RESUMO
Avian influenza viruses of subtype H9N2 that are found worldwide are occasionally transmitted to humans and pigs. Furthermore, by co-circulating with other influenza subtypes, they can generate new viruses with the potential to also cause zoonotic infections, as observed in 1997 with H5N1 or more recently with H7N9 and H10N8 viruses. Comparative analysis of the adaptive mutations in polymerases of different viruses indicates that their impact on the phylogenetically related H9N2 and H7N9 polymerases is higher than on the non-related H7N7 and H1N1pdm09 polymerases. Analysis of polymerase reassortants composed of subunits of different viruses demonstrated that the efficient enhancement of polymerase activity by H9N2-PB2 does not depend on PA and PB1. These observations suggest that the PB2 subunit of the H9N2 polymerase has a high adaptive potential and may therefore be an important pandemic risk factor.
Assuntos
Vírus da Influenza A Subtipo H9N2/enzimologia , Influenza Aviária/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Adaptação Biológica , Animais , Aves , Feminino , Humanos , Vírus da Influenza A Subtipo H9N2/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , RNA Polimerase Dependente de RNA/genética , Vírus Reordenados/enzimologia , Vírus Reordenados/genética , Suínos , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
The segmented genome of influenza viruses is translocated into the nucleus to initiate transcription and replication. The gene segments are present as viral ribonucleoprotein (vRNP) particles composed of RNA, multiple copies of the nucleoprotein (NP), and the polymerase subunits PB1, PB2 and PA. The PB2 subunit and each NP monomer contain a nuclear localisation signal (NLS) that binds to importin-α. To throw light on the role of the NLSs of NP and PB2 in nuclear transport, we have analysed the effect of mutation D701N, responsible for the exposure of the NLS domain of PB2, on the intracellular localisation of vRNPs. We show that exposure of PB2 NLS significantly enhances the amount of vRNPs present in the nucleus. These observations suggest that entry of vRNPs into the nucleus depends on controlled interplay of the NLSs of PB2 and NP with the nuclear import machinery.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H9N2/genética , RNA Polimerase Dependente de RNA/genética , Ribonucleoproteínas/metabolismo , Proteínas Virais/genética , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Mutação/genética , Sinais de Localização Nuclear/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , RNA Viral/genética , Replicação Viral/genéticaRESUMO
The cytoplasmic RNA helicase RIG-I mediates innate sensing of RNA viruses. The genomes of influenza A virus (FLUAV) are encapsidated by the nucleoprotein and associated with RNA polymerase, posing potential barriers to RIG-I sensing. We show that RIG-I recognizes the 5'-triphosphorylated dsRNA on FLUAV nucleocapsids but that polymorphisms at position 627 of the viral polymerase subunit PB2 modulate RIG-I sensing. Compared to mammalian-adapted PB2-627K, avian FLUAV nucleocapsids possessing PB2-627E are prone to increased RIG-I recognition, and RIG-I-deficiency partially restores PB2-627E virus infection of mammalian cells. Heightened RIG-I sensing of PB2-627E nucleocapsids correlates with previously established lower affinity of 627E-containing PB2 for nucleoprotein and is increased by further nucleocapsid instability. The effect of RIG-I on PB2-627E nucleocapsids is independent of antiviral signaling, suggesting that RIG-I-nucleocapsid binding alone can inhibit infection. These results indicate that RIG-I is a direct avian FLUAV restriction factor and highlight nucleocapsid disruption as an antiviral strategy.
Assuntos
RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , Nucleocapsídeo/imunologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58 , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleocapsídeo/genética , Nucleocapsídeo/fisiologia , Orthomyxoviridae , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Receptores Imunológicos , Replicação ViralRESUMO
UNLABELLED: Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza viruses. Here, we analyzed the role of the serine protease TMPRSS2, which activates HA in the human respiratory tract, in pathogenesis in a mouse model. Replication of the human H7N9 isolate A/Anhui/1/13 and of human H1N1 and H3N2 viruses was compared in TMPRSS2 knockout (TMPRSS2(-/-)) and wild-type (WT) mice. Knockout of TMPRSS2 expression inhibited H7N9 influenza virus replication in explants of murine tracheas, bronchi, and lungs. H1N1 virus replication was also strongly suppressed in airway explants of TMPRSS2(-/-) mice, while H3N2 virus replication was only marginally affected. H7N9 and H1N1 viruses were apathogenic in TMPRSS2(-/-) mice, whereas WT mice developed severe disease with mortality rates of 100% and 20%, respectively. In contrast, all H3N2 infected TMPRSS2(-/-) and WT mice succumbed to lethal infection. Cleavage analysis showed that H7 and H1 are efficiently activated by TMPRSS2, whereas H3 is less susceptible to the protease. Our data demonstrate that TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 and H1N1 influenza virus in mice. In contrast, replication of H3N2 virus appears to depend on another, not yet identified protease, supporting the concept that human influenza viruses differ in protease specificity. IMPORTANCE: Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza virus, but little is known about its relevance for pathogenesis in mammals. Here, we show that knockout mice that do not express the HA-activating protease TMPRSS2 are resistant to pulmonary disease with lethal outcome when infected with influenza A viruses of subtypes H7N9 and H1N1, whereas they are not protected from lethal H3N2 virus infection. These findings demonstrate that human influenza viruses differ in protease specificity, and that expression of the appropriate protease in respiratory tissues is essential for pneumotropism and pathogenicity. Our observations also demonstrate that HA-activating proteases and in particular TMPRSS2 are promising targets for influenza therapy.
