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 Yi, M.
Right arrow Articles by Lemon, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yi, M.
Right arrow Articles by Lemon, S. M.

 Previous Article  |  Next Article 

Journal of Virology, February 2002, p. 1171-1180, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1171-1180.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells

MinKyung Yi and Stanley M. Lemon*

Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019

Received 10 April 2001/ Accepted 30 October 2001

Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3Dpol (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5' noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555-0133. Phone: (409) 772-4579. Fax: (409) 772-9598. E-mail: smlemon{at}utmb.edu.

Journal of Virology, February 2002, p. 1171-1180, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1171-1180.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Yu, X., Sainz, B. Jr., Uprichard, S. L. (2009). Development of a Cell-Based Hepatitis C Virus Infection Fluorescent Resonance Energy Transfer Assay for High-Throughput Antiviral Compound Screening. Antimicrob. Agents Chemother. 53: 4311-4319 [Abstract] [Full Text]  
  • Yang, Y., Yi, M., Evans, D. J., Simmonds, P., Lemon, S. M. (2008). Identification of a Conserved RNA Replication Element (cre) within the 3Dpol-Coding Sequence of Hepatoviruses. J. Virol. 82: 10118-10128 [Abstract] [Full Text]  
  • Kusov, Y., Kanda, T., Palmenberg, A., Sgro, J.-Y., Gauss-Muller, V. (2006). Silencing of Hepatitis A Virus Infection by Small Interfering RNAs.. J. Virol. 80: 5599-5610 [Abstract] [Full Text]  
  • Li, K., Chen, Z., Kato, N., Gale, M. Jr., Lemon, S. M. (2005). Distinct Poly(I-C) and Virus-activated Signaling Pathways Leading to Interferon-{beta} Production in Hepatocytes. J. Biol. Chem. 280: 16739-16747 [Abstract] [Full Text]  
  • Bukh, J., Pietschmann, T., Lohmann, V., Krieger, N., Faulk, K., Engle, R. E., Govindarajan, S., Shapiro, M., St. Claire, M., Bartenschlager, R. (2002). Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. USA 99: 14416-14421 [Abstract] [Full Text]  
  • Gauss-Muller, V., Kusov, Y. Y. (2002). Replication of a hepatitis A virus replicon detected by genetic recombination in vivo. J. Gen. Virol. 83: 2183-2192 [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»