Role of ESCRT-I in retroviral budding.
Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.
Martin-Serrano
Juan
J
Aaron Diamond AIDS Research Center and The Rockefeller University, New York, New York 10016, USA.
Zang
Trinity
T
Bieniasz
Paul D
PD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D056827:Endosomal Sorting Complexes Required for Transport; D015497:HIV-1 / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D051379:Mice; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D014771:Virion / Q000378:metabolism; D000099177:Tsg101 Protein
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein; C102954:VPS28 protein, S cerevisiae