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. 1985 Jan;53(1):266–272. doi: 10.1128/jvi.53.1.266-272.1985

Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

C D Morrow, J Lubinski, J Hocko, G F Gibbons, A Dasgupta
PMCID: PMC255028  PMID: 2981346

Abstract

The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.

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Selected References

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  1. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  2. Baron M. H., Baltimore D. Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA. Cell. 1982 Oct;30(3):745–752. doi: 10.1016/0092-8674(82)90279-3. [DOI] [PubMed] [Google Scholar]
  3. Baron M. H., Baltimore D. Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro. J Virol. 1982 Sep;43(3):969–978. doi: 10.1128/jvi.43.3.969-978.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baron M. H., Baltimore D. In vitro copying of viral positive strand RNA by poliovirus replicase. Characterization of the reaction and its products. J Biol Chem. 1982 Oct 25;257(20):12359–12366. [PubMed] [Google Scholar]
  5. Baron M. H., Baltimore D. Purification and properties of a host cell protein required for poliovirus replication in vitro. J Biol Chem. 1982 Oct 25;257(20):12351–12358. [PubMed] [Google Scholar]
  6. Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
  7. Butterworth B. E., Grunert R. R., Korant B. D., Lonberg-Holm K., Yin F. H. Replication of rhinoviruses. Arch Virol. 1976;51(3):169–189. doi: 10.1007/BF01318022. [DOI] [PubMed] [Google Scholar]
  8. Caliguiri L. A., Tamm I. The role of cytoplasmic membranes in poliovirus biosynthesis. Virology. 1970 Sep;42(1):100–111. doi: 10.1016/0042-6822(70)90242-4. [DOI] [PubMed] [Google Scholar]
  9. Dasgupta A. Antibody to host factor precipitates poliovirus RNA polymerase from poliovirus-infected HeLa cells. Virology. 1983 Jul 15;128(1):252–259. doi: 10.1016/0042-6822(83)90336-7. [DOI] [PubMed] [Google Scholar]
  10. Dasgupta A., Baron M. H., Baltimore D. Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2679–2683. doi: 10.1073/pnas.76.6.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dasgupta A., Hollingshead P., Baltimore D. Antibody to a host protein prevents initiation by the poliovirus replicase. J Virol. 1982 Jun;42(3):1114–1117. doi: 10.1128/jvi.42.3.1114-1117.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dasgupta A. Purification of host factor required for in vitro transcription of poliovirus RNA. Virology. 1983 Jul 15;128(1):245–251. doi: 10.1016/0042-6822(83)90335-5. [DOI] [PubMed] [Google Scholar]
  13. Dasgupta A., Zabel P., Baltimore D. Dependence of the activity of the poliovirus replicase on the host cell protein. Cell. 1980 Feb;19(2):423–429. doi: 10.1016/0092-8674(80)90516-4. [DOI] [PubMed] [Google Scholar]
  14. Flanegan J. B., Baltimore D. Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. J Virol. 1979 Jan;29(1):352–360. doi: 10.1128/jvi.29.1.352-360.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flanegan J. B., Baltimore D. Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A). Proc Natl Acad Sci U S A. 1977 Sep;74(9):3677–3680. doi: 10.1073/pnas.74.9.3677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Flanegan J. B., Van Dyke T. A. Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro. J Virol. 1979 Oct;32(1):155–161. doi: 10.1128/jvi.32.1.155-161.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gauntt C. J. Synthesis of ribonucleic acids in KB cells infected with rhinovirus type 14. J Gen Virol. 1973 Nov;21(2):253–267. doi: 10.1099/0022-1317-21-2-253. [DOI] [PubMed] [Google Scholar]
  18. Griffith M. M., Gauntt C. J. Rhinovirus type 14 RNA polymerase complexes. Arch Virol. 1975;47(1):11–20. doi: 10.1007/BF01315588. [DOI] [PubMed] [Google Scholar]
  19. Koliais S. I., Dimmock N. J. Replication of rhinovirus RNA. J Gen Virol. 1973 Jul;20(1):1–15. doi: 10.1099/0022-1317-20-1-1. [DOI] [PubMed] [Google Scholar]
  20. Koliais S. I., Dimmock N. J. Rhinovirus RNA polymerase: products and kinetics of appearance in human diploid cells. J Virol. 1974 Nov;14(5):1035–1039. doi: 10.1128/jvi.14.5.1035-1039.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Macnaughton M. R. The structure and replication of rhinoviruses. Curr Top Microbiol Immunol. 1982;97:1–26. doi: 10.1007/978-3-642-68318-3_1. [DOI] [PubMed] [Google Scholar]
  22. Mildvan A. S., Loeb L. A. The role of metal ions in the mechanisms of DNA and RNA polymerases. CRC Crit Rev Biochem. 1979;6(3):219–244. doi: 10.3109/10409237909102564. [DOI] [PubMed] [Google Scholar]
  23. Morrow C. D., Dasgupta A. Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA. J Virol. 1983 Nov;48(2):429–439. doi: 10.1128/jvi.48.2.429-439.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Morrow C. D., Hocko J., Navab M., Dasgupta A. ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein. J Virol. 1984 May;50(2):515–523. doi: 10.1128/jvi.50.2.515-523.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sethi S. K., Schwerdt C. E. Studies on the biosynthesis and characterization of rhinovirus ribonucleic acid. Virology. 1972 Apr;48(1):221–229. doi: 10.1016/0042-6822(72)90129-8. [DOI] [PubMed] [Google Scholar]
  26. Spector D. H., Baltimore D. Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs. J Virol. 1975 Jun;15(6):1418–1431. doi: 10.1128/jvi.15.6.1418-1431.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Dyke T. A., Flanegan J. B. Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. J Virol. 1980 Sep;35(3):732–740. doi: 10.1128/jvi.35.3.732-740.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van Dyke T. A., Rickles R. J., Flanegan J. B. Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase. J Biol Chem. 1982 Apr 25;257(8):4610–4617. [PubMed] [Google Scholar]
  29. Yin F. H., Knight E., Jr In vivo and in vitro synthesis of human rhinovirus type 2 ribonucleic acid. J Virol. 1972 Jul;10(1):93–98. doi: 10.1128/jvi.10.1.93-98.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yin F. H., Lomax N. B. Host range mutants of human rhinovirus in which nonstructural proteins are altered. J Virol. 1983 Nov;48(2):410–418. doi: 10.1128/jvi.48.2.410-418.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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