This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nomaguchi, M.
Right arrow Articles by Padmanbhan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nomaguchi, M.
Right arrow Articles by Padmanbhan, R.

 Previous Article  |  Next Article 

Journal of Virology, August 2003, p. 8831-8842, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8831-8842.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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,{dagger} Matt Ackermann,1 Changsuek Yon,2 Shihyun You,1,{ddagger} and R. Padmanbhan1,2*

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, A~G, 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Med-Dent Bldg., Room SW-309, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20057. Phone: (202) 687-2092. Fax: (202) 687-1800. E-mail: rp55{at}georgetown.edu.

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

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

Journal of Virology, August 2003, p. 8831-8842, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8831-8842.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Teramoto, T., Kohno, Y., Mattoo, P., Markoff, L., Falgout, B., Padmanabhan, R. (2008). Genome 3'-end repair in dengue virus type 2. RNA 14: 2645-2656 [Abstract] [Full Text]  
  • Chinnaswamy, S., Yarbrough, I., Palaninathan, S., Kumar, C. T. R., Vijayaraghavan, V., Demeler, B., Lemon, S. M., Sacchettini, J. C., Kao, C. C. (2008). A Locking Mechanism Regulates RNA Synthesis and Host Protein Interaction by the Hepatitis C Virus Polymerase. J. Biol. Chem. 283: 20535-20546 [Abstract] [Full Text]  
  • Yap, T. L., Xu, T., Chen, Y.-L., Malet, H., Egloff, M.-P., Canard, B., Vasudevan, S. G., Lescar, J. (2007). Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution. J. Virol. 81: 4753-4765 [Abstract] [Full Text]  
  • Malet, H., Egloff, M.-P., Selisko, B., Butcher, R. E., Wright, P. J., Roberts, M., Gruez, A., Sulzenbacher, G., Vonrhein, C., Bricogne, G., Mackenzie, J. M., Khromykh, A. A., Davidson, A. D., Canard, B. (2007). Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5. J. Biol. Chem. 282: 10678-10689 [Abstract] [Full Text]  
  • Clyde, K., Kyle, J. L., Harris, E. (2006). Recent Advances in Deciphering Viral and Host Determinants of Dengue Virus Replication and Pathogenesis. J. Virol. 80: 11418-11431 [Full Text]  
  • Medin, C. L., Fitzgerald, K. A., Rothman, A. L. (2005). Dengue Virus Nonstructural Protein NS5 Induces Interleukin-8 Transcription and Secretion. J. Virol. 79: 11053-11061 [Abstract] [Full Text]  
  • Tilgner, M., Shi, P.-Y. (2004). Structure and Function of the 3' Terminal Six Nucleotides of the West Nile Virus Genome in Viral Replication. J. Virol. 78: 8159-8171 [Abstract] [Full Text]  
  • Zhang, G., Zhang, J., Simon, A. E. (2004). Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon. J. Virol. 78: 7619-7633 [Abstract] [Full Text]  
  • Choi, K. H., Groarke, J. M., Young, D. C., Kuhn, R. J., Smith, J. L., Pevear, D. C., Rossmann, M. G. (2004). The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc. Natl. Acad. Sci. USA 101: 4425-4430 [Abstract] [Full Text]  
  • Nomaguchi, M., Teramoto, T., Yu, L., Markoff, L., Padmanabhan, R. (2004). Requirements for West Nile Virus (-)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli. J. Biol. Chem. 279: 12141-12151 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»