The Phe105 Loop of Alix Bro1 Domain Plays a Key Role in HIV-1 Release

SummaryAlix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical “boomerang” folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix

The Nucleocapsid Region of HIV-1 Gag Cooperates with the PTAP and LYPXnL Late Domains to Recruit the Cellular Machinery Necessary for Viral Budding

HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPXnL, 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 (Broi) are sufficient to bind Gag. Broi 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 Broi and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPXnL/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 LYPXnL–mediated budding

Mannose Receptor 1 Restricts HIV Particle Release from Infected Macrophages

Human mannose receptor 1 (hMRC1) is expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages (MDMs) and is involved in the endocytic uptake of HIV-1 into these cells. Here, we identify hMRC1 as an antiviral factor that inhibits virus release through a bone marrow stromal antigen 2 (BST-2)-like mechanism. Virions produced in the presence of hMRC1 accumulated in clusters at the cell surface but were fully infectious. HIV-1 counteracted the effect by transcriptional silencing of hMRC1. The effect of hMRC1 was not virus isolate specific. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1 antiviral activity, suggesting the involvement of additional cellular factor(s) in the process. Our data reveal an antiviral mechanism that is active in primary human macrophages and is counteracted by HIV-1 through downregulation of hMRC1