The human T-cell leukemia virus type 1 (HTLV-1) Gag polyprotein contains two adjacent proline-rich motifs (sequence PPPYEPTAP) in the C terminus of the matrix domain. Proline-to-alanine mutations were introduced into either or both motifs of HTLV-1 to determine the effect on the release of HTLV-1 virus-like particles from 293T cells. The release of both single mutants was significantly reduced, whereas a double mutation in both motifs abolished the release of the HTLV-1 particles. Two-hybrid and in vitro binding assays showed that the HTLV-1 Gag polyprotein binds both Tsg101 and Nedd4 proteins. The interaction with HTLV-1 Gag required the central WW domain of Nedd4 and the ubiquitin enzyme variant (UEV) domain of Tsg101. We expressed various fragments of Nedd4 and Tsg101 proteins in 293T cells and tested for their ability to interfere with virion release mediated by the HTLV-1 Gag-Pro polyprotein. Fragments consisting of the N-terminal UEV domain of Tsg101 and the central WW and C-terminal Hect domains of Nedd4 protein all caused transdominant inhibition of HTLV-1 particle release. Similarly, inhibition of the proteasome significantly decreased HTLV-1 particle release. Furthermore, the WW domain overexpression caused an early arrest of HTLV-1 particle morphogenesis before the membrane is deformed into the typical half-shell structure. This result suggests that Nedd4 is involved early in budding of HTLV-1.
A single letter (V for valine) was omitted from the L domain sequence motif, given as PPPYEPTAP in error, both in the title (which has been corrected above) and the body of the article. The correct sequence of the motif is PPPYVEPTAP, as was presented in Fig. 1. The sequences of the viral DNAs have been reexamined to confirm the presence of the valine codon in all constructs, both wild type and mutant. The conclusions of the paper are not affected.
The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in "lump-like" structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.Retroviral Gag polyproteins contain all the information necessary to carry out the processes of assembly, budding, and release of virus-like particles similar in size and morphology to those of infectious viral particles (54). To achieve efficient budding and exocytosis of virus from the cell, retroviral Gag polyproteins require an interaction, via their late-domain motifs, with proteins that function in the class E protein-sorting (Vps) pathway (38). The class E Vps proteins are organized into three endosomal complexes termed ESCRT-I, -II, and -III (for endosomal sorting complex required for transport) (3, 4, 24); these multiprotein complexes are believed to function normally at the surface of endosomal membranes where they are recruited by a protein called the hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) protein via its interaction with components of the ESCRT-I complex, Tsg101 and HCRP1/Vps37A (7,9,17,25,48). Hrs seems to play the role of a scaffold protein that sets the stage for a chain of multiple interactions that take place at the endosomal membrane (25, 42). These events appear to be triggered by the recognition of ubiquitin that has been conjugated to cellular cargo en route to the lysosome (11,25,42,44). The disassembly of these complexes and the release of ubiquitin are then catalyzed by an AAA ATPase called Vps4 (5, 6). Retroviral Gag proteins contain so-called late or L domains that recruit the ESCRT complexes by binding directly to Vps proteins or indirectly through proteins involved in the regulation of the Vps pathway. Thus, the human immunodefic...
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