Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase
| Autor: | Martina Blaschke, Bruno Coutard, Alexander E. Gorbalenya, Bruno Canard, Jacques Rohayem, Stephen W. B. Fullerton, Paul A. Tucker, Julia Gebhardt |
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| Rok vydání: | 2007 |
| Předmět: |
Models
Molecular Polyadenylation viruses Molecular Sequence Data Immunology RNA-dependent RNA polymerase Crystallography X-Ray Microbiology Sapovirus Viral Proteins chemistry.chemical_compound Virology RNA polymerase Magnesium Amino Acid Sequence Polymerase Manganese Binding Sites Molecular Structure biology Structure and Assembly RNA Templates Genetic RNA-Dependent RNA Polymerase biology.organism_classification Molecular biology Caliciviridae Lagovirus chemistry Insect Science biology.protein RNA Viral |
| Zdroj: | Journal of Virology. 81:1858-1871 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.01462-06 |
| Popis: | 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 3D pol -like protein. Enzymatically active sapovirus 3D pol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3D pol 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 3D pol prefers Mn 2+ over Mg 2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3D pol 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 3D pol an attractive target for developing drugs to control calicivirus infection in humans. |
| Databáze: | OpenAIRE |
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