Attenuation of influenza A virus mRNA levels by promoter mutations.
We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G-->A-U double mutation at positions 11 and 12' at the 3' and 5' ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.
Fodor
E
E
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Palese
P
P
Brownlee
G G
GG
García-Sastre
A
A
D000818:Animals; D002417:Cattle; D002460:Cell Line; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000254:growth & development; D016296:Mutagenesis; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D018075:RNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D012261:Ribonucleoproteins; D011061:Poly A; D009439:Neuraminidase