Friday, April 4, 2025
4/4/2025
Basis of Serinc-Independent Enhancement of Infectivity by HIV-1Nef - Project Summary HIV-1 Nef is an accessory protein that contributes to replication fitness, virion-infectivity, immune evasion, and virulence. Over three decades of research have yielded substantial progress in our understanding of these properties at many levels. Still, an important gap persists: how Nef stimulates virion infectivity and viral propagation remains incompletely explained. Initial research on the infectivity-effect led to the realization that the viral receptor, CD4, is a potent inhibitor that Nef counteracts. But Nef stimulates infectivity even in the absence of CD4. This gap was filled - partly - by the SERINC proteins, particularly SERINC5, which like CD4 is a membrane protein and infectivity-inhibitor that is counteracted by Nef. But recently, we and others have observed that in at least two T cell lines, and probably in primary CD4-positive T cells, SERINC-counteraction is insufficient to account for the activity of Nef. We propose a high-risk/high-reward study aimed at identifying the missing protein or pathway that accounts for Nef's contribution to viral infectivity and fitness. In SA1 we will identify the missing factor or pathway modulated by Nef. We propose to use proximity- proteomics as an open-ended search tool. The goal is to find the missing protein or pathway that accounts for Nef's ability to enhance viral replication and infectivity in a CD4- and SERINC-independent manner. We previously used this approach to identify novel targets and cofactors of another viral accessory protein, Vpu. In that instance, we used a Vpu-APEX2 fusion protein to biotinylate cellular proteins in a small radius around Vpu, and we identified and measured them by quantitative mass spectrometry. A key to the analysis of hundreds of proteins was the comparison of Vpu mutants whose properties and activities were known. We plan the same approach here with Nef. Importantly, a key Nef-mutant we will analyze is selectively impaired in the SERINC- and CD4-independent enhancement of infectivity by Nef. This mutant will enable us to look specifically for proteins and pathways that support the phenotype we seek to explain. Moreover, proximity proteomics will enable us to search agnostically for either a restriction factor that Nef antagonizes or a dependency-factor that Nef stimulates. Candidates will be validated virologically in T cell culture systems. In SA2 we will identify the membrane-trafficking cofactor(s) that supports this Nef activity. Independent of the above proteomic approach, we will test specific hypotheses regarding clathrin adaptors as Nef cofactors. While the clathrin adaptor AP2 is critical to the modulation of CD4 and SERINC5 by Nef, our mutational data suggest that the SERINC-independent effect of Nef on viral infectivity is more likely to rely on AP1. We aim to test this hypothesis using knockout or knockdown approaches in T cell culture systems. When these studies are complete, we expect to have identified a novel basis of Nef's virologic activity.
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