A hairpin loop at the 5 ' end of influenza a virus virion RNA is required for synthesis of poly(A)(+) mRNA in vitro

We present evidence, based on extensive mutagenesis, that a hairpin loop at the 5' end of influenza A virus virion RNA (vRNA) is required for the synt hesis of polyadenylated mRNA from model vRNA templates in vitro. The hairpi n loop, which we term the vRNA 5' hook, contains a stem of 2 bp formed by t he second and third residues pairing with the ninth and eighth residues, re spectively, and a 4-nucleotide loop composed of the intervening residues 4 to 7. Disruption of the base pairs of the vRNA 5' hook by introducing point mutations prevented polyadenylation, except in two mutants where a G-U bas e pair reformed. The polyadenylation activity of point mutants could be res cued by constructing double mutants with reformed base pairs in the stem of the vRNA 5' hook. These results suggest that base pairing rather than a pa rticular nucleotide sequence was critical. We also show that mutation of th e analogous region in the 3' arm of vRNA did not interfere with the synthes is of polyadenylated mRNA, suggesting that a hook structure in the 3' arm i s not required for transcription of polyadenylated mRNA in vitro.