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Journal of Virology, September 2007, p. 10172-10187, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.00531-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Interaction between the Cellular Protein eEF1A and the 3'-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis{triangledown}

William G. Davis,1 Jerry L. Blackwell,1,{dagger} Pei-Yong Shi,2 and Margo A. Brinton1*

Department of Biology, Georgia State University, Atlanta, Georgia 30302,1 Wadsworth Center, New York Department of Health, Albany, New York 122082

Received 13 March 2007/ Accepted 22 June 2007

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

* Corresponding author. Mailing address: Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010. Phone: (404) 413-5388. Fax: (404) 413-5301. E-mail: mbrinton{at}gsu.edu

{triangledown} Published ahead of print on 11 July 2007.

{dagger} Present address: Emory Vaccine Center, Emory University, Atlanta, GA 30329.

Journal of Virology, September 2007, p. 10172-10187, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.00531-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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