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De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters

Masako Nomaguchi,^1,2, Matt Ackermann,¹ Changsuek Yon,² Shihyun You,^1, and R. Padmanbhan^1,2*

Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160,¹ Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, D.C. 20057²

Received 12 December 2002/ Accepted 16 May 2003

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40°C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 µM), the polymerase, and the template RNA. Even when the 3'-terminal sequence of template RNA was mutated from -GGUUCU-3' to -GGUUUU-3', a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3'-terminal dinucleotide CU of the template RNA indicated that change of the 3'-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3'-terminal nucleotide, from highest to lowest, is U, AG, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.

Permanent address: KYURIN Corporation, Kitakyushu, Japan 806-0046.

Present address: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10021.

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