Determining the role of AnnexinA2 in HIV-1 and SARS-CoV-2 assembly

Human Immunodeficiency Virus Type 1 (HIV-1) and Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) are two enveloped RNA viruses that affect significant proportions of the global population. HIV- 1 and SARS-CoV-2 rely on lipid rafts to facilitate viral entry, assembly, and release, but the exact mechanisms for how this occurs remain unclear. Lipid rafts represent distinct regions within cellular membranes that are enriched in cholesterol, glycophospholipids, and protein receptors. Disrupting lipid-raft formation pharmacologically reduces both HIV-1 and SARS-CoV-2 infection. Our long-term goal is to determine how viruses hijack cellular proteins to disarm host defenses and co-opt cellular pathways to promote infection. The objective in this application is to determine how the lipid-raft scaffold protein AnnexinA2 facilitates HIV-1 and SARS-CoV-2 assembly. Our preliminary data suggest that AnnexinA2 interacts with two distinct viral membrane proteins: HIV-1 Nef and SARS-CoV-2 Spike. Our central hypothesis is that AnnexinA2 facilitates the localization of Nef and Spike to lipid-raft compartments, thereby augmenting viral assembly and infectivity. The rationale for this project is that identifying specific mechanisms by which HIV-1 and SARS-CoV-2 exploit AnnexinA2 to enhance viral infection will open new aspects of viral biology and offer new therapeutic strategies. We aim to test our hypothesis by pursuing two specific aims: 1) Identify how AnnexinA2 facilitates HIV-1 infection; and 2) Determine how AnnexinA2 promotes SARS-CoV-2 replication. In Aim 1, we will validate whether Nef directly binds to AnnexinA2, determine whether the Nef/AnnexinA2 interaction correlates with the enhanced prevalence of HIV-1, map the molecular determinants required for interaction, and determine whether AnnexinA2 enriches Nef in lipid-rafts at the cell surface. Additionally, we will knockout AnnexinA2 via CRISPR/Cas9 in primary cells to determine whether Nef requires AnnexinA2 to enhance infectivity. For Aim 2, we will produce SARS-CoV-2 Virus Like Particles (SC2-VLPs) in both the presence and absence of AnnexinA2 to determine its role in facilitating SARS-CoV-2 assembly. We will also validate the SARS-CoV-2 Spike/AnnexinA2 interaction and delineate whether AnnexinA2 recruits Spike to lipid rafts at the ER-Golgi Intermediate Compartment (ERGIC). The proposed research is innovative and significant because it will generate novel models of retrovirus and coronavirus biology, vertically advancing both fields and providing novel avenues for further mechanistic and therapeutic investigation.