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. 1974 Nov;14(5):1049–1055. doi: 10.1128/jvi.14.5.1049-1055.1974

In Vitro Replication of Cowpea Mosaic Virus RNA I. Isolation and Properties of the Membrane-Bound Replicase 1

Pim Zabel a, Hannemarie Weenen-Swaans a, Albert Van Kammen a
PMCID: PMC355618  PMID: 4431078

Abstract

A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 × SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 × SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20S, with a shoulder at 16S to 17S. A minor part of the double-stranded RNA sedimented below 10S. Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26S and 34S.

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Selected References

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

  1. Assink A. M., Swaans H., Van Kammen A. The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves. Virology. 1973 Jun;53(2):384–391. doi: 10.1016/0042-6822(73)90218-3. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C. Optimal conditions for counting of precipitated 3H-RNA on glass-fiber filters. Anal Biochem. 1970 Sep;37(1):178–182. doi: 10.1016/0003-2697(70)90275-7. [DOI] [PubMed] [Google Scholar]
  3. Bradley D. W., Zaitlin M. Replication of tobacco mosaic virus. II. The in vitro synthesis of high molecular weight virus-specific RNAs. Virology. 1971 Jul;45(1):192–199. doi: 10.1016/0042-6822(71)90126-7. [DOI] [PubMed] [Google Scholar]
  4. Byfield J. E., Scherbaum O. H. A rapid radioassay technique for cellular suspensions. Anal Biochem. 1966 Dec;17(3):434–443. doi: 10.1016/0003-2697(66)90179-5. [DOI] [PubMed] [Google Scholar]
  5. Hadidi A., Fraenkel-Conrat H. Characterization and specificity of soluble RNA polymerase of brome mosaic virus. Virology. 1973 Apr;52(2):363–372. doi: 10.1016/0042-6822(73)90331-0. [DOI] [PubMed] [Google Scholar]
  6. Kummert J., Semal J. Properties of single-stranded RNA synthesized by a crude RNA polymerase fraction from barley leaves infected with brome mosaic virus. J Gen Virol. 1972 Jul;16(1):11–20. doi: 10.1099/0022-1317-16-1-11. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. May J. T., Gilliland J. M., Symons R. H. Properties of a plant virus-induced RNA polymerase in particulate fractions of cucumbers infected with cucumber mosaic virus. Virology. 1970 Aug;41(4):653–664. doi: 10.1016/0042-6822(70)90430-7. [DOI] [PubMed] [Google Scholar]
  9. Mouches C., Bove C., Bove J. M. Turnip yellow mosaic virus-RNA replicase: partial purification of the enzyme from the solubilized enzyme-template complex. Virology. 1974 Apr;58(2):409–423. doi: 10.1016/0042-6822(74)90076-2. [DOI] [PubMed] [Google Scholar]
  10. Perry R. P., La Torre J., Kelley D. E., Greenberg J. R. On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. Biochim Biophys Acta. 1972 Mar 14;262(2):220–226. doi: 10.1016/0005-2787(72)90236-5. [DOI] [PubMed] [Google Scholar]
  11. Romero J., Jacquemin J. M. Relation between virus-induced RNA polymerase activity and the synthesis of broadbean mottle virus in broadbean. Virology. 1971 Sep;45(3):813–815. doi: 10.1016/0042-6822(71)90202-9. [DOI] [PubMed] [Google Scholar]
  12. Semal J., Hamilton R. I. RNA synthesis in cell-free extracts of barley leaves infected with bromegrass mosaic virus. Virology. 1968 Oct;36(2):293–302. doi: 10.1016/0042-6822(68)90147-5. [DOI] [PubMed] [Google Scholar]
  13. Semal J., Kummert J. Virus-induced RNA polymerase and synthesis of bromegrass mosaic virus in barley. J Gen Virol. 1970;7(2):173–176. doi: 10.1099/0022-1317-7-2-173. [DOI] [PubMed] [Google Scholar]
  14. Semal J. Properties of the products of UTP incorporation by cell-free extracts of leaves infected with bromegrass mosaic virus or with broadbean mottle virus. Virology. 1970 Feb;40(2):244–250. doi: 10.1016/0042-6822(70)90399-5. [DOI] [PubMed] [Google Scholar]
  15. Zaitlin M., Duda C. T., Petti M. A. Replication of tobacco mosaic virus. V. Properties of the bound and solubilized replicase. Virology. 1973 Jun;53(2):300–311. doi: 10.1016/0042-6822(73)90207-9. [DOI] [PubMed] [Google Scholar]
  16. van Griensven L. J., van Kammen A., Rezelman G. Characterization of the double-stranded RNA isolated from cowpea mosaic virus-infected Vigna leaves. J Gen Virol. 1973 Mar;18(3):359–367. doi: 10.1099/0022-1317-18-3-359. [DOI] [PubMed] [Google Scholar]
  17. van Griensven L. J., van Kammen A. The isolation of ribonuclease-resistant RNA induced by cowpea mosaic virus: evidence for two double-stranded RNA components. J Gen Virol. 1969 Apr;4(3):423–428. doi: 10.1099/0022-1317-4-3-423. [DOI] [PubMed] [Google Scholar]
  18. van Kammen A. Purification and properties of the components of cowpea mosaic virus. Virology. 1967 Apr;31(4):633–642. doi: 10.1016/0042-6822(67)90192-4. [DOI] [PubMed] [Google Scholar]

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