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Characterization of HIV-1 host interactions by advanced fluorescence microscopy

Defense Thèse de doctorat : Univ. Genève, 2012 - Sc. 4407 - 2012/02/24
Abstract HIV-1 is responsible for the current AIDS epidemic that caused 30 million dead so far. Albeit an overall stabilization of incidence rates and declining new infections, AIDS represents a major health and economic problem to some developing countries. HIV-1 infection mainly occurs through unprotected sexual intercourse and subsequent mucosal transmission. In mucosal tissue dendritic cells (DC) can capture HIV-1 through cell surface receptors (e.g. the C-type lectin DC-SIGN), degrade virions and present viral antigens to T-cells. Remaining intact virions are transferred to CD4+ T-cells at cell-to-cell contacts, called infectious synapses. Both DC-SIGN signaling and actin-containing cellular protrusions are implicated in HIV-1 cell-to-cell transfer, but their definitive role in highly relevant immature DC T-cell transfer remains unclear. HIV-1 replication in CD4+ T-cells and macrophages leads to general immune activation, T-cell depletion and final failure of the immune system. In contrast, most SIV infections in their natural hosts are nonpathogenic. Primate lentiviruses use their Nef proteins to manipulate the communication of T cells with antigen-presenting cells (APC) through cell-contacts named immunological synapses. The interaction of individual HIV-1 virions with cellular proteins during entry and assembly occurs at scales below the diffraction limit of light that is ~200nm. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules in intact cells. The cellular factor tetherin directly interacts with budding HIV-1 virions and inhibits their release. The restriction mechanism at the molecular scale remains largely unclear. Establishment of quantitative multicolor super-resolution microscopy enabled the visualization of HIV-1 virions, assembly sites and the cellular organization and restriction mechanism of tetherin. New quantitative tools were established to determine sizes of tetherin clusters, number of tetherin molecules and to test models for tetherin recruitment to HIV-1 budding sites. Live-cell fluorescence and electron microscopy together with genetic and pharmacological approaches were used to show that HIV-1 activates Cdc42 via DC-SIGN signalling to induces membrane extensions in immature dendritic that play important roles in cells to cell-to-cell transfer. The effect of primate Nef proteins on immunological synapses between infected primary T-cells and APCs was characterized by immunofluorescence and high resolution confocal laser scanning microscopy. The inability to disrupt the immunological synapse between infected human T cells and APCs distinguishes HIV-1 from most other primate lentiviruses and could explain important differences in lentiviral pathogenesis. In this thesis, super-resolution and confocal fluorescence microscopy allowed a detailed characterization of HIV-1 tetherin interaction, infectious and immunological synapses that play important roles in HIV-1 transmission and pathogenesis.
URN: urn:nbn:ch:unige-188414
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LEHMANN, Martin. Characterization of HIV-1 host interactions by advanced fluorescence microscopy. Université de Genève. Thèse, 2012. https://archive-ouverte.unige.ch/unige:18841

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Deposited on : 2012-03-19

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