, whilst only 20% progress rapidly to AIDS. Understanding mechanisms of delayed HIV-2 disease progression could provide new insights into HIV control. The Caio Community Cohort was established in Guinea-Bissau in the setting of high HIV-2 prevalence. This thesis investigates the role of polymorphic host restriction factors of the TRIM family in HIV-2 outcome. TRIM proteins are a family of E3 ubiquitin-ligases, where closely-related TRIM5α and TRIM22 are thought to inhibit HIV-1 transcription, uncoating and budding. There was an association between TRIM5α amino acid substitution R136Q and reduced HIV-2 viral load/prolonged survival. Conversely, P479L was enriched among HIV-2 infected participants and progressors with CD4+ T cell decline. TRIM22 was highly polymorphic in this cohort, revealing three novel coding variants. Although most substitutions were located in the putative virus-interacting PRYSPRY domain, two in the coiled-coil, D155N and R242T, showed significant and divergent associations with survival. R242T was enriched in HIV-2 infected participants, who progressed to death at twice the rate of wild-type controls. In silico studies predicted D282, D360, and R321 of TRIM22 to be highly conserved, exposed residues, for which polymorphisms would be deleterious. When aligned with sequences from the potent HIV-1 restriction factor, rhesus macaque TRIM5α, TRIM22 substitutions R321K, T415I, and D360Y were spatially relevant to residues involved in HIV-1 restriction. The role of TRIM22 in HIV restriction was supported by in vitro pilot studies showing that TRIM22 was upregulated by HIV-1 infection in a lymphoid cell line and co-localised with the HIV-1 capsid protein p24. Overexpression of TRIM22 resulted in the restriction of VSV-G pseudotyped HIV-1 and SIVmac. The R242T substitution diminished TRIM22's restriction of HIV-1 and SIVmac: protein analysis suggested that this may be due to the inability of the R242T mutant to fully dimerise.

No description available.

Viral protein U (Vpu) is an accessory protein of HIV‐1 that efficiently targets BST2/Tetherin, a cellular restriction factor that acts as molecular anchor impeding the release of various enveloped viruses from the cell surface. The recently discovered natural receptor of BST2 is ILT7, a molecule exclusively expressed at the surface of the professional type 1 interferon (IFN‐1) producing cells, plasmacytoid dendritic cells (pDCs). The interaction between BST2 and ILT7 has been reported to efficiently induce a repression of IFN­‐1 secretion by pDCs. Here, we investigated the impact of Vpu mediated antagonism of BST2, in regards to this newly described immune function of BST2. Using a system of CD4+ T cell lines infected with wild type or Vpu‐deficient HIV-­1 cultured with peripheral blood mononuclear cells or purified pDCs, we report that the presence of Vpu efficiently reduces IFN-­1 production from sensing pDCs. Furthermore, we observed that this Vpu effect is dependent on the availability of BST2 molecules at the surface of the infected cells, since the Vpu's immunoregulation is abrogated when blocking any potential BST2 trans interaction with anti­‐BST2 antibodies. Similarly, depleting ILT7 from pDCs by means of small interfering RNA treatment equally negates the downregulation of pDC IFN-­1 secretion by Vpu. Finally, the use of recombinant soluble ILT7 competes with pDC‐bound ILT7 for the free BST2 and similarly results in high IFN-­1 production, causing an identical phenotype. Overall, our results demonstrate that Vpu heightens ILT7 activation and subsequent repression of IFN‐1 production by pDCs in response to HIV­‐1 infected CD4+ T cells by promoting it's trans interaction with infected T cell bound BST2, through a yet uncharacterized mechanism. By allowing efficient particle release and restraining pDCs antiviral functions, Vpu exerts a double role on BST2 that seems crucial for the replication and dissemination of HIV‐1. / La protéine accessoire U (Vpu) du VIH‐1 cible efficacement BST2/Tetherin, facteur de restriction restreignant la relâche de divers virus enveloppés à même la surface cellulaire. Le récepteur naturel de BST2 récemment découvert est ILT7, une protéine exclusivement exprimée à la surface des cellules produisant l'essentiel de l'interféron de type 1 (IFN-­1) lors d'infections virales, les cellules dendritiques plasmacytoïdes (pDCs). L'interaction entre BST2 et ILT7 réprime la production d'IFN‐1 des pDCs. Considérant le potentiel immunorégulateur de BST2 récemment décrit, nous avons entrepris d'évaluer cet aspect de l'antagonisme de Vpu sur BST2. À l'aide d'un système de co­‐culture entre une lignée de cellules T CD4+ infectée avec un virus exprimant ou n'exprimant pas Vpu et les cellules mononucléées du sang périphérique ou de pDCs purifiés, nous avons observé que Vpu est responsable d'une atténuation majeure de la sécrétion d'interféron de type 1 (IFN-­1) produite en réponse aux cellules infectées. La présence de molécules de BST2 de surface libres est essentielle à ce processus, puisque le bloc de toute interaction en trans de BST2 par des anticorps polyclonaux α­‐BST2 abroge l'effet de Vpu. Similairement, Vpu ne peut exercer cet effet lorsque ILT7 est déplété dans les pDCs à l'aide de petits ARN interférents. Enfin, l'introduction de protéines recombinantes solubles d'ILT7 dans le système de co-culture semble prévenir l'effet inhibiteur de Vpu, suggérant que Vpu exploite l'interaction de BST2 avec ILT7 pour moduler la sécrétion d'IFN-­1 des pDCs. En conclusion, nos résultats démontrent que Vpu exerce un contrôle sophistiqué de la production d'IFN‐1 par les pDCs en réponse aux lymphocytes T CD4+ infectés par le VIH-­1. Il semble ainsi que l'action de Vpu favorise, par un mécanisme encore méconnu, l'activation d'ILT7 à travers BST2. En effet, cette fonction de Vpu semble tout aussi dépendante de BST2 que de l'ILT7. En favorisant la relâche virale et en menant à l'inhibition de la réponse antivirale des pDCs, la régulation ciblée de BST2 par Vpu est non seulement cruciale à la dissémination du virus, mais aussi à sa réplication.

The shortage of human tissues and organs for allotransplantation can be overcome by xenotransplantation. As a source of organs, the miniature pig is convenient. However, the presence of pathogens transmissible to the recipients, especially porcine endogenous retrovirus (PERV), represents a threat for successfull xenotransplantation. Infectious PERVs contain three classes of envelope glycoprotein. Two classes, PERV-A and PERV-B are polytropic, they can infect human, pig and mink cells in vitro. PERV-C is evolutionary young, ecotropic isolate that can infect pig only. We previously detected a new full-lenght, but replication-defective PERV-A isolate dubbed (MAMBA) with high transcriptional activity in Large-White pig from a Czech breed. To support our results with PERV-MAMBA epigenetic regulation in pig tissues, in vitro DNA methylation essay was accomplished. Methylated or non-methylated reporter plasmids containing provirus 5' LTR were transfected into 293T cells and luciferase activity was measured. In both cases, methylated LTR decreased significantly expression of luciferase. Thus, PERV LTR-driven transcription is sensitive to DNA methylation. We also used PERV-A MAMBA provirus to study recombination between two pig endogenous retroviruses. We prepared 293T and BeWo cell clones harboring PERV-A...

La protéine Vif (Viral infectivity factor) joue un rôle essentiel dans la réplication virale et dans le pouvoir infectieux du VIH-1. Elle est aussi qualifiée de « protéine de contre-défense » car elle neutralise un facteur cellulaire à action antivirale, APOBEC3 en recrutant le complexe de la E3 ubiquitine ligase formé de CBFβ/EloB/EloC/Cul5/Rbx2, qui va entrainer sa polyubiquitinilation et sa dégradation. APOBEC3 comprend une famille de protéines présentes dans les mammifères et capable d'induire des mutations dans l'ADN viral.La structure tridimensionnelle des molécules et complexes macromoléculaires étant intimement liée à la fonction, il est primordial de connaître la place d’A3G/F dans le complexe Vif-CBFβ-EloB-EloC ainsi que les détails des interactions entre Vif et A3G/F, ceci dans une perspective thérapeutique.La co-expression des différentes protéines de ce complexe a été réalisée dans deux systèmes d’expression : bactérien (E. coli) et eucaryote (BHK21) dû au manque de solubilité d’APOBEC et de la difficulté de sa cristallisation. Une fois les protéines exprimées et le complexe Vif-CBFβ-EloB-EloC-A3G/F formé dans les deux systèmes, des tests de cristallisation et des études structurales seront réalisés pour caractériser la structure et la fonction des protéines.Dans E. coli, j’ai réussi à obtenir le complexe Vif/CBFβ/EloB/EloC/Cul5 in vivo et in vitro de manière soluble, pure, monodisperse ainsi qu’en quantité adéquate avec les études biophysiques envisagées. Par contre, je n’ai pas réussi à surexprimer A3G. En passant dans les cellules de hamster BHK21 et en appliquant la technique du virus de la vaccine, nous avons réussi à obtenir le complexe Vif-CBFβ-EloB-EloC-A3G/F mais en quantité insuffisante pour la cristallisation.Les perspectives de ce projet seront d’améliorer : (1) les rendements du complexe pour des tests de cristallisation et (2) la pureté du complexe pour réaliser la cryo-microscopie électronique. / APOBEC3 are human antiviral cytidine deaminase and RNA/DNA-editing enzymes that belong to the innate immune defense system, targeting retroviruses or retrotransposons. Among all APOBEC3 proteins, hA3B, hA3C, hA3DE, hA3F and hA3G are able to interfere negatively with HIV-1 infectivity: they induce a deamination of dC to dU in the minus strand DNA, resulting in G to A hypermutation in the plus strand DNA. This hypermutation results either into a degradation of U-rich DNA strands by the uracyl-DNA glycosylase or into the production of aberrant viral protein. In addition, a deaminase-independent mechanism is able to inhibit the HIV-1 reverse transcription through a yet unknown mechanism.To evade the host defense system, HIV expresses the virion infectivity factor, Vif, which causes the degradation of APOBEC3G by the proteasome by recruiting the E3 ubiquitin ligase complex composed of the CBFβ, EloB, EloC, Cullin5, Rbx2 proteins. The goal of this study will be the determination of the X-ray structure of the Vif/APOBEC-3 complex. Understanding this molecular recognition might be useful to direct structure-based design of anti-HIV drugs that act by inhibiting the action of Vif and lead to new anti-HIV drugs.To overcome the solubility problems of Vif and APOBEC3G/F, a co-expression strategy had been applied with the different E3 ubiquitin ligase proteins both in prokaryotic (E. coli) and eukaryotic (BHK21) systems. Once the complex obtained, we will perform several structural and biophysical studies as well as crystallization trials.In E. coli, I managed to obtain the Vif/CBFβ/EloB/EloC/Cul5 complex in vivo and in vitro, soluble, monodisperse and in good quantities for structural studies. The protein A3G was not obtained in E. coli even by co-expression with the complex.On the other side, I succeeded in obtaining the polyprotein Vif-CBFβ-EloB-EloC-A3G/F complex in the hamster cells (BHK21) by applying the vaccinia virus strategy. Optimizing the yield and the purity is necessary for crystallization and more structural studies.