Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus.

A Kato,A Ishihama,A Honda,K Mizumoto,K Kawakami

doi:10.1016/s0021-9258(18)67540-4

Abstract

The influenza virus-associated RNA polymerase cleaves capped RNA in an endonucleolytic manner and the transcription is initiated by the addition of GMP, the first substrate to be polymerized under the direction of viral RNA template, onto 3'-termini of resulting capped RNA fragments. In the presence of high concentrations of GTP as a sole substrate, multiple GMP residues were polymerized onto the primers. By the addition of the second substrate CTP, excess GMP residues, other than the 1st residue, were removed prior to elongation. The result may suggest that the RNA-dependent RNA polymerase carries a proofreading function.

Highlights

Theinfluenzavirus-associated RNA polymerase cleaves capped RNAin an endonucleolytic mannaenrd the transcriptionis initiated by the addition of GMP, the first substrate to be polymerized under the direction of viral RNA template, onto 3’-termionfiresulting capped RNA fragments
Alkalinephosphatase was a product of Boehringer, while polynucleotide phosphoryiase of Micrococcus luteus was from P-L Biochemicals
In the presenceof high concentrations of G T P as a sole substrate; 1-5 GMP residues are incorporated into a primer

Summary

MATERIALS ANDMETHODS

Virus-Influenza virus A/PR/8/34 (H1N1) was used throughout this study. Virus was grown in allantoic sacs of 10-day-oldembryonated eggs for 48 h a t 35.5 “C. Vaccinia virus was supplied by Dr T. Triphospha ~was from New England Nuclear Research Products. Ture were treated with influenza virus-associated capped RNA endonuclease and fractionated by 20%polyacrylamide gel electrophoresis in the presence of 8 M urea. Cleavage products formed discrete bands on gels, which were eluted by soaking in 0.5 M ammonium acetate and 1 mM EDTA. A n a ~ yosf ~ ~ l e o t ~ s - ~ oanndool-igonucleotideswere analyzed by either thin layer chromatography on a polyethyleneimine sheet or electrophoresis on a DEAE-cellulose sheet according to Honda et al [10].~ hromato ~ a p hwyas developed with LiCl solution while electrophoresis was performed in acetic acid/pyridine solution

ProofreaFduinngction of InfluVePRnioNrzluaAysmerase

Sample Number

Sample number

Proofreading Function of Influenza VirRuNs A Polymerase rn

DISCUSSION