THE RNA-POLYMERASE OF INFLUENZA-VIRUS, BOUND TO THE 5' END OF VIRION RNA, ACTS IN CIS TO POLYADENYLATE MESSENGER-RNA

We previously demonstrated, by limited mutagenesis, that conserved seq uence elements within the 5' end of influenza virus virion RNA (vRNA) are required for the polyadenylation of mRNA in vitro. To further char acterize the nucleotide residues at the 5' end of vRNA which might be involved in polyadenylation, a complete set of short and long model vR NA-like templates with mutations at nucleotides 1' to 13' (prime notat ion denotes numbering from the 5' end) of vRNA were synthesized and tr anscribed in vitro. The products mere assayed for mRNA production with both reverse transcription-PCR and [alpha-P-32]ATP incorporation assa ys. Results from these independent assays showed that vRNA templates w ith point mutations at positions 2', 3', 7' to 9', and 11' to 13' synt hesized polyadenylated transcripts inefficiently compared with those w ith mutations at positions 1', 4' to 6', and 10', Positions 2', 3', 7' to 9', and 11' are known to be involved in RNA polymerase binding. Fu rthermore, residues at positions 11' to 13' are known to be involved i n base pairing between the 3' and 5' ends of vRNA. These findings demo nstrate that the RNA polymerase has to bind to the 5' end of the templ ate vRNA, which must then interact with the 3' end of the same templat e for polyadenylation to occur. These results support a model in which a cis-acting RNA polymerase is required for the polyadenylation of in fluenza virus.