EWI-2 Inhibits Cell-Cell Fusion at the Virological Presynapse

Cell-to-cell transfer of virus particles through the virological synapse (VS) is a highly efficient mode of HIV-1 transmission. Formation of the VS, a transient multiform adhesion structure, is mediated through an interaction between the HIV-1 envelope glycoprotein (Env) and the viral receptor CD4 on the surface of infected cell and target cell, respectively. Given that Env, unlike many other viral fusogens, can mediate the merger of membranes at neutral pH, the close encounter of infected and uninfected cells would seem prone to result in cell-cell fusion and thus the formation of syncytia. However, while it is being recognized now that small, T cell-based syncytia are indeed a defining feature of the natural history of HIV-1, the majority of VSs nevertheless resolve without fusion, thus securing continued virus spread. Gag, the main viral structural component, is partially responsible for restraining Env and preventing it from becoming fusogenic before being incorporated into particles. In addition, a few cellular factors, including tetraspanins and ezrin, have also been shown to inhibit Env’s activity while this fusogen is still part of the producer cell.Here, we identify EWI-2, a protein that was previously shown to associate with the tetraspanins CD9 and CD81 and also with ezrin, as a host factor that contributes to the inhibition of Env­mediated cell-cell fusion. Using fluorescence microscopy, flow cytometry, and TZM-bl fusion assays, we show that EWI-2, comparable to tetraspanins, while overall being downregulated upon HIV-1 infection, accumulates at the producer cell side of the VS (i.e. the presynapse), where it contributes to the fusion-preventing activities of the other viral and cellular components.