tetano
Editor, Senior Moderator
Virology. 2012 Jul 27. [Epub ahead of print]
Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.
Noble E, Cox A, Deval J, Kim B.
Source
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue Box 672, Rochester, NY 14642, USA.
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.
Copyright ? 2012 Elsevier Inc. All rights reserved.
PMID:
22841552
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22841552
Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.
Noble E, Cox A, Deval J, Kim B.
Source
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue Box 672, Rochester, NY 14642, USA.
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.
Copyright ? 2012 Elsevier Inc. All rights reserved.
PMID:
22841552
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22841552