Abstract
Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell–cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell–cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell–cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely.
Highlights
HIV-1 spreads between T cells primarily through two modes of transmission: the release of cell-free virus particles followed by their uptake by cells expressing the viral receptor/co-receptor, and the cell-to-cell transmission of particles to an adjacent cell via the virological synapse (VS), i.e., when infected and uninfected cells transiently align
While we know that small, T cell-based syncytia arise early in HIV-1 infection and can spread the virus by cell–cell contact [7,8,9,10,11,12], the majority of infected T cells observed in lymphoid tissue are mononucleated, documenting that most HIV-1 VSs result in complete cell separation and the generation of a new, productively infected cell
Because EWI-2 is known to associate with ezrin and CD81 [25,27], two cellular factors that accumulate at the producer cell side of the virological synapse (VS) [24,54], we first sought to determine whether this protein localizes to the VS
Summary
HIV-1 spreads between T cells primarily through two modes of transmission: the release of cell-free virus particles followed by their uptake by (more or less distantly located) cells expressing the viral receptor/co-receptor, and the cell-to-cell transmission of particles to an adjacent cell via the virological synapse (VS), i.e., when infected and uninfected cells transiently align. While we know that small, T cell-based syncytia arise early in HIV-1 infection and can spread the virus by cell–cell contact [7,8,9,10,11,12], the majority of infected T cells observed in lymphoid tissue are mononucleated, documenting that most HIV-1 VSs result in complete cell separation and the generation of a new, productively infected cell This is likely due to tight regulation at the VS that acts to prevent excessive syncytium formation (reviewed in [13,14])
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