50
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

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

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          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.

          Author Summary

          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.

          Related collections

          Most cited references59

          • Record: found
          • Abstract: found
          • Article: not found

          Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting.

          The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.

            Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.

              The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2009
                March 2009
                13 March 2009
                : 5
                : 3
                : e1000339
                Affiliations
                [1 ]Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
                [2 ]SAIC at NCI-Frederick, Frederick, Maryland, United States of America
                King's College London School of Medicine, United Kingdom
                Author notes

                Conceived and designed the experiments: VD FB. Performed the experiments: VD MPJ GAJ JAJ. Analyzed the data: VD FB. Wrote the paper: FB. Performed electron microscopy experiments: JdlC KN.

                Article
                08-PLPA-RA-0862R4
                10.1371/journal.ppat.1000339
                2651531
                19282983
                269f1417-fd82-468d-93da-c6b0d052cfa7
                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                History
                : 4 August 2008
                : 12 February 2009
                Page count
                Pages: 17
                Categories
                Research Article
                Virology/Immunodeficiency Viruses
                Virology/Virion Structure, Assembly, and Egress

                Infectious disease & Microbiology
                Infectious disease & Microbiology

                Comments

                Comment on this article