HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX nL, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro i) are sufficient to bind Gag. Bro i interferes with HIV-1 release in an NC–dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Bro i and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX nL/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1–CHMP4 complex required for LYPX nL–mediated budding.
Human immunodeficiency virus type 1 (HIV-1) assembles its structural protein Gag into a viral shell at the plasma membrane. Gag is divided into several regions, each with its own distinct function(s). Within the p6 region of Gag, there are two short peptide sequences, called Late (L) domains, that serve to recruit cellular proteins Tsg101 and Alix. In an uninfected cell, these proteins facilitate membrane dynamics during vesicle budding into cellular compartments called endosomes. Upon infection, HIV-1 hijacks these proteins and makes use of the machinery to facilitate viral budding at the plasma membrane. Our study shows that, in addition to binding the p6 region, Alix also interacts with the Nucleocapsid (NC) region of Gag. Importantly, we show that when HIV-1 buds via the Alix-driven pathway, this interaction with NC is essential for recruiting host proteins necessary for HIV-1 release. Moreover, we show that a non-functional fragment of Alix inhibits Tsg101-mediated HIV-1 release in ways similar to those caused by mutations in the NC domain of Gag. Collectively, our findings favor a model in which the p6-located L domain motifs require cooperation with NC to facilitate HIV-1 release.