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Journal of Virology, September 2009, p. 8327-8339, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00586-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structural and Functional Elements of the Promoter Encoded by the 5' Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome{triangledown}

Raghavendran Kulasegaran-Shylini,2 Svetlana Atasheva,3 David G. Gorenstein,2 and Ilya Frolov1*

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019,1 Department of Biochemistry and Molecular Biology and the Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1157,2 Department of Microbiology, University of Alabama, Birmingham, Alabama 35294-21703

Received 22 March 2009/ Accepted 3 June 2009

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5' untranslated region (5'UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5'-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5'UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5'-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem's stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5'UTR either containing 5'-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5' terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3' terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.

* Corresponding author. Present address: Department of Microbiology, BBRB 373/Box 3, University of Alabama, 1530 Third Avenue South, Birmingham, AL 35294-2170. Phone: (205) 996-8957. Fax: (205) 996-4008. E-mail: ivfrolov{at}UAB.edu

{triangledown} Published ahead of print on 10 June 2009.

Journal of Virology, September 2009, p. 8327-8339, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00586-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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