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Journal of Virology, August 2006, p. 7450-7458, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.00358-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The NS5A Protein of Bovine Viral Diarrhea Virus Contains an Essential Zinc-Binding Site Similar to That of the Hepatitis C Virus NS5A Protein

Department of Infectology, The Scripps Research Institute, Scripps-Florida, Jupiter, Florida 33458,¹ Gilead Sciences, Inc., Foster City, California 94404,² Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10021³

Received 21 February 2006/ Accepted 20 May 2006

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx¹⁷CxCx²⁰C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx²²CxCx²⁴C and likely comprises a three-stranded antiparallel ß-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.

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