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Journal of Virology, August 2004, p. 8159-8171, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8159-8171.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Structure and Function of the 3' Terminal Six Nucleotides of the West Nile Virus Genome in Viral Replication

Mark Tilgner1 and Pei-Yong Shi1,2*

Wadsworth Center, New York State Department of Health,1 Department of Biomedical Sciences, University at Albany, State University of New York, Albany, New York 122012

Received 19 January 2004/ Accepted 1 April 2004

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCUOH-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i) The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii) The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CUOH, are absolutely essential for viral replication. (iii) The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv) The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v) Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.

* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, Albany, NY 12208. Phone: (518) 473-7487. Fax: (518) 473-1326. E-mail: ship{at}wadsworth.org.

Journal of Virology, August 2004, p. 8159-8171, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8159-8171.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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