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Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.
Noble, Erin; Cox, Andrew; Deval, Jerome; Kim, Baek.
Afiliação
  • Noble E; Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, USA.
Virology ; 433(1): 27-34, 2012 Nov 10.
Article em En | MEDLINE | ID: mdl-22841552
ABSTRACT
The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5' end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Vírus da Influenza A / Proteínas Virais / RNA Polimerase Dependente de RNA / Capuzes de RNA / RNA Viral / Endonucleases Idioma: En Ano de publicação: 2012 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Vírus da Influenza A / Proteínas Virais / RNA Polimerase Dependente de RNA / Capuzes de RNA / RNA Viral / Endonucleases Idioma: En Ano de publicação: 2012 Tipo de documento: Article