Document Detail


Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.
MedLine Citation:
PMID:  22841552     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Erin Noble; Andrew Cox; Jerome Deval; Baek Kim
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-07-28
Journal Detail:
Title:  Virology     Volume:  433     ISSN:  1096-0341     ISO Abbreviation:  Virology     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-09-17     Completed Date:  2013-01-04     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0110674     Medline TA:  Virology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  27-34     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Biological Assay
Cations, Divalent
Coenzymes / metabolism
Endonucleases / chemistry,  genetics,  metabolism*
Fluorescence Resonance Energy Transfer
Humans
Influenza A virus / enzymology*,  genetics
Magnesium / metabolism
Manganese / metabolism
Protein Multimerization
Protein Structure, Secondary
Protein Structure, Tertiary
RNA Caps / chemistry*,  genetics
RNA Replicase / chemistry,  genetics,  metabolism*
RNA, Viral / chemistry*,  genetics
Recombinant Proteins / chemistry,  genetics,  metabolism
Substrate Specificity
Viral Proteins / chemistry,  genetics,  metabolism*
Grant Support
ID/Acronym/Agency:
266200700008C//PHS HHS; HHSN266200700008C/AI/NIAID NIH HHS; T32 DE007202/DE/NIDCR NIH HHS; T32 DE007202/DE/NIDCR NIH HHS; T32GM07356/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Cations, Divalent; 0/Coenzymes; 0/PA protein, influenza viruses; 0/RNA Caps; 0/RNA, Viral; 0/Recombinant Proteins; 0/Viral Proteins; 42Z2K6ZL8P/Manganese; EC 2.7.7.48/RNA Replicase; EC 3.1.-/Endonucleases; I38ZP9992A/Magnesium
Comments/Corrections

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