RESUMO
BACKGROUND: The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila. RESULTS: In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1DeltaPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1DeltaPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release. CONCLUSION: Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , HIV-1/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação/genética , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , HIV-1/genética , Células HeLa , Humanos , Imunoprecipitação , Mutação , Ligação Proteica , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Montagem de Vírus , Replicação ViralRESUMO
The HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1(NL4-3)-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Membrana Nuclear/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Apoptose , Caspase 3/genética , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Sobrevivência Celular , HIV-1/metabolismo , Células HeLa/virologia , Humanos , Células Jurkat/virologia , Transfecção , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
Mutational analysis of the four conserved proline residues in human immunodeficiency virus type 1 (HIV-1) Vpr reveals that only Pro-35 is required for efficient replication of R5-tropic, but not of X4-tropic, viruses in human lymphoid tissue (HLT) cultivated ex vivo. While Vpr-mediated apoptosis and G(2) cell cycle arrest, as well as the expression and subcellular localization of Vpr, were independent, the capacity for encapsidation of Vpr into budding virions was dependent on Pro-35. (1)H nuclear magnetic resonance data suggest that mutation of Pro-35 causes a conformational change in the hydrophobic core of the molecule, whose integrity is required for the encapsidation of Vpr, and thus, Pro-35 supports the replication of R5-tropic HIV-1 in HLT.