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Journal of Virology, September 1999, p. 7349-7356, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein

Liz Medcalf, Emma Poole, Debra Elton, and Paul Digard*

Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom

Received 11 March 1999/Accepted 25 May 1999

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30°C. This suggests that the RNA-binding activity of NP is required for replicative transcription.

* Corresponding author. Mailing address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom. Phone: 44-1223-336918. Fax: 44-1223-336926. E-mail: pd1{at}mole.bio.cam.ac.uk.

Journal of Virology, September 1999, p. 7349-7356, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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