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Characterization of HIV-1 Nef and Vpu function with a focus on T cell polarity

Lamas Murua, Miguel

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Expression of accessory proteins during human immunodefiency virus-1 (HIV-1) life cycle is essential to ensure efficient viral replication and spread. Despite of being dispensable for HIV-1 infection of cell lines in vitro, use of primary target cells and in vivo experiments proved them to be critical key pathogenicity factors. Along with counteraction of restriction factors to facilitate immune response escape, they play an important role as manipulators of host cell fundamental biological processes to favor replication. This work focuses on the study of the accessory proteins. Both these proteins are known to strongly alter intracellular vesicular transport. As a consequence, the composition of the plasma membrane is modified upon their expression, a phenomenon studied in the first part of this thesis. Here, it has been demonstrated the specificity of the Nef- and Vpu-mediated downregulation of tetraspanins, the most drastically affected host receptors, and identification of molecular determinants of both proteins necessary for this activity was pursued. It was also studied the ability of Vpu to retarget the T lymphocyte master regulator Lck, a well described phenomenon induced by Nef. Another yet unstudied field for Vpu examined herein was the induction of extracellular vesicle release. This work revealed that Vpu, like Nef, enhances extracellular vesicle release when expressed by T lymphocytes with comparable or even higher efficiency. The second part of this thesis addressed the effect of these proteins on T cell polarity disruption in the context of T cell migration. Unlike Vpu, Nef has been reported to inhibit F-actin cytoskeleton rearrangements. This requires the interaction of Nef with PAK2 which facilitates a specificity switch of the host kinase resulting in hyperphosphorylation and thus inactivation of the actin severing factor cofilin. As a consequence, the motility of HIV-infected T lymphocytes is hampered. A previous report showed that in murine CD4+ T cells, the central Nef-mediated block in vivo and ex vivo is at the level of transendothelial and subendothelial migration. Whereas the latter is dependent on Nef-PAK2 on actin dynamics, the effect on transmigration involves other, yet uncharacterized protein interactions and is remarkably paralleled by a pronounced reduction of cell polarization. This thesis shows that Nef also hinders polarization of HIV-1 infected primary human CD4+ T lymphocytes. Vpu was entirely unrequired for this effect. Nef-expression in human T cell lines caused a marked polarization defect conserved among Nef proteins from HIV-1, -2 and simian immunodeficiency virus. The interaction surface of Nef with the Nef-associated kinase complex (NAKC) was found to be the essential molecular determinant for polarization disruption by Nef, both when overexpressed or in HIV-1 infection. Pharmacological inhibition of NAKC components evidenced the involvement of protein kinase C (PKC) in this Nef activity. This work also details setting up of experimental conditions to study the possible implication of this Nef amino acid stretch on transendothelial migration, that is on diapedesis ex vivo. Altogether, the results presented here underline the importance of the studied redundant functions between Nef and Vpu in HIV-1 infection and the relevance of these accessory proteins as pathogenicity factors. Also, they highlight the role of Nef as the main HIV-1 negative regulator of T lymphocyte migration.

Item Type: Dissertation
Supervisor: Fackler, Prof. Dr. Oliver T.
Date of thesis defense: 9 November 2016
Date Deposited: 28 Nov 2016 07:38
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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