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Journal of Virology, November 2008, p. 10657-10670, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00991-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutation of Mapped TIA-1/TIAR Binding Sites in the 3' Terminal Stem-Loop of West Nile Virus Minus-Strand RNA in an Infectious Clone Negatively Affects Genomic RNA Amplification{triangledown}

Mohamed M. Emara, Hsuan Liu, William G. Davis, and Margo A. Brinton*

Department of Biology, Georgia State University, Atlanta, Georgia 30302

Received 12 May 2008/ Accepted 20 August 2008

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(–)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(–)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(–)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (–)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(–)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

* 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 3 September 2008.

Journal of Virology, November 2008, p. 10657-10670, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00991-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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