RESUMO
The influenza virus RNA polymerase synthesizes three types of RNA: genomic vRNA, anti-genomic cRNA and mRNA. Both vRNA and cRNA are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mRNAs are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mRNA, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mRNA. We found that viral mRNA associates with the viral non-structural protein 1 (NS1), cellular poly(A)-binding protein 1 (PABP1), the 20 kDa subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eIF4E. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mRNP) complex. We propose a model in which viral mRNAs, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mRNAs for splicing, nuclear export and translation.
Assuntos
Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , RNA Viral/metabolismo , Linhagem Celular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.
Assuntos
Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Proteínas não Estruturais Virais/genéticaRESUMO
RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.
Assuntos
RNA Helicases DEAD-box/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Infecções por Vírus de RNA/imunologia , RNA Viral/imunologia , Animais , Linhagem Celular , Proteína DEAD-box 58 , Cães , Humanos , Interferons/imunologia , Camundongos , Vírus de RNA/fisiologia , Receptores de Superfície Celular , Receptores Imunológicos , Replicação ViralRESUMO
SC35M is a mouse-adapted variant of the highly pathogenic avian influenza virus SC35. We have previously shown that interspecies adaptation is mediated by mutations in the viral polymerase and that it is paralleled by the acquisition of high pathogenicity for mice. In the present study, we have compared virus spread and organ tropism of SC35 and SC35M in mice. We show that SC35 virus causes mild bronchiolitis in these animals, whereas infection with the mouse-adapted SC35M virus leads to severe hemorrhagic pneumonia with dissemination to other organs, including the brain. In SC35M-infected animals, viral RNA and viral antigen were detected in monocytes and macrophages, and SC35M, unlike SC35, replicated in lymphocyte and macrophage cultures in vitro. SC35M did not induce an adequate cytokine response but, unlike SC35, caused severe lymphopenia in mice. These observations suggest that the high efficiency of the SC35M polymerase is responsible for infection and depletion of lymphocytes and other white blood cells, which results in immune suppression and systemic virus spread.
