Nucleic Acids Research, 2003, Vol. 31, No. 6 1624-1632
© 2003 Oxford University Press
Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA
Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK, 1 Roche Bioscience, Palo Alto, CA 94304, USA and 2 Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
*To whom correspondence should be addressed. Tel: +44 1223 339554; Fax: +44 1223 337610; Email: Lst21{at}cam.ac.uk
Present address:
M.-T. Michael Lee, Howard Hughes Medical Institute, Duke University Medical Centre, Durham, NC 27710, USA
The current model for influenza virus mRNA transcription involves the sequential interaction of the viral polymerase with the 5'- and 3'-ends of vRNA, with each RNA–protein interaction triggering a polymerase function necessary for cap-primed transcription. Here we show that the order in which this ternary complex is assembled is in fact important. Polymerase bound simultaneously to a pre-annealed duplex of the 5'- and 3'-ends of vRNA had greatly increased levels of primer binding and endonuclease activities compared to a sequentially assembled complex. Increased primer binding was due to the activation of a high affinity binding site with a preference for primer length RNAs. This correlated with enhanced levels of cap-primed transcription. Polymerase that was bound initially to just 5' vRNA had low primer binding activity, but was endonucleolytically active. Neither activity was significantly increased by the subsequent addition of 3' vRNA, and this sequentially assembled complex had correspondingly low mRNA transcription activity. Nevertheless, both routes of assembly led to complexes that were highly competent for dinucleotide ApG-primed transcription. Therefore, polymerase complexes assembled on pre-annealed 5' and 3' terminal viral RNA sequences have distinct properties from those assembled by sequential loading of polymerase onto the 5'-end followed by the 3'-end. This suggests a mechanism by which the virus couples transcription initiation and termination during mRNA transcription.
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