Recruitment of Env to the HIV-1 T cell virological synapse by targeted and sustained Env recycling

HIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Using surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, we observed targeted accumulation and sustained recycling of Env between the endocytic recycling compartment (ERC) and the VS. We observed dynamic exchange of Env at the VS while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse indicate that retention of Env is not likely to be maintained by entrapment into an immobile Gag lattice or through immobilizing interactions with CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus.