RESUMO
A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.
Assuntos
Compartimento Celular , Membrana Celular/metabolismo , Endocitose , Produtos do Gene env/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Antígenos CD4/genética , Antígenos CD4/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Clatrina , Invaginações Revestidas da Membrana Celular , Regulação Viral da Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/ultraestrutura , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/ultraestrutura , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Relação Estrutura-Atividade , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/ultraestruturaRESUMO
Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.
Assuntos
Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Proteínas Oncogênicas de Retroviridae/ultraestrutura , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Lipossomos/química , Microscopia Eletrônica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/genéticaRESUMO
We observed expression of envelope gp46 protein on an HTLV-II-producing T-cell line (Mo) cells by an immunoelectron microscopical method using a monoclonal antibody against HTLV-II gp46. gp46 reactivity was observed on virus-like particles, extracellular vesicles, cell membrane, and partially in nuclear membrane and endoplasmic reticulum.