Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dikeakos, Jimmy D.

Abstract

HIV-1 Nef is a key pathogenic protein, allowing HIV-1 to evade the host immune system by downregulating MHC-I and CD4. Furthermore, it was recently discovered that Nef counteracts the host factor SERINC5 to increase HIV-1 infectivity, but the mechanistic details of the Nef:SERINC5 interaction still need to be explored. Throughout this dissertation, I will explore the hypothesis that the genetic diversity that defines HIV-1 has a pronounced effect on the HIV-1 protein Nef, altering its function between and within group M subtypes. To address this hypothesis I investigated how MHC-I and CD4 downregulation differ among all non-recombinant group M subtypes. These studies revealed subtype-specific differences in Nef function that were associated with differences in Nef expression between subtypes. Further investigation revealed unique subcellular distribution of Nef within the rarely studied subtypes G and H. A low expressing Nef isolate of the globally predominant subtype C was then analyzed using mutational and stability studies, identifying a previously undefined region in an alpha helix of Nef that is essential for protein expression and function. Moreover, the molecular details of the recently defined Nef-mediated SERINC5 downregulation are elucidated. The microscopy technique of bimolecular fluorescence complementation was used to demonstrate an in cellulo Nef:SERINC5 interaction, implicating key Nef protein interaction motifs. The Nef:SERINC5 complex was then mapped throughout the cell, highlighting the ability of Nef to hijack protein trafficking machinery, shuttling SERINC5 to degradative compartments to favour HIV-1 replication. Mutational analysis of SERINC5 shed light on the genetic determinants of the anti-infectivity of this poorly understood HIV-1 restriction factor. From there, these findings were placed in the context of the HIV-1 epidemic, investigating the conservation and variation in SERINC5 downregulation among Nef isolates from subtype C and A endemic regions.

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