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Journal of Virology, June 2004, p. 6263-6270, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6263-6270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Influenza Virus cRNA Promoter and Identification of Nucleotides Critical for Replication

Mandy Crow ,{dagger},{ddagger} Tao Deng,{dagger} Mark Addley, and George G. Brownlee*

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Received 26 December 2003/ Accepted 27 January 2004

Replication of the influenza A virus virion RNA (vRNA) requires the synthesis of full-length cRNA, which in turn is used as a template for the synthesis of more vRNA. A "corkscrew" secondary-structure model of the cRNA promoter has been proposed recently. However the data in support of that model were indirect, since they were derived from measurement, by use of a chloramphenicol acetyltransferase (CAT) reporter in 293T cells, of mRNA levels from a modified cRNA promoter rather than the authentic cRNA promoter found in influenza A viruses. Here we measured steady-state cRNA and vRNA levels from a CAT reporter in 293T cells, directly measuring the replication of the authentic influenza A virus wild-type cRNA promoter. We found that (i) base pairing between the 5' and 3' ends and (ii) base pairing in the stems of both the 5' and 3' hairpin loops of the cRNA promoter were required for in vivo replication. Moreover, nucleotides in the tetraloop at positions 4, 5, and 7 and nucleotides forming the 2-9 base pair of the 3' hairpin loop were crucial for promoter activity in vivo. However, the 3' hairpin loop was not required for polymerase binding in vitro. Overall, our results suggest that the corkscrew secondary-structure model is required for authentic cRNA promoter activity in vivo, although the precise role of the 3' hairpin loop remains unknown.

* Corresponding author. Mailing address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. Phone: (01865) 275559. Fax: (01865) 275556. E-mail: George.Brownlee{at}path.ox.ac.uk.

{dagger} M.C. and T.D. contributed equally to this work.

{ddagger} Present address: UMIST, Manchester, M60 1QD United Kingdom.

Journal of Virology, June 2004, p. 6263-6270, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6263-6270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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