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 Gorchakov, R.
Right arrow Articles by Frolov, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gorchakov, R.
Right arrow Articles by Frolov, I.

 Previous Article  |  Next Article 

Journal of Virology, January 2004, p. 61-75, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.61-75.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Selection of Functional 5' cis-Acting Elements Promoting Efficient Sindbis Virus Genome Replication

Rodion Gorchakov,1 Richard Hardy,2 Charles M. Rice,3 and Ilya Frolov1*

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019,1 Department of Biology, Indiana University, Bloomington, Indiana 47405,2 Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021-63993

Received 15 July 2003/ Accepted 19 September 2003

The 5' portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5' 200 bases of the SIN genome RNA, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5' end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5' terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5' end of the original SFV 5' sequence or its "deleted" versions. Early pseudorevertants had heterogeneous 5' termini that were inefficient for replication relative to the parental SIN 5' sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5'-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5'-terminal sequences always began with 5'-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5' modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1019. Phone: (409) 772-2327. Fax: (409) 772-5065. E-mail: ivfrolov{at}UTMB.edu.

Journal of Virology, January 2004, p. 61-75, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.61-75.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kulasegaran-Shylini, R., Atasheva, S., Gorenstein, D. G., Frolov, I. (2009). Structural and Functional Elements of the Promoter Encoded by the 5' Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome. J. Virol. 83: 8327-8339 [Abstract] [Full Text]  
  • Li, M.-L., Stollar, V. (2007). Distinct Sites on the Sindbis Virus RNA-Dependent RNA Polymerase for Binding to the Promoters for the Synthesis of Genomic and Subgenomic RNA. J. Virol. 81: 4371-4373 [Abstract] [Full Text]  
  • TORTORICI, M. A., SHAPIRO, B. A., PATTON, J. T. (2006). A base-specific recognition signal in the 5' consensus sequence of rotavirus plus-strand RNAs promotes replication of the double-stranded RNA genome segments. RNA 12: 133-146 [Abstract] [Full Text]  
  • Alvarez, D. E., Lodeiro, M. F., Luduena, S. J., Pietrasanta, L. I., Gamarnik, A. V. (2005). Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome. J. Virol. 79: 6631-6643 [Abstract] [Full Text]  
  • Hardy, R. W., Rice, C. M. (2005). Requirements at the 3' End of the Sindbis Virus Genome for Efficient Synthesis of Minus-Strand RNA. J. Virol. 79: 4630-4639 [Abstract] [Full Text]  
  • Ryman, K. D., Meier, K. C., Nangle, E. M., Ragsdale, S. L., Korneeva, N. L., Rhoads, R. E., MacDonald, M. R., Klimstra, W. B. (2005). Sindbis Virus Translation Is Inhibited by a PKR/RNase L-Independent Effector Induced by Alpha/Beta Interferon Priming of Dendritic Cells. J. Virol. 79: 1487-1499 [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»