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Journal of Virology, September 2009, p. 9031-9044, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00850-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription{triangledown} ,{dagger}

Ilya Frolov,1 Natalia Garmashova,2 Svetlana Atasheva,1 and Elena I. Frolova1*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-2170,1 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-10192

Received 27 April 2009/ Accepted 18 June 2009

Sindbis virus nonstructural protein 2 (SINV nsP2) is an important determinant of virus pathogenesis and downregulation of virus-induced cell response. This protein efficiently inhibits transcription of cellular messenger and ribosomal RNAs and, thus, is capable of inhibiting the activation of genes whose products are involved in development of the antiviral response. Alphavirus nsP2 has a number of predicted functional domains, some of which were confirmed by crystal structure. Our current study demonstrated that none of the putative or known structural domains alone or their combinations was capable of functioning in transcription inhibition. By using random, transposon-mediated mutagenesis, we generated a library of SINV nsP2 variants having short peptide insertions and selected those that lost the ability to inhibit cellular transcription and cause a cytopathic effect. Insertions abrogating the nuclear functions of the protein were found in the three different functional nsP2 domains. Some of the mutated protein variants retained the enzymatic functions required for replication of the viral genome. Such viruses were capable of efficient, productive replication in cells defective in interferon (IFN) signaling but were attenuated and incapable of spreading in cells with an intact type I IFN response. These results revealed new information about the structure of SINV nsP2 and interaction of its domains.

* Corresponding author. Mailing address: Department of Microbiology, University of Alabama at Birmingham, 1530 Third Avenue South, BBRB 373/Box 3 Birmingham, AL 53294-2170. Phone: (205) 996-8958. Fax: (205) 996-4008. E-mail: efrolova{at}uab.edu

{triangledown} Published ahead of print on 1 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, September 2009, p. 9031-9044, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00850-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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