This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lo, M. K. Articles by Shi, P.-Y. Search for Related Content PubMed PubMed Citation Articles by Lo, M. K. Articles by Shi, P.-Y.

Functional Analysis of Mosquito-Borne Flavivirus Conserved Sequence Elements within 3' Untranslated Region of West Nile Virus by Use of a Reporting Replicon That Differentiates between Viral Translation and RNA Replication

Wadsworth Center, New York State Department of Health,¹ Department of Biomedical Sciences, University at Albany, State University of New York, Albany, New York²

Received 21 April 2003/ Accepted 20 June 2003

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A Renilla luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3' untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5' region of the capsid gene (5'CS) abolished RNA replication. Restoration of the 5'CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5'CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.

This article has been cited by other articles:

• Zhang, B., Dong, H., Zhou, Y., Shi, P.-Y. (2008). Genetic Interactions among the West Nile Virus Methyltransferase, the RNA-Dependent RNA Polymerase, and the 5' Stem-Loop of Genomic RNA. J. Virol. 82: 7047-7058 [Abstract] [Full Text]  
• Suzuki, R., Fayzulin, R., Frolov, I., Mason, P. W. (2008). Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate. J. Virol. 82: 6942-6951 [Abstract] [Full Text]  
• Tesfay, M. Z., Yin, J., Gardner, C. L., Khoretonenko, M. V., Korneeva, N. L., Rhoads, R. E., Ryman, K. D., Klimstra, W. B. (2008). Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism. J. Virol. 82: 2620-2630 [Abstract] [Full Text]  
• Ivanyi-Nagy, R., Lavergne, J.-P., Gabus, C., Ficheux, D., Darlix, J.-L. (2008). RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae. Nucleic Acids Res 36: 712-725 [Abstract] [Full Text]  
• Davis, W. G., Blackwell, J. L., Shi, P.-Y., Brinton, M. A. (2007). Interaction between the Cellular Protein eEF1A and the 3'-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis. J. Virol. 81: 10172-10187 [Abstract] [Full Text]  
• Mori, Y., Yamashita, T., Tanaka, Y., Tsuda, Y., Abe, T., Moriishi, K., Matsuura, Y. (2007). Processing of Capsid Protein by Cathepsin L Plays a Crucial Role in Replication of Japanese Encephalitis Virus in Neural and Macrophage Cells. J. Virol. 81: 8477-8487 [Abstract] [Full Text]  
• Tajima, S., Nukui, Y., Takasaki, T., Kurane, I. (2007). Characterization of the variable region in the 3' non-translated region of dengue type 1 virus. J. Gen. Virol. 88: 2214-2222 [Abstract] [Full Text]  
• Deas, T. S., Bennett, C. J., Jones, S. A., Tilgner, M., Ren, P., Behr, M. J., Stein, D. A., Iversen, P. L., Kramer, L. D., Bernard, K. A., Shi, P.-Y. (2007). In Vitro Resistance Selection and In Vivo Efficacy of Morpholino Oligomers against West Nile Virus. Antimicrob. Agents Chemother. 51: 2470-2482 [Abstract] [Full Text]  
• Silva, P. A. G. C., Molenkamp, R., Dalebout, T. J., Charlier, N., Neyts, J. H., Spaan, W. J. M., Bredenbeek, P. J. (2007). 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. 88: 1738-1747 [Abstract] [Full Text]  
• Davis, C. T., Galbraith, S. E., Zhang, S., Whiteman, M. C., Li, L., Kinney, R. M., Barrett, A. D. T. (2007). A Combination of Naturally Occurring Mutations in North American West Nile Virus Nonstructural Protein Genes and in the 3' Untranslated Region Alters Virus Phenotype. J. Virol. 81: 6111-6116 [Abstract] [Full Text]  
• Pijlman, G. P., Kondratieva, N., Khromykh, A. A. (2006). Translation of the Flavivirus Kunjin NS3 Gene in cis but Not Its RNA Sequence or Secondary Structure Is Essential for Efficient RNA Packaging. J. Virol. 80: 11255-11264 [Abstract] [Full Text]  
• Gritsun, T. S., Gould, E. A. (2006). Direct repeats in the 3' untranslated regions of mosquito-borne flaviviruses: possible implications for virus transmission.. J. Gen. Virol. 87: 3297-3305 [Abstract] [Full Text]  
• Gritsun, T. S., Gould, E. A. (2006). The 3' untranslated regions of Kamiti River virus and Cell fusing agent virus originated by self-duplication. J. Gen. Virol. 87: 2615-2619 [Abstract] [Full Text]  
• Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., Deas, T. S., Zhou, Y., Li, H., Shi, P.-Y. (2006). West nile virus 5'-cap structure is formed by sequential Guanine N-7 and ribose 2'-o methylations by nonstructural protein 5.. J. Virol. 80: 8362-8370 [Abstract] [Full Text]  
• Filomatori, C. V., Lodeiro, M. F., Alvarez, D. E., Samsa, M. M., Pietrasanta, L., Gamarnik, A. V. (2006). A 5' RNA element promotes dengue virus RNA synthesis on a circular genome. Genes Dev. 20: 2238-2249 [Abstract] [Full Text]  
• Puig-Basagoiti, F., Tilgner, M., Forshey, B. M., Philpott, S. M., Espina, N. G., Wentworth, D. E., Goebel, S. J., Masters, P. S., Falgout, B., Ren, P., Ferguson, D. M., Shi, P.-Y. (2006). Triaryl pyrazoline compound inhibits flavivirus RNA replication.. Antimicrob. Agents Chemother. 50: 1320-1329 [Abstract] [Full Text]  
• Kofler, R. M., Hoenninger, V. M., Thurner, C., Mandl, C. W. (2006). Functional Analysis of the Tick-Borne Encephalitis Virus Cyclization Elements Indicates Major Differences between Mosquito-Borne and Tick-Borne Flaviviruses.. J. Virol. 80: 4099-4113 [Abstract] [Full Text]  
• Puig-Basagoiti, F., Deas, T. S., Ren, P., Tilgner, M., Ferguson, D. M., Shi, P.-Y. (2005). High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery. Antimicrob. Agents Chemother. 49: 4980-4988 [Abstract] [Full Text]  
• Whitby, K., Pierson, T. C., Geiss, B., Lane, K., Engle, M., Zhou, Y., Doms, R. W., Diamond, M. S. (2005). Castanospermine, a Potent Inhibitor of Dengue Virus Infection In Vitro and In Vivo. J. Virol. 79: 8698-8706 [Abstract] [Full Text]  
• Chiu, W.-W., Kinney, R. M., Dreher, T. W. (2005). Control of Translation by the 5'- and 3'-Terminal Regions of the Dengue Virus Genome. J. Virol. 79: 8303-8315 [Abstract] [Full Text]  
• Deas, T. S., Binduga-Gajewska, I., Tilgner, M., Ren, P., Stein, D. A., Moulton, H. M., Iversen, P. L., Kauffman, E. B., Kramer, L. D., Shi, P.-Y. (2005). Inhibition of Flavivirus Infections by Antisense Oligomers Specifically Suppressing Viral Translation and RNA Replication. J. Virol. 79: 4599-4609 [Abstract] [Full Text]  
• Kinney, R. M., Huang, C. Y.-H., Rose, B. C., Kroeker, A. D., Dreher, T. W., Iversen, P. L., Stein, D. A. (2005). Inhibition of Dengue Virus Serotypes 1 to 4 in Vero Cell Cultures with Morpholino Oligomers. J. Virol. 79: 5116-5128 [Abstract] [Full Text]  
• Gehrke, R., Heinz, F. X., Davis, N. L., Mandl, C. W. (2005). Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon. J. Gen. Virol. 86: 1045-1053 [Abstract] [Full Text]  
• Bryant, J. E., Vasconcelos, P. F. C., Rijnbrand, R. C. A., Mutebi, J. P., Higgs, S., Barrett, A. D. T. (2005). Size Heterogeneity in the 3' Noncoding Region of South American Isolates of Yellow Fever Virus. J. Virol. 79: 3807-3821 [Abstract] [Full Text]  
• Shirato, K., Miyoshi, H., Goto, A., Ako, Y., Ueki, T., Kariwa, H., Takashima, I. (2004). Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. J. Gen. Virol. 85: 3637-3645 [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]  
• 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]