However, quantification of VLPs budding at the plasma membrane also demonstrated that while budding of VLPs still took place in K41-treated cells, the number of buds per cell was considerably reduced (by about 70%) compared to untreated cells
However, quantification of VLPs budding at the plasma membrane also demonstrated that while budding of VLPs still took place in K41-treated cells, the number of buds per cell was considerably reduced (by about 70%) compared to untreated cells. and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins. The Inolitazone dihydrochloride surfaces of cells, even of those with no manifest polarization, can be transiently partitioned into discrete domains for the execution of specific functions. This principle was elegantly demonstrated some 20 years ago in a study that investigated the directionality of virus budding (46). It was shown that influenza virus, an enveloped virus that buds from the apical side of polarized epithelial cells, is released from free surfaces of such cells before they form polarized monolayers. In contrast, vesicular stomatitis virus, which buds at the basolateral side of a cell monolayer, was found to be released at cell-cell contact sites within aggregates composed of only a few, Rabbit Polyclonal to MRPS31 nonpolarized cells. Release from either surface likely secures efficient spread of these viruses. It was presumed that cytoskeletal changes, together with a Inolitazone dihydrochloride redistribution of membrane proteins involved in organizing activities at either free cell surfaces or at the adhesion sites, were responsible for the functional separation of the two plasma membrane areas, but the molecular details were not investigated at the time. Dissemination of human immunodeficiency virus type 1 (HIV-1) in infected individuals is thought to be driven both by virions released into the cleft of the so-called virological synapse (22) and by infection of cells with cell-free virus (for a review, see reference43). However, in vitro replication studies clearly show that this virus, like vesicular stomatitis virus, is released preferentially at the basolateral side of epithelial cells and evolved to be transmitted most efficiently at cell-cell interfaces (9,48), for instance, when replicating in lymphocytes (for reviews, see references21and40). One group of membrane proteins that has been implicated in organizing cell surfaces, particularly at sites of cell-cell contact, includes the so-called tetraspanins (recently reviewed in references18,25, and50). It is thought that the propensity of these small proteins to self-aggregate and to associate with other transmembrane proteins allows them to build temporary scaffolds for activities such as antigen presentation. Recently, we demonstrated that tetraspanins, including CD9 and CD63, coaccumulate at the plasma Inolitazone dihydrochloride membrane, thus forming tetraspanin-enriched microdomains (TEMs) (35). Our report also described that newly produced components of HIV-1 colocalize with surface TEMs in epithelial cells and in T lymphocytes. Together with other recent reports which showed budding of HIV-1 through tetraspanin-rich membrane segments in T cells, macrophages, and dendritic cells (2,7,23,53), our data suggested that tetraspanins are constituents of a universal HIV-1 exit gateway. Here we provide further evidence in support of this hypothesis. Incubation of HIV-1-producing cells with the anti-CD9 antibody K41 leads to extensive aggregation of tetraspanins at cell-cell contact sites. This process, which is accompanied by clustering of viral components at the same sites, results in inhibition of HIV-1 release. Additionally, the infectivity of virus released from K41-treated cells is diminished. In contrast, egress of influenza virus is not inhibited by K41. Evidently, influenza virus, whose spread in infected individuals does not depend on transmission at the cell-cell interface, exits through microdomains that are clearly distinct from surface TEMs through which HIV-1 buds. == MATERIALS AND METHODS == == Cell culture, plasmids, transfections, and antibodies. == HeLa, 293T, and CrFK cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Invitrogen). E6-1 Jurkat T cells (NIH AIDS Research and Reference Reagent Program, Rockville, MD) were grown in RPMI supplemented with 10% fetal bovine serum. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Jurkat cells were electroporated using a Bio-Rad Gene Pulser. The following plasmids were used: pNL4-3, pGagopt (codon-optimized Gag), pSRalpha-Env, pGagopt-CFP, and TSG101-YFP (kind gift of W..