Alix Arrives Late During HIV-1 Assembly

Abstract

Human immunodeficiency virus (HIV-1) assembles at the plasma membrane of infected cells and is released after the membrane envelope surrounding the virus is separated from the plasma membrane. Virus assembly is driven by the viral Gag polyprotein. Gag also recruits endosomal sorting complex required for transport (ESCRT) proteins that provide the machinery required for membrane fission. Previous studies analyzing the dynamics of assembly and release of retroviruses at the plasma membrane of living cells using fluorescently labeled proteins have given us great insight into the behavior of HIV-1 and ESCRT proteins. However, the dynamics of the associated ESCRT protein ALIX has not been analyzed for HIV-1. Using a library of linkers to fuse ALIX to green fluorescent proteins (GFP) we identified a construct that functions like wild type in the context of viral budding. Using total internal reflection fluorescence microscopy we successfully followed the assembly kinetics of HIV-1 and ALIX, like other ESCRT proteins, arrived with burst like recruitment near the end of Gag assembly. These results are in contrast to the observed early recruitment of ALIX into EIAV budding sites and open new questions about early recruitment of ESCRT into HIV-1 budding sites