A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK Correspondence George G. Brownlee george.brownlee{at}path.ox.ac.uk The influenza A virus RNA-dependent RNA polymerase consists of three subunits PB1, PB2 and PA. The 5' and 3' terminal sequences of the viral RNA (vRNA) form the viral promoter and are bound by the PB1 subunit. The putative promoter-binding sites of the PB1 subunit have been mapped in previous studies but with contradictory results. The aim of the current study was to investigate the function of two evolutionary conserved regions in PB1 – from aa 233 to 249 and 269 to 281, which lie immediately N- and C-terminal, respectively, of a previously proposed binding site for the 3' end of the vRNA promoter. The previously proposed binding site extended from aa 249 to 256 and centred on two phenylalanine residues (F251 and F254). However, the fact that F251 is required for polymerase activity was not confirmed here. Instead, it was proposed that the 233–249 region contains a new 5' vRNA promoter-binding site, and arginine residues crucial for this activity were characterized. However, residues 269–281 were unlikely to be directly involved in promoter binding. These results are discussed in relation to the previous studies and a new model for vRNA promoter binding to the influenza RNA polymerase is presented. Supplementary figure showing primer extension data of steady-state levels of mRNA and cRNA isolated from cells expressing influenza A virus polymerase and vRNA-like CAT-RNA is available in JGV Online.