Project Summary HIV-1 establishes a chronic infection that the immune system cannot spontaneously clear. The virus has a remarkable capacity to evade immune responses and generates a high sequence diversity that tolerates defective genes. We and others have found that viral dissemination takes place both in vitro and in vivo through cell-cell contacts, called virological synapses (VS), which can help mask immune detection of infected cells, and promote viral quasispecies diversity that enable escape. Our data indicate that HIV Env, the central viral protein involved in VS formation and viral entry, is regulated during the process of cell-to-cell transmission and assumes distinct conformations on the cell surface versus the virus particle. We examine how low abundance, rapid turnover and heterogeneity of processing of Env at the cell surface contributes to diminished antigenicity of infected cells as compared to abundant cell-free virus or shed glycoprotein. The studies proposed here test a model whereby the sequence of Env trafficking--to the cell surface, to the recycling endosome, and then to the virus particle--supports distinct antigenic states along this pathway. In the prior study periods we have learned that during VS formation Env works as a cell adhesion receptor between the infected and uninfected cell, prior to its role as viral membrane fusion protein. HIV exploits cell biology including the polarized receptor recruitment and viral endocytosis into the target cell, to enhance cell-to-cell transmission. The T cell VS is critical for viral spread in cell culture and functions in vivo in lymphoid tissues of humanized mice. Most broadly neutralizing antibodies (bNAbs) are less potent at neutralizing cell-to-cell infection than the same virus in a cell-free form. When tested against transmitted founder clones, bNAbs frequently fail to inhibit 100% of cell-to-cell infection at maximum concentration, i.e. display reduced efficacy. In the continuation of these studies, we will define the cellular mechanisms underlying the reduced potency and efficacy of neutralizing antibodies against the VS. We will also test a model for how the multicopy transmission of HIV through VS contributes to maintaining a diverse swarm of mutated sequences, or quasispecies, that promotes immune escape. We hypothesize that cell-to-cell HIV-1 transmission is a pivotal immune evasion and escape strategy that drives viral persistence.