Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dikeakos, Jimmy D.

Abstract

The HIV-1 accessory proteins Nef and Vpu have no known enzymatic activity, yet these viral proteins are critical for HIV-1 viral spread and pathogenicity. To mediate this, these viral proteins bind to and hijack host cellular proteins, including proteins implicated in the membrane trafficking machinery. Key to the effects of these proteins is their ability to alter the subcellular localization of a plethora of cell surface receptors, including the key immune cell receptorsCD28 and MHC-I.However,the mechanism utilized by Nef and Vpu to modulate these proteins is not fully understood. Herein, we demonstrated that Nef and Vpu mediate a decrease in both cell surface and total CD28 protein levels in CD4+T cells. We have implicated the endosomal degradation machinery in this process, as we observed that in the presence of these viral proteins CD28 localizes to LAMP-1 positive compartments in an endosomal acidification-dependent manner. Moreover, in investigating the mechanisms utilized by Nef and Vpu to mediate CD28 downregulation we implicated the Nef LL165and DD175and Vpu S52/56motifs, which interact with specific cellular membrane trafficking machinery, in CD28 downregulation. Furthermore, upon infection with viruses encoding or lacking Nef and Vpu, we observed differential effects on cellular activation upon stimulation. In seeking to understand the mechanism of MHC-I downregulation by Nef, we examined the roles of membrane trafficking mediators in the sorting nexin family of proteins. Namely, we identified a novel direct Nef binding partner, SNX18, and mapped this interaction with Nef to the SH3 domain of SNX18 and a polyproline PxxP75motif on Nef. Additionally, using shRNA knock-downs we have implicated SNX18 in Nef-mediated MHC-I downregulation, a key mechanism for HIV-1 viral immune evasion. Overall, this work sheds light on the mechanism of Nef- and Vpu-mediated mis-localization of host cellular proteins, processes key to the pathogenic effects of these viral proteins.

Available for download on Sunday, March 01, 2020

Included in

Virology Commons

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