Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase

• Published in: Virology (New York, N.Y.) Vol. 433; no. 1; pp. 27 - 34
• Main Authors: Noble, Erin, Cox, Andrew, Deval, Jerome, Kim, Baek
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.11.2012
• Subjects: Biological Assay; Cations; Cations, Divalent; Coenzymes - metabolism; Cofactors; DNA; Endonuclease; Endonucleases - chemistry; Endonucleases - genetics; Endonucleases - metabolism; Fluorescence Resonance Energy Transfer; Genomes; Humans; Infectious Disease; Influenza A; Influenza A Virus; Influenza A virus - enzymology; Influenza A virus - genetics; Magnesium - metabolism; Manganese - metabolism; mRNA; Nucleotide sequence; Polymerase; Primers; Protein Multimerization; Protein structure; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA Caps - chemistry; RNA Caps - genetics; RNA Replicase - chemistry; RNA Replicase - genetics; RNA Replicase - metabolism; RNA, Viral - chemistry; RNA, Viral - genetics; Secondary structure; Substrate Specificity; Transcription; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Polymerase; Endonuclease; Influenza A Virus
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/j.virol.2012.07.008

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.