Purification and Molecular Structure of RNA Polymerase from Influenza Virus A/PRS1
| Autor: | Akiko Yokoiyama, Akira Ishihama, Mark Krystal, Debi P. Nayak, Jun Mukaigawa, Susumu Ueda, Kyosuke Nagata, Ayae Honda, Atsushi Kato |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Gel electrophoresis
Differential centrifugation biology viruses Orthomyxoviridae RNA-dependent RNA polymerase RNA General Medicine biology.organism_classification Biochemistry Molecular biology chemistry.chemical_compound chemistry RNA polymerase biology.protein Molecular Biology Polymerase Ribonucleoprotein |
| Zdroj: | The Journal of Biochemistry. 107:624-628 |
| ISSN: | 1756-2651 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a123097 |
| Popis: | The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA. |
| Databáze: | OpenAIRE |
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