RNA-POLYMERASE I-MEDIATED EXPRESSION OF INFLUENZA VIRAL-RNA MOLECULES

RNA polymerase I transcription has been used for expression of influen za vRNA molecules, with influenza hemagglutinin or other cDNAs precise ly inserted between mouse rDNA promoter and terminator sequences. In i n vitro studies generation of HA vRNA transcripts in high rates and co rrect formation of their 5' ends as well as their 3' ends has been ach ieved for such hybrid DNA templates. For in vivo expression studies, t he HA coding region was replaced by chloramphenicol acetyltransferase (CAT), also in vRNA antisense orientation, with both influenza termina l sequences beyond start and stop codons being retained on the resulti ng transcript. Following transfection with precisely constructed hybri d DNA templates and depending on infection with influenza virus, CAT a ctivity could be demonstrated. Templates resulting in 3' extended vRNA molecules did not give this result. vRNA-CAT molecules were not only recognized by influenza viral RNA polymerase for synthesis of plus str and mRNAs, but also were packaged into progeny virus particles, as sho wn by CAT activity in infected cells after passaging of virus containi ng supernatants. (C) 1994 Academic Press, Inc.