%0 Generic
%A Lamas Murua, Miguel
%D 2016
%F heidok:22180
%R 10.11588/heidok.00022180
%T Characterization of HIV-1 Nef and Vpu function  with a focus on T cell polarity
%U https://archiv.ub.uni-heidelberg.de/volltextserver/22180/
%X 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.