UC San Diego

The Human Immunodeficiency Virus (HIV) Nef protein has evolved multiple methods to evade the host immune system. Reduction in cell-surface major histocompatibility complex I (MHC-I) is one such method; this enables virally- infected cells to evade lysis by cytotoxic T lymphocytes (CTL). In addition, Nef also performs functions related to HIV infectivity and replication. The main objective of this dissertation is to delineate the mechanism by which Nef downregulates MHC-I, and how this function may be sacrificed in order to optimize infectivity and replication. I first focused on the optimization of Nef functions after sexual transmission by studying the modulation of MHC-I and CD4 by Nef clones from phylogenetically- and epidemiologically-linked transmission partners. Flow cytometry of T cells transfected with these Nef clones showed that both CD4 and MHC-I downregulation are important to the establishment of a successful infection in a new host and neither function is expendible. Next I addressed how Nef modulates MHC-I trafficking in the cell by examining the interactions of Nef, MHC-I, and the non-clathrin endocytic pathway. I studied the role of the GTP cycle of the ADP-ribosylation factor 6 (ARF6) in the Nef-mediated downregulation of MHC- I. Immunofluorescence microscopy of cells transfected with both Nef and epitope-labeled ARF6 were examined for colocalization. Additionally, T cells transfected with Nef, and mutants of ARF6 that interfered with the ARF6-GTP cycle, were examined for cell-surface MHC-I by flow cytometry. While disruptions of the ARF6-GTP cycle did appear to interfere with MHC-I cell-surface expression, this result was not related to MHC-I downregulation by Nef. Finally, I changed the focus of the mechanistic studies to the intracellular endosomal system, specifically to adaptor protein-1(AP-1)-containing vesicles. Studying protein-protein interactions, I found that Nef and MHC-I can synergize to create a novel AP-1 interaction surface, and I determined residues in Nef, MHC -I, and the subunit of AP-1 involved in this interaction. In summary, my dissertation research has contributed to a greater understanding of not only the importance of MHC-I downregulation by HIV-1 Nef, but also to its mechanism in the cell