The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding.
Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.
Stuchell
Melissa D
MD
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132-3201, USA.
Garrus
Jennifer E
JE
Müller
Barbara
B
Stray
Kirsten M
KM
Ghaffarian
Sanaz
S
McKinnon
Rena
R
Kräusslich
Hans-Georg
HG
Morham
Scott G
SG
Sundquist
Wesley I
WI
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000378:metabolism*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics; D014779:Virus Replication; D000099177:Tsg101 Protein
D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D015683:Gene Products, gag; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein; C115245:SRN2 protein, S cerevisiae; C102954:VPS28 protein, S cerevisiae