After 24 h, cells were labeled with 2 M eFluor 670 dye (eBioscience, Inc.) and combined at a 1:1 ratio with activated uninfected target cells from autologous donors. in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to computer virus particle maturation but which can still engage in cell-to-cell contamination was tested for the ability to resist neutralizing antibodies. The CT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated contamination, and had reduced or no effect on cell-free contamination, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of important neutralizing epitopes during cell-to-cell contamination and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations uncovered around the infected cell surface to enhance protection against VS-mediated HIV-1 spread. INTRODUCTION The ability of HIV-1 to evade neutralizing antibody responses represents a major obstacle to the creation of an effective vaccine. The failure of HIV-1 vaccines is usually often attributed to the high sequence variability and conformational plasticity of the major neutralizing antigen, envelope glycoprotein (Env) (13, 39). The functional Env subunit is usually a trimeric spike made of gp120-gp41 heterodimers, which mediate viral access during contamination with both cell-free and cell-associated viral sources (5, 17). Cell-free contamination of CD4+ T cells entails the release of viral particles from a productively infected cell, fluid-phase particle diffusion, viral attachment, and access into an uninfected cell (28). Direct T cell-to-T cell contamination occurs through contact between an infected donor T cell and an uninfected target T cell, resulting in the formation of an infectious cell-cell adhesion referred to as a virological synapse (VS) (17, 18). During VS-mediated contamination, it has been proposed that computer virus particles may bud into the synapse and fuse directly with the plasma membrane at the synaptic space (34). However, a number of studies MULK support a model for access following VS formation involving two actions: (i) coreceptor-independent, coordinated transfer of viral particles into GDC0994 (Ravoxertinib) the target cell endocytic compartment (cell-to-cell transfer) followed by (ii) coreceptor-dependent fusion of the viral and cellular membranes within the endocytic compartment (VS-mediated contamination) (3, 7, 29, 34). In support of this model, T cell VS was found to transfer into target cells immature HIV-1 particles which undergo viral membrane fusion only after proteolytic maturation of the viral core (7). In cell-free computer virus particles, the gp41 cytoplasmic tail (CT) controls Env fusogenicity through inside-out allosteric mechanisms (16, 25, 45). These studies show that during computer virus particle production, the conversation of gp41 CT with Pr55Gag maintains Env in a prefusogenic conformation. After computer virus budding, cleavage of Pr55Gag and subsequent particle maturation relieve the inhibitory function of gp41 to activate Env fusogenicity. Thus, the gp41 CT plays an GDC0994 (Ravoxertinib) important role in regulating the fusogenic potential of Env during the computer virus life cycle. On cell-free HIV-1, the gp41 CT is usually important in regulating the exposure of both neutralizing and nonneutralizing epitopes around the Env ectodomain of mature computer virus particles (19). The fusogenicity of Env and the exposure of CD4-induced (CD4i) epitopes are enhanced in gp41 CT truncation mutants when tested with pseudovirion contamination assays and cell-cell fusion assays (9, 46). During VS-mediated contamination, the cell-surface Env functions first as a cell adhesion molecule and then as the viral membrane fusion apparatus (15). In this pathway, Env does not mediate membrane fusion until after the computer virus particle has undergone maturation (7). While the gp41 CT is not required for VS formation or subsequent contamination (5, 10, 23), it does enhance the efficiency of cell-to-cell contamination in nonpermissive cell types (10). A number of broadly neutralizing monoclonal antibodies (MAbs) and peptide inhibitors have been tested for their ability to block cell-to-cell HIV-1 transfer or VS-mediated contamination (5, 11, 21, 22, 33). To date, only antibodies that block Env-CD4 interaction have been shown to inhibit both cell-to-cell transfer and subsequent VS-mediated contamination. Other neutralizing MAbs and access inhibitors have been found to block contamination from cell-associated HIV-1 after the transfer of computer virus across the VS. Using an indirect assay to measure increased HIV-1 DNA following coculture of donor and target cells, one GDC0994 (Ravoxertinib) study reported that VS-mediated contamination could be inhibited by all neutralizing antibodies tested (21). Other studies have found that sera from HIV-1-positive patients are much less effective at blocking cell-to-cell transfer (5, 7) and VS-mediated contamination (15) than cell-free GDC0994 (Ravoxertinib) HIV-1 contamination. These studies on patient sera suggest that quantitative differences in neutralization sensitivity are likely to be.