Transient bicistronic vRNA segments for indirect selection of recombinant influenza viruses.

The 5'- and 3'-terminal regions of influenza vRNA molecules are known to constitute the promoter structure upon association with viral RNA polymerase in an activated complementary conformation. An inherent requirement for their location at the very ends of the vRNA molecules always has been implied because of that natural structure, but this study demonstrates that one or both of the promoter sequences may be relocated into vRNA-internal positions and still retain their polymerase-binding function. External extensions of vRNA molecules employed include either single-stranded RNA sequences </=750 nucleotides in length or complementary, and hence potentially double-stranded sequences, or promoter duplications. 5' RACE analyses of internally promoted cRNA and mRNA molecules prove initiation to occur at exactly the 3' standard template position 1, as defined by the regular promoter structure. Thereby any template extensions are lost from the resulting RNA molecules and progeny virions. These observations have been used to construct bicistronic vRNAs with an additional 3'-promoter sequence located between the two reading frames. During propagation, these spontaneously give rise to monocistronic vRNAs upon internal initiation reactions. Accordingly designed bicistronic vRNAs can be employed for indirectly selecting any foreign gene encoded in the resulting monocistronic vRNA for incorporation into recombinant influenza viruses.