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Journal of Virology, January 2005, p. 696-704, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.696-704.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases

Anja Seybert,1,{dagger} Clara C. Posthuma,2 Leonie C. van Dinten,2,{ddagger} Eric J. Snijder,2 Alexander E. Gorbalenya,2 and John Ziebuhr1*

Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany,1 Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands2

Received 29 June 2004/ Accepted 18 August 2004

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

* Corresponding author. Mailing address: Institute of Virology and Immunology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany. Phone: 49-931-20149928. Fax: 49-931-20149553. E-mail: j.ziebuhr{at}mail.uni-wuerzburg.de.

{dagger} Present address: European Molecular Biology Laboratory, Heidelberg, Germany.

{ddagger} Present address: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

Journal of Virology, January 2005, p. 696-704, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.696-704.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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