Functional and evolutionary dynamics of HIV-1 Nef: Contributions to viral virulence and immune evasion

Human Immunodeficiency Virus Type 1 (HIV-1) expresses viral proteins that mediate disease progression. My thesis focuses on the HIV-1 accessory protein Nef, a viral protein that facilitates host immune evasion and enhances viral infectivity in people with HIV-1 (PWH). These functions are mediated by Nef’s ability to modulate cell surface expression of host human proteins, including Major Histocompatibility Complex Class I (MHC-I), Cluster of Differentiation 4 (CD4), and Serine Incorporator 5 (SERINC5). In my first aim, a bioinformatics pipeline was developed to create a time-scaled phylogeny relating extant primate nef sequences. The nef sequences of all ancestors involved in the phylogeny towards HIV-1 group M were then reconstructed. All Nef ancestors were assessed for SERINC5, CD4, and CD3ζ downregulation to validate the accuracy of the reconstruction and to test how these functions evolved. Functional characterization of all reconstructed Nef ancestors involved in the phylogeny towards HIV-1 group M allowed for estimations of how Nef-mediated SERINC5, CD4, and CD3ζ downregulation changed throughout its evolutionary history, thereby demonstrating this pipeline as a viable strategy to estimate how other viral functions may have evolved. In the second aim, I defined Nef-mediated SERINC5 and CD4 downregulation in a cohort of PWH. I found that two primary Nef proteins drastically differed in their SERINC5 downregulatory capabilities yet retained the ability to downregulate CD4. My studies mapped the Nef region responsible for this functional uncoupling to the highly conserved C-terminal dileucine motif. Identification of this polymorphism provides clues regarding the biochemical nature of the Nef:SERINC5 interaction and the pathways Nef hijacks to antagonize SERINC5. In the third aim, I explored whether Nef-mediated MHC-I downregulation contributes to the size of the replication-competent latent viral reservoir (RC-LVR) in PWH on long-term antiretroviral therapy (ART). Nef-dependent MHC-I downregulation was significantly inversely correlated with the rate of change of the RC-LVR, suggesting that efficient evasion of the cytotoxic T cell (CTL) response contributes to the maintenance of the RC-LVR in PWH. Overall, these studies provide a framework for understanding how Nef functions evolved and how these functions contribute to pathogenesis and virulence in PWH.