Wednesday, February 5, 2025
2/5/2025
Project Summary/Abstract BST-2, also known as tetherin, inhibits HIV-1 release from the plasma membrane of infected cells. HIV-1 Vpu neutralizes this restriction through not-mutually exclusive mechanisms, including degradation/downregulation, sequestration, and displacement. However, the relative importance of each of these anti-tetherin mechanisms in virus replication and transmission remains to be defined. Our long-term goal is to understand the roles of Vpu and tetherin in HIV-1 pathogenesis. Specifically, we aim to understand the mechanisms of how host cell proteins are involved in the Vpu-tetherin interaction. Recent studies from our laboratory demonstrate that a specific host cell protein, filamin A (FLNa) can modulate the antiviral activity of tetherin, and the particle release-promoting activity of Vpu. These discoveries suggest that, an FLNa-dependent trafficking pathway of tetherin is the essential step for Vpu to overcome the restriction imposed by tetherin. In this proposal, we will further understand the details of how FLNa regulates the association of Vpu to tetherin. In Aim 1, we will determine the mechanism by which FLNa regulates the antiviral activity of tetherin. Using live cell imaging and flow cytometry, we will examine the effects of FLNa on trafficking dynamics of cell-surface tetherin and intracellular tetherin. By examining the role of FLNa in the antiviral activity of tetherin against different HIV-1 subtypes, we will define the action spectrum of FLNa. By performing rescue experiments coupled with site- directed mutagenesis, we will test the specificity of different FLNa domains in the antiviral activity of tetherin. In Aim 2, we will determine the mechanism by which FLNa modulates the activity of Vpu to relieve tetherin restriction. We will examine the role of FLNa in the anti-tetherin activity of Vpu derived from different genetic subtypes. Using isothermal titration calorimetry (ITC) and microscale thermophoresis (MST), we will quantify the FLNa-Vpu interaction. Using quantitative IP assays and quantitative confocal microscopy, we will examine potential roles of FLNa in the Vpu-tetherin interaction and in the Vpu-tetherin colocalization. Finally, we will evaluate the importance of FLNa in each of three different mechanisms involved in Vpu-mediated tetherin antagonism. In Aim 3, we will focus on human primary macrophages to define the potential role of FLNa in the association of Vpu to tetherin. We will determine the effects of FLNa in HIV-1 infected macrophages on virus release and virus transmission from macrophages to CD4+ T cells. Taken together, these studies will provide important insights into the Vpu-tetherin interaction in HIV-1 replication, transmission, and pathogenesis.
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