HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 13, 12141-12151, March 26, 2004

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 279/13/12141    most recent M310839200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nomaguchi, M. Articles by Padmanabhan, R. Search for Related Content PubMed PubMed Citation Articles by Nomaguchi, M. Articles by Padmanabhan, R. Social Bookmarking                      What's this?

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli^*

Masako Nomaguchi, Tadahisa Teramoto, Li Yu¶, Lewis Markoff¶, and R. Padmanabhan||

From the Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, D. C. 20057 and the ^¶Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (–)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Received for publication, October 1, 2003 , and in revised form, December 16, 2003.

^* This work was supported by United States Public Health Service Grant AI-32078. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

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

^|| To whom correspondence should be addressed: Dept. of Microbiology & Immunology, Georgetown University Medical Center, SW309-MedDent Bldg., 3900 Reservoir Rd., Washington, D. C. 20057. E-mail: rp55{at}georgetown.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				P. Schlick, C. Taucher, B. Schittl, J. L. Tran, R. M. Kofler, W. Schueler, A. von Gabain, A. Meinke, and C. W. Mandl Helices {alpha}2 and {alpha}3 of West Nile Virus Capsid Protein Are Dispensable for Assembly of Infectious Virions J. Virol., June 1, 2009; 83(11): 5581 - 5591. [Abstract] [Full Text] [PDF]

				M. M. Emara, H. Liu, W. G. Davis, and M. A. Brinton Mutation of Mapped TIA-1/TIAR Binding Sites in the 3' Terminal Stem-Loop of West Nile Virus Minus-Strand RNA in an Infectious Clone Negatively Affects Genomic RNA Amplification J. Virol., November 1, 2008; 82(21): 10657 - 10670. [Abstract] [Full Text] [PDF]

				P. A. G. C. Silva, R. Molenkamp, T. J. Dalebout, N. Charlier, J. H. Neyts, W. J. M. Spaan, and P. J. Bredenbeek Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3' stem-loop structure is not required for replication J. Gen. Virol., June 1, 2007; 88(6): 1738 - 1747. [Abstract] [Full Text] [PDF]

				Y. Zhou, D. Ray, Y. Zhao, H. Dong, S. Ren, Z. Li, Y. Guo, K. A. Bernard, P.-Y. Shi, and H. Li Structure and Function of Flavivirus NS5 Methyltransferase J. Virol., April 15, 2007; 81(8): 3891 - 3903. [Abstract] [Full Text] [PDF]

				D. Ray, A. Shah, M. Tilgner, Y. Guo, Y. Zhao, H. Dong, T. S. Deas, Y. Zhou, H. Li, and P.-Y. Shi West nile virus 5'-cap structure is formed by sequential Guanine N-7 and ribose 2'-o methylations by nonstructural protein 5. J. Virol., September 1, 2006; 80(17): 8362 - 8370. [Abstract] [Full Text] [PDF]

				C. V. Filomatori, M. F. Lodeiro, D. E. Alvarez, M. M. Samsa, L. Pietrasanta, and A. V. Gamarnik A 5' RNA element promotes dengue virus RNA synthesis on a circular genome Genes & Dev., August 15, 2006; 20(16): 2238 - 2249. [Abstract] [Full Text] [PDF]

				R. M. Kofler, V. M. Hoenninger, C. Thurner, and C. W. Mandl Functional Analysis of the Tick-Borne Encephalitis Virus Cyclization Elements Indicates Major Differences between Mosquito-Borne and Tick-Borne Flaviviruses. J. Virol., April 1, 2006; 80(8): 4099 - 4113. [Abstract] [Full Text] [PDF]

				K. Clyde and E. Harris RNA Secondary Structure in the Coding Region of Dengue Virus Type 2 Directs Translation Start Codon Selection and Is Required for Viral Replication J. Virol., March 1, 2006; 80(5): 2170 - 2182. [Abstract] [Full Text] [PDF]

				F. Puig-Basagoiti, T. S. Deas, P. Ren, M. Tilgner, D. M. Ferguson, and P.-Y. Shi High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery Antimicrob. Agents Chemother., December 1, 2005; 49(12): 4980 - 4988. [Abstract] [Full Text] [PDF]