RESUMO
Several viruses encode ion channels that both modulate the trafficking of envelope glycoprotein(s) and stimulate the release of virions from cells. HIV-1 Vpu enhances virion release and inhibits the endosomal accumulation of the viral structural protein Gag. We investigated whether Vpu affects the subcellular distribution of Env as well as Gag. Env and Vpu colocalized with each other, in part within the trans-Golgi network. In the absence of Vpu, Env accumulated more extensively within clathrin-coated endosomal structures. These structures had several features consistent with an endosomal viral assembly domain: they contained Gag, including proteolytically processed viral matrix protein; the tetraspanins CD63 and CD81; the adaptor protein complex AP-3; and AIP1/ALIX, a cellular cofactor for viral budding. These endosomes labelled incompletely with Env derived from the cell surface, suggesting that some Env reaches this compartment without transiting the plasma membrane. Consistent with this, endosomal accumulation of Env was not blocked by dominant-negative Eps15, an inhibitor of AP-2-mediated endocytosis. Although these data are potentially explained by greater endocytosis of mature virions in the absence of Vpu, they also raise the possibility that Vpu inhibits the transport of Env and Gag to late endosomes, leading to viral assembly at the plasma membrane.
Assuntos
Endossomos/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Antígenos CD/metabolismo , Linhagem Celular , Clatrina/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana/análise , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Tetraspanina 28 , Tetraspanina 30 , Proteínas Virais Reguladoras e Acessórias/análise , Produtos do Gene env do Vírus da Imunodeficiência Humana/análise , Produtos do Gene gag do Vírus da Imunodeficiência Humana/análiseRESUMO
The HIV-1 accessory protein Vpu counteracts a host factor that restricts virion release from infected cells. Here we show that the interferon-induced cellular protein BST-2/HM1.24/CD317 is such a factor. BST-2 is downregulated from the cell surface by Vpu, and BST-2 is specifically expressed in cells that support the vpu phenotype. Exogenous expression of BST-2 inhibits HIV-1 virion release, while suppression of BST-2 relieves the requirement for Vpu. Downregulation of BST-2 requires both the transmembrane/ion channel domain and conserved serines in the cytoplasmic domain of Vpu. Endogenous BST-2 colocalizes with the HIV-1 structural protein Gag in endosomes and at the plasma membrane, suggesting that BST-2 traps virions within and on infected cells. The unusual structure of BST-2, which includes a transmembrane domain and a lumenal GPI anchor, may allow it to retain nascent enveloped virions on cellular membranes, providing a mechanism of viral restriction counteracted by a specific viral accessory protein.
Assuntos
Antígenos CD/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas Virais Reguladoras e Acessórias/fisiologia , Vírion/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Regulação para Baixo , Endossomos/metabolismo , Proteínas Ligadas por GPI , Humanos , Interferons/biossíntese , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The cytoplasmic domain of the HIV-1 Env glycoprotein (gp41) contains sequences that affect the trafficking of Env within the host cell. We previously showed that the membrane-proximal tyrosine-based adaptor protein (AP)-binding signal of gp41 (Y712XXL) is required for optimal viral infectivity and entry into target cells. Because these effects were not attributable to an effect on the incorporation of Env into virions, we hypothesized that they involved targeting of viral assembly to specific endosomal membranes that conferred greater fusogenicity. To further elaborate this hypothesis, we mutated the C-terminal leucine-based AP-binding signal of gp41 (LL855/856). In contrast to Env Y712, the leucine signal was dispensable for viral infectivity in both single cycle assays and during spreading infections within cultures of peripheral blood mononuclear cells (PBMCs). To test the hypothesis that these AP-binding motifs target Env to endosomes during viral morphogenesis, we compared the subcellular localization of wild-type Env to mutants of the Y712 and LL855/856 signals. The results failed to support the hypothesis that these signals target viral assembly to specific endosomal membranes. Strikingly, in the context of a C2-V3 region that confers macrophage-tropism, mutation of Y712 no longer markedly affected viral infectivity in either single cycle assays or during spreading infection within PBMCs, and it did not impair viral entry. These data indicate that the importance of the tyrosine-based sorting signal in gp41 for optimal viral infectivity depends on sequences in gp120. This observation is consistent with the hypothesis that the Y712 residue is part of the ectodomain of gp41 in virion-associated Env. We speculate that as part of the ectodomain, Y712 could affect specifically the conformation of the more positively charged CXCR4-tropic V3 loop in a manner that augments viral fusogenicity and infectivity.
Assuntos
Membrana Celular/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/fisiologia , HIV-1/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Motivos de Aminoácidos , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Células HeLa , Humanos , Sinais Direcionadores de Proteínas/genética , Tirosina/metabolismo , Vírion/metabolismoRESUMO
The nef gene contributes to the replication of primate lentiviruses by altering the trafficking of cellular proteins involved in adaptive immunity (class I and II major histocompatibility complex [MHC]) and viral transmission (CD4 and DC-SIGN). A conserved acidic leucine-based sequence (E(160)xxxLL) within human immunodeficiency virus type 1 (HIV-1) Nef binds to the cellular adaptor protein (AP) complexes, which mediate protein sorting into endosomal vesicles. The leucine residues in this motif are required for the down-regulation of CD4 and for the up-regulation of DC-SIGN and the invariant chain of MHC class II, but the role of the acidic residue is unclear. Here, substitution of E160 with uncharged residues impaired the ability of Nef to up-regulate the expression of the invariant chain and DC-SIGN at the cell surface, whereas substitution with a basic residue was required for a similar effect on the down-regulation of CD4. All substitutions of E160 relieved the Nef-mediated block to transferrin uptake. E160 was required for the efficient interaction of Nef with AP-1 and AP-3 and for the stabilization of these complexes on endosomal membranes in living cells. Systematic mutation of the ExxxLL sequence together with correlation of binding and functional data leads to the hypotheses that AP-1 and AP-3 are major cofactors for the effect of Nef on the trafficking of transferrin, are less important but contribute to the modulation of the invariant chain and DC-SIGN, and are least critical for the modulation of CD4. The data suggest that the E160 residue plays a differential role in the modulation of leucine-dependent Nef-targets and support a model in which distinct AP complexes are used by Nef to modulate different cellular proteins.
Assuntos
Produtos do Gene nef/metabolismo , HIV-1/fisiologia , Transporte Proteico , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Antígenos CD4/análise , Moléculas de Adesão Celular/análise , Linhagem Celular , Membrana Celular/metabolismo , Endossomos/química , Produtos do Gene nef/química , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Membranas Intracelulares/metabolismo , Lectinas Tipo C/análise , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores de Superfície Celular/análise , Transferrina/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.