Bimolecular Fluorescence Complementation Reveals that HIV-1 Nef Oligomerization is Essential for CD4 Downregulation and Viral Replication

HIV-1 Nef is a small myristoylated protein capable of interaction with a diverse array of host cell signaling molecules. Multi-faceted in its function, Nef is a critical accessory factor, essential for high-titer viral replication and AIDS progression. Despite its essential role, the molecular mechanisms of Nef-mediated HIV pathogenicity are not fully understood. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher order oligomers in HIV-infected cells. The studies summarized below investigated the oligomeric status of the HIV-1 nef gene product and its role relative to Nef-mediated function. We explored the formation of Nef oligomers in live cells by adapting a bimolecular fluorescence complementation (BiFC) assay, a well-defined system in which dimeric protein interactions are observed in live cells. Using this assay, we provided the first direct evidence for Nef oligomerization in vivo. We then assessed the generality of oligomerization by a group of Nef alleles broadly representative of all major HIV-1 subtypes and found oligomerization was highly conserved across all subtypes examined. We then used our BiFC system to define residues previously suggested via X-ray crystallographic studies to comprise the Nef dimerization interface. Using a systematic strategy for the mutagenic profiling of the oligomerization interface, we discovered two classes of residues were critical to Nef oligomerization. BiFC was completely abolished when either all four key hydrophobic interactions were simultaneously removed or when ionic interactions mediated by D123 and R105 were disrupted. Finally, we utilized Nef mutants identified in the mutagenic profiling of the oligomerization interface to explore the effects of oligomeric disruption on Nef function. Screening a panel of Nef mutants with varying degrees of oligomeric disruption we discovered, surprisingly, despite the varying effects on oligomerization, all of these mutants were shown to dramatically disrupt Nef-induced CD4 downregulation and viral replication. Taken together, the studies presented in this dissertation advance the field of HIV research by furthering our understanding of the regulation of Nef-mediated downregulation of CD4 and enhancement of HIV replication as well as validating the Nef oligomerization interface as a potential target for anti-retroviral drug design