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J. Biol. Chem., Vol. 278, Issue 34, 31722-31730, August 22, 2003

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Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA^*

Kostas Lymperopoulos  , Christoph Wirblich , Ian Brierley ¶ and Polly Roy  || **

From the Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom, the ^¶Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, United Kingdom, and the ^||Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.

Received for publication, January 31, 2003 , and in revised form, May 12, 2003.

^* This work was supported in part by Biotechnology and Biological Sciences Research Council Grant 43/P13500, Wellcome Trust Grant 063995/Z/01/Z, and National Institutes of Health Grant R01 AI45000-01AI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Graduate Student in the Dept. of Biochemistry, University of Oxford.

^** To whom correspondence should be addressed: Dept. of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Tel.: 44-20-7927-2324; Fax: 44-20-7927-2839; E-mail: polly.roy{at}lshtm.ac.uk.

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