This Article
Right arrow Full Text
Right arrow Full Text (PDF)
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 Gu, B.
Right arrow Articles by Sarisky, R. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gu, B.
Right arrow Articles by Sarisky, R. T.

 Previous Article  |  Next Article 

Journal of Virology, May 2003, p. 5352-5359, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5352-5359.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Replication Studies Using Genotype 1a Subgenomic Hepatitis C Virus Replicons

Baohua Gu,1* Adam T. Gates,1 Olaf Isken,2 Sven-Erik Behrens,2 and Robert T. Sarisky1

Department of Virology, The Metabolic and Viral Diseases Center of Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426,1 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191112

Received 19 December 2002/ Accepted 23 January 2003

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-{alpha}), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-{alpha} may be attributed directly to the type 1a polymerase.

* Corresponding author. Mailing address: Department of Virology, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Rd., UP1450, Collegeville, PA 19426-0989. Phone: (484) 802-3315. Fax: (610) 917-4617. E-mail: baohua_gu-1{at}comcast.net.

Journal of Virology, May 2003, p. 5352-5359, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5352-5359.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • McCown, M. F., Rajyaguru, S., Kular, S., Cammack, N., Najera, I. (2009). GT-1a or GT-1b Subtype-Specific Resistance Profiles for Hepatitis C Virus Inhibitors Telaprevir and HCV-796. Antimicrob. Agents Chemother. 53: 2129-2132 [Abstract] [Full Text]  
  • Noppornpanth, S., Lien, T. X., Poovorawan, Y., Smits, S. L., Osterhaus, A. D. M. E., Haagmans, B. L. (2006). Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus.. J. Virol. 80: 7569-7577 [Abstract] [Full Text]  
  • Ludmerer, S. W., Graham, D. J., Boots, E., Murray, E. M., Simcoe, A., Markel, E. J., Grobler, J. A., Flores, O. A., Olsen, D. B., Hazuda, D. J., LaFemina, R. L. (2005). Replication Fitness and NS5B Drug Sensitivity of Diverse Hepatitis C Virus Isolates Characterized by Using a Transient Replication Assay. Antimicrob. Agents Chemother. 49: 2059-2069 [Abstract] [Full Text]  
  • O'Boyle, D. R. II, Nower, P. T., Lemm, J. A., Valera, L., Sun, J.-H., Rigat, K., Colonno, R., Gao, M. (2005). Development of a Cell-Based High-Throughput Specificity Screen Using a Hepatitis C Virus-Bovine Viral Diarrhea Virus Dual Replicon Assay. Antimicrob. Agents Chemother. 49: 1346-1353 [Abstract] [Full Text]  
  • Kato, T., Date, T., Miyamoto, M., Zhao, Z., Mizokami, M., Wakita, T. (2005). Nonhepatic Cell Lines HeLa and 293 Support Efficient Replication of the Hepatitis C Virus Genotype 2a Subgenomic Replicon. J. Virol. 79: 592-596 [Abstract] [Full Text]  
  • Evans, M. J., Rice, C. M., Goff, S. P. (2004). Genetic Interactions between Hepatitis C Virus Replicons. J. Virol. 78: 12085-12089 [Abstract] [Full Text]  
  • Yi, M., Lemon, S. M. (2004). Adaptive Mutations Producing Efficient Replication of Genotype 1a Hepatitis C Virus RNA in Normal Huh7 Cells. J. Virol. 78: 7904-7915 [Abstract] [Full Text]  
  • Moradpour, D., Evans, M. J., Gosert, R., Yuan, Z., Blum, H. E., Goff, S. P., Lindenbach, B. D., Rice, C. M. (2004). Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes. J. Virol. 78: 7400-7409 [Abstract] [Full Text]  
  • Ma, Y., Shimakami, T., Luo, H., Hayashi, N., Murakami, S. (2004). Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture. J. Biol. Chem. 279: 25474-25482 [Abstract] [Full Text]  
  • Mehta, A. S., Gu, B., Conyers, B., Ouzounov, S., Wang, L., Moriarty, R. M., Dwek, R. A., Block, T. M. (2004). {alpha}-Galactosylceramide and Novel Synthetic Glycolipids Directly Induce the Innate Host Defense Pathway and Have Direct Activity against Hepatitis B and C Viruses. Antimicrob. Agents Chemother. 48: 2085-2090 [Abstract] [Full Text]  
  • Date, T., Kato, T., Miyamoto, M., Zhao, Z., Yasui, K., Mizokami, M., Wakita, T. (2004). Genotype 2a Hepatitis C Virus Subgenomic Replicon Can Replicate in HepG2 and IMY-N9 Cells. J. Biol. Chem. 279: 22371-22376 [Abstract] [Full Text]  
  • Shimakami, T., Hijikata, M., Luo, H., Ma, Y. Y., Kaneko, S., Shimotohno, K., Murakami, S. (2004). Effect of Interaction between Hepatitis C Virus NS5A and NS5B on Hepatitis C Virus RNA Replication with the Hepatitis C Virus Replicon. J. Virol. 78: 2738-2748 [Abstract] [Full Text]  
  • McCormick, C. J., Challinor, L., Macdonald, A., Rowlands, D. J., Harris, M. (2004). Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system. J. Gen. Virol. 85: 429-439 [Abstract] [Full Text]  
  • Frick, D. N., Rypma, R. S., Lam, A. M. I., Gu, B. (2004). The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently. J. Biol. Chem. 279: 1269-1280 [Abstract] [Full Text]  
  • Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. (2004). Hepatitis C Virus Subgenomic Replicons in the Human Embryonic Kidney 293 Cell Line. J. Virol. 78: 491-501 [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»