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

Virology. 2012 Nov 10;433(1):27-34. doi: 10.1016/j.virol.2012.07.008. Epub 2012 Jul 28.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biological Assay
  • Cations, Divalent
  • Coenzymes / metabolism
  • Endonucleases / chemistry
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Influenza A virus / enzymology*
  • Influenza A virus / genetics
  • Magnesium / metabolism
  • Manganese / metabolism
  • Protein Multimerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA Caps / chemistry*
  • RNA Caps / genetics
  • RNA, Viral / chemistry*
  • RNA, Viral / genetics
  • RNA-Dependent RNA Polymerase / chemistry
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Cations, Divalent
  • Coenzymes
  • PA protein, influenza viruses
  • RNA Caps
  • RNA, Viral
  • Recombinant Proteins
  • Viral Proteins
  • Manganese
  • RNA-Dependent RNA Polymerase
  • Endonucleases
  • Magnesium