Formation of Syncytia Is Repressed by Tetraspanins in Human Immunodeficiency Virus Type 1-Producing Cells

In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is incorporated into released virus particles and enables the virus to attach to and fuse with target cells in order to initiate the infectious cycle. Before Env mediates the fusion of viral and cellular membranes, i.e., while it is still incorporated in the plasma membrane of the infected cell, it drives the adhesion between virus producer cell and target cells, which gives rise to the formation of the so-called virological synapse (VS) (21, 24, 35, 36). The VS shares certain characteristics with the immunological synapse, including an accumulation of specific cellular membrane proteins and lipids (see, e.g., reference 5), and it provides efficient and secure transfer of virus particles from infected to uninfected cells (8). Importantly, the two adhering cells, like the pre- and postsynaptic cells that form an immunological synapse, typically do not fuse during such cell-to-cell transfer events. At first glance this seems surprising, as HIV-1 Env, unlike many other viral envelope proteins, can induce membrane fusion at physiological pH. Also, adhesion of producer and target cell, which can be initiated when the uropod of the infected cell contacts the uninfected cell (8), followed by reorganization of the cytoskeleton (25) and formation of full-fledged synapses, can extend over minutes (see, e.g., reference 20). This process should allow enough time to trigger cell-cell fusion. However, it is now well established that newly synthesized Env is efficiently internalized upon its arrival at the host cell plasma membrane, unless it is recruited into budding structures by viral Gag (see, e.g., reference 11; also discussed in references 3 and 6). Further, and likely also contributing to the prevention of producer-target cell fusion, immature Gag at the host cell plasma membrane represses Env-driven fusion, and this repression is lost only once Gag is processed in released virions (9, 22, 23, 31, 50). Finally, because syncytia are clearly not required for the transmission of virus from cell to cell in vitro and are possibly detrimental to virus spread in vivo, we hypothesize that HIV-1 cooperates with cellular membrane proteins to prevent cell-cell fusion.Members of a group of cellular proteins known as tetraspanins play an important role as regulators of cellular fusion processes, including myotube formation and fertilization (28, 30, 44; reviewed in, e.g., reference 17). As membrane organizers, these proteins homo- and heteromultimerize and associate with other cellular proteins to form variably sized but discrete microdomains, the so-called tetraspanin-enriched microdomains (TEMs) (29) (also called TERMs 1] or TEAs 12]). Knowledge of the molecular mechanisms through which tetraspanins regulate the fusion of cellular membranes is still lacking, though the available evidence strongly suggests (i) that these proteins are not themselves fusogens but rather that they coordinate the fusion activity of other cellular proteins and (ii) that they can act both as positive and negative regulators of cellular fusion processes. For instance, several in vivo studies unequivocally showed that CD9 expression in oocytes is essential for sperm-egg fusion (27, 28, 30), but CD9 and CD81 ablation in monocytes enhances the formation of multinucleated phagocytes that are involved in immune defense against certain microbes (45). Interestingly, the same two tetraspanins are also known to regulate virus-induced fusion processes. CD9 is involved in regulating cell-cell fusion driven by canine distemper virus, as the anti-CD9 antibody K41 inhibits syncytium formation by this virus (42), and CD81 is a necessary cofactor for infection of cells by hepatitis C virus (see, e.g., references 2 and 52). Finally, tetraspanins on uninfected (target) cells inhibit HIV-1-induced cell-cell fusion (14). This fusion regulation is likely due to interactions of CD9 and CD81 with CD4 and coreceptors at the surface of target cells, though the tetraspanin CD63 has also been implicated in the trafficking of CXCR4 to the plasma membrane (51).Because tetraspanins in HIV-1-producing cells are enriched at budding sites (4, 10, 13, 15, 33, 46, 49) and at the VS (26), we hypothesized that they regulate Env-driven fusion at the VS. Here, we document that tetraspanins in HIV-1-producing cells can indeed restrict syncytium formation. We also define some of the requirements for this fusion inhibition, thus laying the necessary groundwork for future mechanistic analyses. In addition, the characterization of cell-cell fusion regulation parameters in this study will allow the fusion-inhibitory activities to be distinguished from other regulatory functions exerted by tetraspanins, such as the modulation of virion infectivity and the regulation of cell-to-cell transmission of HIV-1.