POLYADENYLATION OF INFLUENZA-VIRUS MESSENGER-RNA TRANSCRIBED IN-VITROFROM MODEL VIRION RNA TEMPLATES - REQUIREMENT FOR 5'-CONSERVED SEQUENCES

Here we report the development of two independent assays which demonst rate for the first time that exogenous model RNA templates based on in fluenza virus virion RNA (vRNA) are transcribed in vitro to produce po lyadenylated mRNA. We investigated the activities of mutated templates with known polymerase binding properties to test our model that polya denylation occurs when a polymerase complex, which is bound to conserv ed 5' sequences of vRNA, prevents read-through of the U track at which polyadenylation subsequently occurs by reiterative copying. Mutated t emplates with perturbed polymerase binding sites (i.e. a deletion muta nt lacking the first 4 5' residues and a U --> A point mutant at the t hird residue initiated transcription in the in vitro assay but failed to produce polyadenylated transcripts, whereas an A --> U point mutant at the fourth residue, which retained polymerase binding properties s imilar to those of the wild type produced polyadenylated transcripts, Our results show that nucleotides within the conserved 5' sequence are required for polyadenylation and support the hypothesis that polymera se binding to 5' sequences of the template is required for mRNA synthe sis.