Abrogating ALIX interactions results in stuttering of the ESCRT machinery

ESCRTs are cellular proteins that catalyze the fission of membranes and play an important role in biology of disease including cancer and infectious virus release1. ESCRT associated protein ALIX plays an essential role in HIV budding2-4, exosome release5,6, down regulation of G-protein coupled receptors7, cytokinesis8,9and multi vesicular budding1. The consensus view is that ALIX plays a role by binding to the viral late domains2-4/Syntenin late domain5/Cepp558,9and helps recruit downstream protein CHMP42,8,10,11which along with VPS4 catalyzes the fission of the membrane12,13. Using live imaging we have visualized the recruitment of ALIX, CHMP4 and VPS4 during budding of HIV with abrogated Gag-ALIX interactions. Based on the canonical view, we were expecting to find reduced recruitment of ALIX under these conditions. Instead we report observing multiple rounds of transient recruitment of ALIX, CHMP4 and VPS4 prior to virion release. We further show that during each, transient recruitment, stoichiometry of all ESCRT components remained the same regardless of mutations abrogating ALIX and Gag interaction. In addition, mutations abrogating interactions between Gag and TSG101 result in recruitment of ESCRTs with a substantial delay while maintaining similar stoichiometry. Our results demonstrate that recruitment of ESCRTs is driven by a robust network of interactions resulting in an “On/Off” switch behavior and ALIX’s interactions with late domains of HIV Gag play a crucial role during final catalysis of membrane fission after assembly of the full ESCRT machinery.