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Journal of Virology, July 2008, p. 6218-6231, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.02624-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A New Role for ns Polyprotein Cleavage in Sindbis Virus Replication {triangledown}

Rodion Gorchakov,1 Elena Frolova,1,2 Stanley Sawicki,3 Svetlana Atasheva,1 Dorothea Sawicki,3 and Ilya Frolov1*

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019,1 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1072,2 Department of Medical Microbiology and Immunology, University of Toledo College of Medicine, Toledo, Ohio 43614-25983

Received 10 December 2007/ Accepted 8 April 2008

One of the distinguishing features of the alphaviruses is a sequential processing of the nonstructural polyproteins P1234 and P123. In the early stages of the infection, the complex of P123+nsP4 forms the primary replication complexes (RCs) that function in negative-strand RNA synthesis. The following processing steps make nsP1+P23+nsP4, and later nsP1+nsP2+nsP3+nsP4. The latter mature complex is active in positive-strand RNA synthesis but can no longer produce negative strands. However, the regulation of negative- and positive-strand RNA synthesis apparently is not the only function of ns polyprotein processing. In this study, we developed Sindbis virus mutants that were incapable of either P23 or P123 cleavage. Both mutants replicated in BHK-21 cells to levels comparable to those of the cleavage-competent virus. They continuously produced negative-strand RNA, but its synthesis was blocked by the translation inhibitor cycloheximide. Thus, after negative-strand synthesis, the ns proteins appeared to irreversibly change conformation and formed mature RCs, in spite of the lack of ns polyprotein cleavage. However, in the cells having no defects in {alpha}/β interferon (IFN-{alpha}/β) production and signaling, the cleavage-deficient viruses induced a high level of type I IFN and were incapable of causing the spread of infection. Moreover, the P123-cleavage-deficient virus was readily eliminated, even from the already infected cells. We speculate that this inability of the viruses with unprocessed polyprotein to productively replicate in the IFN-competent cells and in the cells of mosquito origin was an additional, important factor in ns polyprotein cleavage development. In the case of the Old World alphaviruses, it leads to the release of nsP2 protein, which plays a critical role in inhibiting the cellular antiviral response.

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

{triangledown} Published ahead of print on 16 April 2008.

Journal of Virology, July 2008, p. 6218-6231, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.02624-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Frolov, I., Garmashova, N., Atasheva, S., Frolova, E. I. (2009). Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription. J. Virol. 83: 9031-9044 [Abstract] [Full Text]  
  • Mai, J., Sawicki, S. G., Sawicki, D. L. (2009). Fate of Minus-Strand Templates and Replication Complexes Produced by a P23-Cleavage-Defective Mutant of Sindbis Virus. J. Virol. 83: 8553-8564 [Abstract] [Full Text]  
  • Gorchakov, R., Garmashova, N., Frolova, E., Frolov, I. (2008). Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells. J. Virol. 82: 10088-10101 [Abstract] [Full Text]  


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