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Journal of Virology, February 2007, p. 1858-1871, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.01462-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase{triangledown}

Stephen W. B. Fullerton,1,{dagger} Martina Blaschke,2,{dagger} Bruno Coutard,3 Julia Gebhardt,2 Alexander Gorbalenya,4 Bruno Canard,3 Paul A. Tucker,1 and Jacques Rohayem2*

European Molecular Biology Laboratory, Hamburg, Germany,1 Institut für Virologie, The Calicilab, Medizinisch-Theoretisches Zentrum, Dresden, Germany,2 Laboratoire Architecture et Fonction des Macromolécules Biologiques UMR6098 CNRS and Université de la Méditérannée, Marseilles, France,3 Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands4

Received 11 July 2006/ Accepted 13 November 2006

Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

* Corresponding author. Mailing address: Institut für Virologie, The Calicilab, Fiedlerstr. 42, D-01307 Dresden, Germany. Phone: 49 351 4586200. Fax: 49 351 4586314. E-mail: Jacques.Rohayem{at}mailbox.tu-dresden.de.

{triangledown} Published ahead of print on 22 November 2006.

{dagger} Both authors contributed equally to the study.

Journal of Virology, February 2007, p. 1858-1871, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.01462-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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