A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo.

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.