Abstract
The multifunctional HIV-1 accessory protein Nef plays a crucial role in viral pathogenesis by enabling infected cells to evade host immunity. One of Nef’s prominent functions is the downregulation of major histocompatibility complex class I (MHC-I), disrupting antigen presentation and allowing infected cells to escape immune surveillance by cytotoxic T cells. Additionally, Nef consistently downregulates surface CD4 through clathrin adaptor protein complex 2 (AP2)-dependent endocytosis. Despite CD4 being the primary receptor for HIV infections, its presence post-entry negatively impacts viral replication and transmission. The association between CD4 and the viral Env protein interferes with Env processing, anterograde transport, and new virion release at the cell surface. Surface CD4 can also lead to superinfection, premature cell death, and limited viral replication. Furthermore, CD4-Env binding exposes antigenic Env epitopes, making the infected cell susceptible to antibody-dependent cellular cytotoxicity (ADCC). Notably, defects in nef genes are associated with disease nonprogression— patients infected with such HIV-1 strains do not develop AIDS for decades without antiviral treatment (ART), highlighting Nef’s role in enhancing viral infectivity. Consequently, a promising avenue for developing novel antiretrovirals is the therapeutic inhibition of the viral accessory protein Nef. Based on our research group’s solved structures of Nef-mediated CD4 and MHC-I downregulation (in 2020 and 2012, respectively), I have initiated the design and development of high-throughput screening (HTS) assays. The goal is to identify potent molecules that interfere with Nef’s immune evasion function. These molecules could potentially revitalize immune mechanisms, leading to targeted clearance of the virus. I used fluorescence polarization as the method to develop two HTS-compatible assays and these optimized assays have a good signal-to-noise ratio, excellent tolerance of DMSO and detergent, and the ability to detect competitive inhibition, making it suitable for high-throughput screening. Two related assay development papers have been submitted for publication and are summarized in chapter 2 and 3 of the dissertation. Serinc3 and Serinc5, human transmembrane proteins, act as antiviral restriction factors that inhibit HIV-1 infection. In the absence of viral antagonism, these proteins incorporate into the envelopes of nascent virions, effectively hindering virion fusion with target cells. However, the HIV-1 virus has developed a countermeasure by downregulating Serinc3 and Serinc5 from the cell surface, thereby excluding them from budding virions. This downregulation process is orchestrated by the viral accessory protein Nef, which hijacks clathrin adaptor protein complex 2 (AP-2)-dependent endocytosis. In my research, I explored the molecular determinants of Serinc3 downregulation by Nef. The results revealed that Nef recruits Serinc3 by binding to its N-terminal cytosolic tail. Interestingly, the Nef residues crucial for Serinc3 binding and downregulation significantly overlap with those required for Nef-mediated CD4 downregulation, suggesting notable similarities between the two processes. Additionally, my work sheds light on the conserved substrate-binding pocket of Nef—a molecular hotspot with implications for inhibitor development and antiretroviral drug discovery. The findings from this study have been submitted for publication and are summarized in chapter 4. Regarding CD4 fate after downregulation from the cell surface, it undergoes lysosomal degradation, as observed in cellular environments. However, due to the lack of structural elucidation, hypotheses abound regarding the exact mechanisms by which Nef hijacks AP1 to redirect CD4 toward endosomal compartments. The structure of AP1-Nef-CD4 will significantly enhance our fundamental understanding of how Nef manipulates endocytosis machinery molecules like AP1 and AP2, ultimately redirecting critical molecules along virus-desired pathways. In our work, we successfully solved a high-resolution Cryo-EM structure of Nef-mediated CD4 endosomal retention, revealing the intricate binding dynamics between Nef and CD4 during this process. We showed how Nef stays bound to CD4 while transitioning from cell-surface to early endosome. Interestingly Nef only recruits the σ1 subunit of AP1, thus conveniently can switch between γ1 and γ2 isoforms of AP1. The structural findings have been summarized in chapter 5.