Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse. Author Summary: Newly formed HIV-1 particles assemble at the plasma membrane of virus producing cells. The inner structural protein Gag and the envelope glycoprotein Env, which are both essential components of infectious virus particles, traffic to the membrane via different pathways. Attached to the inner side of the membrane, Gag assembles into spherical particles that incorporate Env proteins in their surrounding lipid envelope. The mechanism of Env incorporation is incompletely understood, however. Here, we have exploited recently developed super-resolution fluorescence microscopy techniques that yield a near-molecular spatial resolution to analyze HIV-1 Gag and Env distribution patterns at the surface of virus producing cells. We observed recruitment of Env to the surroundings of Gag assembly sites, dependent on the presence of its cytoplasmic domain. A large proportion of Env was found in the vicinity of the Gag assembly sites rather than directly co-localizing with it. These results support an indirect mechanism of Env recruitment, presumably mediated through virus induced changes in the environment of the nascent Gag assembly. Furthermore, they suggest a role for the Env protein in HIV-1 transmission that goes beyond its well-characterized function as an entry protein on the viral surface.