RESUMO
The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain 'X' of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232-252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5-8% increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233-252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by â¼ 50%, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Humana/virologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Motivos de Aminoácidos , Animais , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Vírus da Influenza A Subtipo H9N2/química , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , VirulênciaRESUMO
PA-X is a novel protein encoded by PA mRNA and is found to decrease the pathogenicity of pandemic 1918 H1N1 virus in mice. However, the importance of PA-X proteins in current epidemiologically important influenza A virus strains is not known. In this study, we report on the pathogenicity and pathological effects of PA-X deficient 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza H5N1 viruses. We found that loss of PA-X expression in pH1N1 and H5N1 viruses increased viral replication and apoptosis in A549 cells and increased virulence and host inflammatory response in mice. In addition, PA-X deficient pH1N1 and H5N1 viruses up-regulated PA mRNA and protein synthesis and increased viral polymerase activity. Loss of PA-X was also accompanied by accelerated nuclear accumulation of PA protein and reduced suppression of PA on non-viral protein expression. Our study highlights the effects of PA-X on the moderation of viral pathogenesis and pathogenicity.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Virais/genética , Transporte Ativo do Núcleo Celular , Animais , Apoptose/genética , Linhagem Celular , Modelos Animais de Doenças , Deleção de Genes , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Biossíntese de Proteínas , Transporte Proteico , Virulência/genética , Replicação Viral/genéticaRESUMO
Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV-host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.