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1.
Front Microbiol ; 10: 432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30894843

RESUMO

The influenza virus protein PA-X modulates the host immune responses and viral pathogenicity through suppression of host protein expression. The endonuclease active site in the N-terminal region, the basic amino acid cluster in the C-terminal PA-X-specific region, and N-terminal acetylation of PA-X by NatB are important for the shutoff activity of PA-X. Here, we focused on the shutoff activity of PA-X derived from the A/California/04/2009 and A/WSN/33 viruses because these two PA-X proteins differ in their shutoff activity. Mutagenesis analysis revealed that proline and serine at positions 28 and 65, respectively, play a central role in this difference. Furthermore, we found that P28 and S65 also affect the shutoff activity of PA-X derived from other influenza virus subtypes. These data demonstrate that P28 and S65 contribute to enhanced shutoff activity of PA-X.

2.
Cell Rep ; 24(4): 851-860, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30044982

RESUMO

N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.

3.
Virology ; 516: 71-75, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29331676

RESUMO

The influenza A virus protein PA-X comprises an N-terminal PA region and a C-terminal PA-X-specific region. PA-X suppresses host gene expression, termed shutoff, via mRNA cleavage. Although the endonuclease active site in the N-terminal PA region of PA-X and basic amino acids in the C-terminal PA-X-specific region are known to be important for PA-X shutoff activity, other amino acids may also play a role. Here, we used yeast to identify novel amino acids of PA-X that are important for PA-X shutoff activity. Unlike wild-type PA-X, most PA-X mutants predominantly localized in the cytoplasm, indicating that these mutations decreased the shutoff activity of PA-X by affecting PA-X translocation to the nucleus. Mapping of the identified amino acids onto the N-terminal structure of PA revealed that some of them likely contribute to the formation of the endonuclease active site of PA.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Influenza Humana/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas não Estruturais Virais/genética
4.
Cell Host Microbe ; 22(5): 615-626.e8, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29056430

RESUMO

Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.


Assuntos
Furões/virologia , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Animais , Antivirais/farmacologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , Galinhas/virologia , Túnica Conjuntiva/patologia , Túnica Conjuntiva/virologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Influenza Aviária/virologia , Pulmão/patologia , Pulmão/virologia , Macaca/virologia , Camundongos , Neuraminidase/efeitos dos fármacos , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Replicação Viral
5.
Vaccine ; 35(15): 1892-1897, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28285982

RESUMO

Vaccination is the first line of protection against influenza virus infection in humans. Although inactivated and live-attenuated vaccines are available, each vaccine has drawbacks in terms of immunogenicity and safety. To overcome these issues, our group has developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2-expressing cells. Here we generated PB2-KO viruses possessing the hemagglutinin (HA) and neuraminidase (NA) segments from H1N1pdm09 or type B viruses and tested their vaccine potential. The two PB2-KO viruses propagated efficiently in PB2-expressing cells, and expressed chimeric HA as expected. Virus-specific IgG and IgA antibodies were detected in mice immunized with the viruses, and the immunized mice showed milder clinical signs and/or lower virus replication levels in the respiratory tract upon virus challenge. Our results indicate that these PB2-KO viruses have potential as vaccine candidates.


Assuntos
Técnicas de Inativação de Genes , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Proteínas Virais/genética , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza B/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Sistema Respiratório/virologia , Carga Viral
6.
EBioMedicine ; 17: 182-191, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28286060

RESUMO

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development.


Assuntos
Anticorpos Monoclonais/imunologia , Hemaglutininas/imunologia , Vírus da Influenza A/fisiologia , Liberação de Vírus , Animais , Afinidade de Anticorpos , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cães , Epitopos/imunologia , Células HEK293 , Células HeLa , Hemaglutininas/química , Hemaglutininas/genética , Humanos , Vírus da Influenza A/imunologia , Células Madin Darby de Rim Canino , Camundongos , Replicação Viral
7.
J Virol ; 89(16): 8661-5, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26041295

RESUMO

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.


Assuntos
Endonucleases/genética , Vírus da Influenza A/enzimologia , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Endonucleases/metabolismo , Luciferases , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Proteínas não Estruturais Virais/metabolismo
8.
Vet Ital ; 43(1): 55-64, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-20411501

RESUMO

The Animal Products Safety Division (APSD) of Japan was created in the Ministry of Agriculture, Forestry and Fisheries in October 2005 to strengthen the role of the Ministry in animal production food safety. The authors outline the background to the establishment of the APSD and its functions. The APSD is engaged in administration related to risk management during the production stage of terrestrial and aquatic animal products. The APSD endeavours to ensure that risk management measures are taken in accordance with the relevant laws, regulations and administrative notifications and that they are based on risk assessment and science. The APSD works in close collaboration with other national agencies, prefecture governments, foreign governments and international organisations.

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