Interactions of the HIV-1 nef virulence factor with host cell tyrosine kinases of the src and tec families

Current antiretroviral therapies effectively slow AIDS progression and lengthen the life of AIDS patients but sadly, cannot completely cure HIV-positive individuals. The development of drug-resistance in HIV often renders the current anti-HIV therapeutic regimens ineffective and even contraindicated in some cases. Thus there exists an urgent need to identify alternate targets for the discovery and development of newer anti-HIV drugs. A promising approach lies in targeting an underexplored yet critical accessory factor in HIV pathogenesis – Nef, which promotes AIDS progression by binding to a plethora of host cell factors leading to altered cell signaling. Identifying novel host factors that are direct effectors for HIV-1 Nef will enable future drug discovery directed against this key HIV virulence factor.\ud In the first part of my dissertation study, I developed a novel, cell-based approach to explore the scope of Nef-SH3 interactions. Particularly, I explored the interaction of Nef with Tec-family kinases and their relevance to HIV biology. This assay allowed direct visualization of protein-protein interactions between Nef and three Tec family members – Bmx, Btk and Itk in live cells. Interaction occurred between the SH3 domains of the kinases and a conserved polyproline motif on Nef. Allelic variants of Nef representing all the M-group HIV-1 subtypes interacted strongly with Itk demonstrating the highly conserved nature of this interaction. Interaction with Nef induced Itk activation which was reversed by treatment with an Itk inhibitor that also potently blocked Nef-dependent HIV replication. These results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family, and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.\ud In the second part of this study, I validated the biological relevance of a newly determined high resolution crystal structure of Nef in complex with its best characterized kinase binding partner, Hck. Using human and yeast cell-based systems, I have shown by mutagenesis studies that the newly recognized intercomplex contact between Nef R105 and E93 in the RT loop of the SH3 domain is critical to complex formation and function. These results renew our perception of the Nef:Hck binding interface by offering new insight into possible conformations for the active Nef:Hck complex, which is essential for Nef function and further establishes it as a valid druggable target for HIV-1.\ud Taken together, the studies presented in this dissertation deepen our understanding of the interaction between the HIV-1 virulence factor Nef and the Src family kinase, Hck; identify additional novel cytoplasmic tyrosine kinases that are direct SH3-based effectors of HIV-1 Nef and validate a novel virus:host cell interaction as a potential target for therapeutic intervention. Thus, my results not only have a strong public health significance and advance the field of HIV research, but also offer a step forward in our combat against what remains as one of the most relevant public health menaces of today – HIV/AIDS.\u