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J Virol, August 1998, p. 6283-6290, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations

Ervin Fodor,1,dagger Peter Palese,1,* George G. Brownlee,2 and Adolfo García-Sastre1

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029,1 and Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom2

Received 3 February 1998/Accepted 24 April 1998

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-Gright-arrowA-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.

* Corresponding author. Mailing address: Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-7318. Fax: (212) 722-3634. E-mail: ppalese{at}smtplink.mssm.edu.

dagger Permanent address: Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

J Virol, August 1998, p. 6283-6290, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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