Dussupt Vincent, Javid Melodi P, Abou-Jaoudé Georges, Jadwin Joshua A, de La Cruz Jason, Nagashima Kunio, Bouamr Fadila
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Pathog. 2009 Mar;5(3):e1000339. doi: 10.1371/journal.ppat.1000339. Epub 2009 Mar 13.
HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(n)L, 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(n)L/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(n)L-mediated budding.
HIV-1的释放是由Gag蛋白p6区域的两个基序介导的，即PTAP和LYPX(n)L，它们分别招募细胞蛋白Tsg101和Alix。Gag蛋白的核衣壳区域（NC）与Alix的Bro1结构域结合，在HIV-1释放中也起着重要作用，但其潜在机制尚不清楚。在此我们表明，Bro1结构域的前202个残基（Bro(i)）足以与Gag结合。Bro(i)以NC依赖的方式干扰HIV-1的释放，并使病毒出芽停滞在质膜上。在过表达包含Bro1及其相邻V结构域的片段（Bro1-V）后，可观察到类似的出芽中断结构。尽管只有Bro1-V包含与CHMP4的结合决定簇，但Bro(i)和Bro1-V均通过PTAP/Tsg101和LYPX(n)L/Alix途径抑制释放，这表明它们干扰了HIV-1释放中的关键步骤。值得注意的是，我们发现过表达Bro1可挽救缺乏两个L结构域的HIV-1的释放。这种挽救需要Gag中NC结构域的N端区域和Bro1中的CHMP4结合位点。有趣的是，通过过表达Nedd4.2提供与ESCRT机制的联系，可挽救由于NC突变导致的Bro1介导的病毒释放挽救缺陷。我们的数据支持一种模型，其中NC在招募其L结构域活性所需的细胞蛋白方面与PTAP协同作用，并结合LYPX(n)L介导的出芽所需的Bro1-CHMP4复合物。
