The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases
| Autor: | Marie C. Lin, Leo L.M. Poon, Lin Yu Lu, Jian-Dong Huang, J. S. Malik Peiris, Hsiang-Fu Kung, Yu-Bo Chai, Rory M. Watt, Julian A. Tanner |
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| Rok vydání: | 2003 |
| Předmět: |
viruses
Molecular Sequence Data medicine.disease_cause Biochemistry Substrate Specificity chemistry.chemical_compound Chlorocebus aethiops medicine Animals Coronaviridae Vero Cells Molecular Biology Escherichia coli Coronavirus chemistry.chemical_classification Enzyme Catalysis and Regulation Base Sequence biology DNA Helicases Helicase RNA Cell Biology Nucleoside-Triphosphatase biology.organism_classification RNA Helicase A Kinetics Enzyme Severe acute respiratory syndrome-related coronavirus chemistry DNA Viral biology.protein RNA Helicases DNA |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.c300328200 |
| Popis: | The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km . |
| Databáze: | OpenAIRE |
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