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1.
J Proteome Res ; 17(4): 1474-1484, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29558158

RESUMO

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/química , RNA Mensageiro/antagonistas & inibidores , Proteínas não Estruturais Virais/farmacologia , Animais , Fator de Especificidade de Clivagem e Poliadenilação , Interações entre Hospedeiro e Microrganismos , Humanos , Immunoblotting , Imunoprecipitação , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Ligação Proteica , Fatores de Poliadenilação e Clivagem de mRNA
2.
J Proteome Res ; 15(5): 1639-48, 2016 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-27096427

RESUMO

Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC-MS/MS. By employing software to build biological process and protein-protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication.


Assuntos
Enzimas Reparadoras do DNA/fisiologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/química , Proteínas Nucleares/fisiologia , Proteômica/métodos , Fatores de Processamento de RNA/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Cromatografia Líquida , Humanos , Imunoprecipitação , Vírus da Influenza A Subtipo H1N1/fisiologia , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/análise
3.
J Virol ; 90(9): 4696-4705, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26912617

RESUMO

UNLABELLED: The NS1 protein encoded by influenza A virus antagonizes the interferon response through various mechanisms, including blocking cellular mRNA maturation by binding the cellular CPSF30 3' end processing factor and/or suppressing the activation of interferon regulatory factor 3 (IRF3). In the present study, we identified two truncated NS1 proteins that are translated from internal AUGs at positions 235 and 241 of the NS1 open reading frame. We analyzed the cellular localization and function of the N-truncated NS1 proteins encoded by two influenza A virus strains, Udorn/72/H3N2 (Ud) and Puerto Rico/8/34/H1N1 (PR8). The NS1 protein of PR8, but not Ud, inhibits the activation of IRF3, whereas the NS1 protein of Ud, but not PR8, binds CPSF30. The truncated PR8 NS1 proteins are localized in the cytoplasm, whereas the full-length PR8 NS1 protein is localized in the nucleus. The infection of cells with a PR8 virus expressing an NS1 protein containing mutations of the two in-frame AUGs results in both the absence of truncated NS1 proteins and the reduced inhibition of activation of IRF3 and beta interferon (IFN-ß) transcription. The expression of the truncated PR8 NS1 protein by itself enhances the inhibition of the activation of IRF3 and IFN-ß transcription in Ud virus-infected cells. These results demonstrate that truncated PR8 NS1 proteins contribute to the inhibition of activation of this innate immune response. In contrast, the N-truncated NS1 proteins of the Ud strain, like the full-length NS1 protein, are localized in the nucleus, and mutation of the two in-frame AUGs has no effect on the activation of IRF3 and IFN-ß transcription. IMPORTANCE: Influenza A virus causes pandemics and annual epidemics in the human population. The viral NS1 protein plays a critical role in suppressing type I interferon expression. In the present study, we identified two novel truncated NS1 proteins that are translated from the second and third in-frame AUG codons in the NS1 open reading frame. The N-terminally truncated NS1 encoded by the H1N1 PR8 strain of influenza virus that suppresses IRF3 activation is localized primarily in the cytoplasm. We demonstrate that this truncated NS1 protein by itself enhances this suppression, demonstrating that some strains of influenza A virus express truncated forms of the NS1 protein that function in the inhibition of cytoplasmic antiviral events.


Assuntos
Vírus da Influenza A/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Códon de Iniciação , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologia , Interferon beta/genética , Camundongos , Mutação , Fases de Leitura Aberta , Biossíntese de Proteínas , Transporte Proteico , Transcrição Genética , Proteínas não Estruturais Virais/química
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