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J. Biol. Chem., Vol. 280, Issue 29, 27412-27419, July 22, 2005

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Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5'-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase^*

Changsuek Yon, Tadahisa Teramoto, Niklaus Mueller, Jessica Phelan, Vannakambadi K. Ganesh¶, Krishna H. M. Murthy¶, and R. Padmanabhan||

From the Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington D. C. 20057 and ^¶Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3_FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3_FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3_FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, ¹⁸⁴RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.

Received for publication, February 7, 2005 , and in revised form, May 5, 2005.

^* This work was supported from NIAID, National Institutes of Health Grants AI32078, AI54776 and AI45623. 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.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Georgetown University School of Medicine, 3900 Reservoir Rd., Washington, D. C. 20057. Tel.: 202-687-2092; Fax: 202-687-1800; E-mail: rp55{at}georgetown.edu.

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