Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1971 Aug;8(2):190–196. doi: 10.1128/jvi.8.2.190-196.1971

Unilateral Synthesis of Reovirus Double-Stranded Ribonucleic Acid by a Cell-Free Replicase System

S Sakuma 1, Y Watanabe 1
PMCID: PMC356230  PMID: 5115915

Abstract

A large-particle fraction obtained from reovirus-infected L cells contained both replicase and transcriptase activity. The in vitro replicase reaction slowed down soon after initiation, whereas the transcriptase reaction proceeded at an unabated rate. The replicase and transcriptase were both template-bound and could be separated from one another by controlled chymotryptic digestion followed by centrifugation in a CsCl gradient. The transcriptase was recovered as a sharp band (ρ = 1.43) and resembled virus core derived from mature virions. In contrast, replicase activity was distributed throughout the gradient, indicating that replicase is associated with structures of various density in CsCl. In subsequent experiments, the replicase product was found to be indistinguishable from the double-stranded ribonucleic acid (RNA) reovirus genome with respect to its buoyant density in cesium-salt gradients and denaturation-annealing characteristics. A “hybridization-competition” experiment in which the replicase product was denatured and annealed in the presence of an excess of plus-RNA indicated that the in vitro replicase reaction proceeded by means of a unilateral synthesis of minus-RNA upon a preexisting plus-RNA template, presumably of single-stranded form.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banerjee A. K., Shatkin A. J. Transcription in vitro by reovirus-associated ribonucleic acid-dependent polymerase. J Virol. 1970 Jul;6(1):1–11. doi: 10.1128/jvi.6.1.1-11.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellamy A. R., Joklik W. K. Studies on the A-rich RNA of reovirus. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1389–1395. doi: 10.1073/pnas.58.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borsa J., Grover J., Chapman J. D. Presence of nucleoside triphosphate phosphohydrolase activity in purified virions of reovirus. J Virol. 1970 Sep;6(3):295–302. doi: 10.1128/jvi.6.3.295-302.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gomatos P. J. Comparison of the virion polymerase of reovirus with the enzyme purified from reovirus-infected cells. J Virol. 1970 Nov;6(5):610–620. doi: 10.1128/jvi.6.5.610-620.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  6. Levin D. H., Mendelsohn N., Schonberg M., Klett H., Silverstein S., Kapuler A. M., Acs G. Properties of RNA transcriptase in reovirus subviral particles. Proc Natl Acad Sci U S A. 1970 Jul;66(3):890–897. doi: 10.1073/pnas.66.3.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lozeron H. A., Szybalski W. Suppression of RNA precipitation during Cs2SO4 density gradient centrifugation. Biochem Biophys Res Commun. 1966 Jun 13;23(5):612–618. doi: 10.1016/0006-291x(66)90443-8. [DOI] [PubMed] [Google Scholar]
  8. Nonoyama M., Watanabe Y., Graham A. F. Defective virions of reovirus. J Virol. 1970 Aug;6(2):226–236. doi: 10.1128/jvi.6.2.226-236.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Schonberg M., Silverstein S. C., Levin D. H., Acs G. Asynchronous synthesis of the complementary strands of the reovirus genome. Proc Natl Acad Sci U S A. 1971 Feb;68(2):505–508. doi: 10.1073/pnas.68.2.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Shatkin A. J., Sipe J. D. RNA polymerase activity in purified reoviruses. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1462–1469. doi: 10.1073/pnas.61.4.1462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Skehel J. J., Joklik W. K. Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores. Virology. 1969 Dec;39(4):822–831. doi: 10.1016/0042-6822(69)90019-1. [DOI] [PubMed] [Google Scholar]
  12. Watanabe Y., Gauntt C. J., Graham A. F. Reovirus-induced ribonucleic acid polymerase. J Virol. 1968 Sep;2(9):869–877. doi: 10.1128/jvi.2.9.869-877.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Watanabe Y., Graham A. F. Structural units of reovirus ribonucleic acid and their possible functional significance. J Virol. 1967 Aug;1(4):665–677. doi: 10.1128/jvi.1.4.665-677.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Watanabe Y., Kudo H., Graham A. F. Selective inhibition of reovirus ribonucleic acid synthesis by cycloheximide. J Virol. 1967 Feb;1(1):36–44. doi: 10.1128/jvi.1.1.36-44.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Watanabe Y., Millward S., Graham A. F. Regulation of transcription of the Reovirus genome. J Mol Biol. 1968 Aug 28;36(1):107–123. doi: 10.1016/0022-2836(68)90223-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES