RESUMO
The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/virologia , Produtos do Gene gag/metabolismo , HIV/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/fisiologia , Eliminação de Partículas Virais/fisiologia , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Endossomos/ultraestrutura , Endossomos/virologia , Células Eucarióticas/metabolismo , HIV/patogenicidade , HIV/ultraestrutura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Mimetismo Molecular/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Vesículas Transportadoras/virologia , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.