Abstract
HIV-1 Nef (Negative Regulatory Factor) is expressed at high levels in the early stage of the viral life cycle. Its cellular functions are implemented through its interactions with dozens of host cell proteins. Nef, as a crucial factor in HIV-1 pathogenesis is a promising target for antiretroviral drug discovery. Nef inhibition has the unique potential to revitalize host immune mechanisms to target infected cells and thereby possibly clear the infection. While Nef alone lacks druggable sites, the molecular complexes that include Nef with host proteins could possibly contain druggable pockets. Thus, the high-resolution structures of Nef combined with host proteins have tremendous values. The goal of my research is to elucidate the mechanisms of three critical Nef functions using biochemistry and structural biology: CD4 downregulation, Serinc5 downregulation, and modulation of autophagy. Then, these compounds can significantly contribute to the development of Nef inhibitors. While HIV-1 requires CD4+T helper cells, often referred to as CD4 cells, for entry, the presence of surface CD4 will later be problematic for the virus to progress. That is because CD4 plays a pivotal role in the regulation of the immune system. Therefore, downregulation of CD4 is necessary for the maintenance of viral infection and its progression to AIDS. HIV-1uses Nef and Vpu to maintain the Env as a close conformation to prevent the accumulation of the CD4 receptor. It is known that Nef hijacks the clathrin AP2 endocytosis pathway to downregulate CD4 from the cell membrane. However, the exact mechanism is not known. Therefore, we aimed to define the role of HIV-1 Nef in inducing the AP2-dependent CD4 downregulation using biochemical analysis and X-ray crystallography. This work, which was published in 2020, is summarized in Chapter 2.Serinc5 and Serinc3, members of the Serinc protein family, have recently been identified as HIV-1 antagonists with significant antiviral activity against HIV-1inNef-deficient cell cultures. They incorporate into the envelope of new virions and are counteracted by Nef. However, the exact mechanism for HIV-1 Nef-mediated Serinc5 surface downregulation has not been identified. The goal of this project was to characterize the molecular details of how HIV-1 Nef mediates the AP2-dependent Serinc5 downregulation. Chapter 3 of this dissertation summarizes the biochemistry analysis and structural work that I carried out in pursuing this goal. Autophagy serves to restore homeostasis in response to a number of stress conditions. To create chronic infection, the HIV-1 virus has established mechanisms, which help the virus evade autophagy-mediated degradation. The HIV protein Nef is associated with autophagy control, either inhibiting or stimulating the mechanism through direct interaction with autophagy proteins. Beclin1 is a central protein in the regulation of the autophagy pathway. Studies have found a novel role of Beclin1 in controlling viral replication and viral-induced inflammation in HIV-infected microglia. Therefore, knowing the basic molecular mechanism would allow us to understand the activity, and potentially inhibit it. The purpose of this research is to examine the possible interaction between HIV-1 Nef and Beclin1 (BECN1). We also considered the possibility, as suggested by the literature, that Nef modulates autophagy through direct interaction with IRGM. This work is described in Chapter 4.