Abstract
Human immunodeficiency virus 1 (HIV-1) has evolved mechanisms that allow the virus to evade the immune mechanisms and continue replicating and spreading. Nef, an important accessory protein involved in this process, is able to suppress immune mechanisms by hijacking cellular pathways to downregulate specific immune receptors such as MHC-1, CD4, Serinc3 (S3), and Serinc5 (S5). Previous research has demonstrated that Nef, through hijacking clathrin adaptor protein AP2, downregulates S3and S5 to cause their endocytosis and lysosomal degradation, which facilitates the spread of viral infection. The mechanism in which this counteraction is taking place is currently unknown. The goal of this research is to investigate the mechanism of Nef- mediated Serinc5 surface downregulation biochemically and structurally. In studying the Nef-mediated surface downregulation of S5, the binding of AP2 to the fusion of S5 loop9 to Nef (Nef-S5L9) was analyzed. Recent research demonstrated that the binding of AP2 to Nef-S5L9 fusion was improved by the phosphorylation of amino acid position Ser360 on S5L9. In vitro, our pulldown assay showed that the phosphomimatic mutant binds stronger to AP2 than than wildtype Nef-S5L9. The structural mechanism of these constructs is being pursued using crystallography (Chapter II).Following surface downregulation, S5 is transported to the lysosome for degradation. This mechanism is mediated by Nef and is dependent on clathrin AP1. Chapter III of this thesis described our work on using Cryogenic Electron Microscopy (cryo-EM) to study the structural basis of Nef-mediated, AP1 dependent lysosomal delivery of S5.The work produced here not only informed key determinants in Nef-mediated S5 downregulation and lysosomal degradation but also paved the way to solving high-resolution structures to fully elucidate these mechanisms.