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    Replication and sequestration of HIV-1 in macrophages

     

    Leslie Lepont

    September 30th 2:30 pm 2020

    Rosalind Franklin room, 2,nd floor

    Institut Cochin, 22 rue Méchain, Paris, 75014

    Supervisor: Clarisse Berlioz-Torrent

    Team:Host-virus interactions

    Department: Infection, Immunity and Inflammation (3I)

    Abstract:

    Human Immunodeficiency Virus, HIV-1, currently infects 37.9 million of people across the world, counting 32 million of deaths since the beginning of the epidemic. One of the major issues is to purge viral reservoirs, obstacles to complete cure of patients. Those reservoirs are majorly constituted of latently infected CD4 T cells in which the expression of the viral genome is repressed. Macrophages have also been shown to be viral sanctuaries, capable of archiving fully infectious viruses in intracellular vesicles (VCC) for long periods, even in patients on antiretroviral therapy

    VCC are pH neutral structures derived from invaginated membranes. They are enriched in tetraspanins CD81, CD63, CD53, CD36, CD9, HLA-DR, Siglec1 but also in BST2/Tetherin. BST2 is a restriction factor of HIV-1, able to physically tether budding viruses at the membrane impeding their dissemination. Facing this restriction, HIV-1 has evolved, and its viral protein Vpu counteracts BST2 restriction. Indeed, Vpu alters BST2 trafficking, diminishes its cell surface expression by targeting it to lysosomal degradation. Moreover, HIV-1 can engage a non-canonical autophagy process, similar to LC3-associated phagocytosis (LAP) to antagonise BST2. In this process, Vpu binds to LC3C and triggers a LAP-like process leading to surface BST2 sequestration in internal vesicles, decorated by LC3 molecules. Interestingly, we observed that silencing of LC3C in HeLa cells led to virions sequestration into single membrane vesicle very similar to the VCC found in infected macrophages. This observation led us to hypothesise that VCC would be the result of an abortive LAP process. Therefore, my thesis objectives are to characterise molecular mechanisms leading to HIV-1 sequestration in VCC in macrophages. To do so, I addressed two issues: i) define BST2 contribution to VCC formation, ii) monitor the implication of the LAP process in HIV-1 sequestration in VCC. First, we show that Vpu is able to increase viral release and to control VCC volume. These effects of Vpu depends on BST2 expression. Indeed, Vpu decreases BST2 expression, both at cellular and cell surface levels, and excludes BST2 from VCC. This limited amount of BST2 at the cell surface and in VCC favors viral dissemination and reduces the quantity of viruses trapped into VCC, reducing by the way VCC volume. Those Vpu activities rely on the integrity of its transmembrane domain, and on its di-serine S52-S56 and E59xxxLV64 motifs. Furthermore, our study reveals that BST2 silencing does not prevent the formation of VCC, refuting the hypothesis of BST2 being necessary to VCC formation. This first part highlights that BST2 and Vpu act as two antagonist factors, in the control of VCC volume in macrophages. In the second part of my thesis, we evaluated the contribution of Vpu-induced LAP-like pathway to viral sequestration in macrophages. Indeed, our preliminary results suggest a role for those autophagy proteins in viral sequestration in human macrophages. Further studies are ongoing in the lab to determine the exact contribution of these ATG proteins in the formation and persistence of these HIV sanctuaries. 

     

    Key Words: HIV-1 / human primary macrophages / restriction factor / BST2 / Vpu / VCC / viral sequestration / LAP / LC3 / ATG

    Human Immunodeficiency Virus, HIV-1, currently infects 37.9 million of people across the world, counting 32 million of deaths since the beginning of the epidemic. One of the major issues is to purge viral reservoirs, obstacles to complete cure of patients. Those reservoirs are majorly constituted of latently infected CD4 T cells in which the expression of the viral genome is repressed. Macrophages have also been shown to be viral sanctuaries, capable of archiving fully infectious viruses in intracellular vesicles (VCC) for long periods, even in patients on antiretroviral therapy. 

    VCC are pH neutral structures derived from invaginated membranes. They are enriched in tetraspanins CD81, CD63, CD53, CD36, CD9, HLA-DR, Siglec1 but also in BST2/Tetherin. BST2 is a restriction factor of HIV-1, able to physically tether budding viruses at the membrane impeding their dissemination. Facing this restriction, HIV-1 has evolved, and its viral protein Vpu counteracts BST2 restriction. Indeed, Vpu alters BST2 trafficking, diminishes its cell surface expression by targeting it to lysosomal degradation. Moreover, HIV-1 can engage a non-canonical autophagy process, similar to LC3-associated phagocytosis (LAP) to antagonise BST2. In this process, Vpu binds to LC3C and triggers a LAP-like process leading to surface BST2 sequestration in internal vesicles, decorated by LC3 molecules. Interestingly, we observed that silencing of LC3C in HeLa cells led to virions sequestration into single membrane vesicle very similar to the VCC found in infected macrophages. This observation led us to hypothesise that VCC would be the result of an abortive LAP process. Therefore, my thesis objectives are to characterise molecular mechanisms leading to HIV-1 sequestration in VCC in macrophages. To do so, I addressed two issues: i) define BST2 contribution to VCC formation, ii) monitor the implication of the LAP process in HIV-1 sequestration in VCC. First, we show that Vpu is able to increase viral release and to control VCC volume. These effects of Vpu depends on BST2 expression. Indeed, Vpu decreases BST2 expression, both at cellular and cell surface levels, and excludes BST2 from VCC. This limited amount of BST2 at the cell surface and in VCC favors viral dissemination and reduces the quantity of viruses trapped into VCC, reducing by the way VCC volume. Those Vpu activities rely on the integrity of its transmembrane domain, and on its di-serine S52-S56 and E59xxxLV64 motifs. Furthermore, our study reveals that BST2 silencing does not prevent the formation of VCC, refuting the hypothesis of BST2 being necessary to VCC formation. This first part highlights that BST2 and Vpu act as two antagonist factors, in the control of VCC volume in macrophages. In the second part of my thesis, we evaluated the contribution of Vpu-induced LAP-like pathway to viral sequestration in macrophages. Indeed, our preliminary results suggest a role for those autophagy proteins in viral sequestration in human macrophages. Further studies are ongoing in the lab to determine the exact contribution of these ATG proteins in the formation and persistence of these HIV sanctuaries. 

     

    Key Words: HIV-1 / human primary macrophages / restriction factor / BST2 / Vpu / VCC / viral sequestration / LAP / LC3 / ATG