MUTATIONAL ANALYSIS OF THE RNA-FORK MODEL OF THE INFLUENZA-A VIRUS VRNA PROMOTER IN-VIVO

The genome of influenza A virus consists of eight negative-stranded RN A segments which have partially complementary non-coding terminal sequ ences, Previous transcription studies of the virion RNA promoter in vi tro have shown that the 5' terminus forms an integral part of the prom oter and an 'RNA-fork' model has been proposed far the initiation of t ranscription, According to this model part of the promoter is formed b y an RNA-duplex which involves complementary residues 10 to 12 of the 3' end and residues 11' to 13' of the 5' end. With a reverse genetics system, based on the chloramphenicol acetyltransferase (CAT) gene, we have now tested this part of the promoter in vivo. Single mutations of the conserved residues at positions 11 and 12 of the 3' terminus and at positions 12' and 13' of the 5' terminus abolished promoter activit y. The introduction of complementary mutations into both termini parti ally restored activity. On the other hand, mutations at positions 10 o f the 3' terminus and 11' of the 5' terminus inhibited activity indepe ndently of whether a basepair was formed or not. Thus, at these positi ons, the nature of the residues is apparently more important than thei r ability to form base-pairs. These results extend our previous virion 'RNA-fork' model and are consistent with in vitro findings that the 5 ' terminus is involved in the initiation of transcription.