NONCONSERVED NUCLEOTIDES AT THE 3'-END AND 5'-END OF AN INFLUENZA-A VIRUS-RNA PLAY AN IMPORTANT ROLE IN VIRAL-RNA REPLICATION

The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packag ing of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form ''panhandle'' or '' fork'' structures by partial complementarity, are important for the tr anscriptional activity of the viral RNA polymerase. In contrast, the n onconserved noncoding nucleotides located close to the open reading fr ame of the viral RNAs had not been implicated in RNA transcription. Us ing a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these noncons erved noncoding regions. Deletion either of the nucleotide residues be tween the poly(U) stretch and the stop codon at the 5' end or of the n ucleotides between position 15 and the start codon at the 3' end did n ot affect the amount of NA-RNA species found in virions or infected ce lls. However, a combination of deletions at both the 3' and the 5' end s decreased by 60 times the levels of NA-specific viral RNA found in i nfected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady- state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of influenza AN/WSN/33 virus also s howed a reduction in the NA-specific viral RNA in virions and in infec ted cells. The present results demonstrate that the nonconserved nucle otides at the 3' and 5' ends of the NA-RNA segment of influenza A viru s play an important role in the replication of this segment. (C) 1996 Academic Press, Inc.