We previously demonstrated, by limited mutagenesis, that conserved sequence 
elements within the 5' end of influenza virus virion RNA (vRNA) are required for 
the polyadenylation of mRNA in vitro. To further characterize the nucleotide 
residues at the 5' end of vRNA which might be involved in polyadenylation, a 
complete set of short and long model vRNA-like templates with mutations at 
nucleotides 1' to 13' (prime notation denotes numbering from the 5' end) of vRNA 
were synthesized and transcribed in vitro. The products were assayed for mRNA 
production with both reverse transcription-PCR and [alpha-32P]ATP incorporation 
assays. Results from these independent assays showed that vRNA templates with 
point mutations at positions 2', 3', 7' to 9', and 11' to 13' synthesized 
polyadenylated transcripts inefficiently compared with those with 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. Furthermore, residues at positions 11' 
to 13' are known to be involved in base pairing between the 3' and 5' ends of 
vRNA. These findings demonstrate that the RNA polymerase has to bind to the 5' 
end of the template vRNA, which must then interact with the 3' end of the same 
template for polyadenylation to occur. These results support a model in which a 
cis-acting RNA polymerase is required for the polyadenylation of influenza 
virus.