Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1996 Sep;70(9):6227–6234. doi: 10.1128/jvi.70.9.6227-6234.1996

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

T A Osman 1, K W Buck 1
PMCID: PMC190647  PMID: 8709249

Abstract

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.

Full Text

The Full Text of this article is available as a PDF (789.1 KB).

Selected References

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

  1. Barton D. J., Black E. P., Flanegan J. B. Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA. J Virol. 1995 Sep;69(9):5516–5527. doi: 10.1128/jvi.69.9.5516-5527.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bates H. J., Farjah M., Osman T. A., Buck K. W. Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus. J Gen Virol. 1995 Jun;76(Pt 6):1483–1491. doi: 10.1099/0022-1317-76-6-1483. [DOI] [PubMed] [Google Scholar]
  3. Behrens S. E., Tomei L., De Francesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 1996 Jan 2;15(1):12–22. [PMC free article] [PubMed] [Google Scholar]
  4. Carr J. P., Marsh L. E., Lomonossoff G. P., Sekiya M. E., Zaitlin M. Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):397–404. doi: 10.1094/mpmi-5-397. [DOI] [PubMed] [Google Scholar]
  5. Cho M. W., Richards O. C., Dmitrieva T. M., Agol V., Ehrenfeld E. RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol. J Virol. 1993 Jun;67(6):3010–3018. doi: 10.1128/jvi.67.6.3010-3018.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davies C., Symons R. H. Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3. Virology. 1988 Jul;165(1):216–224. doi: 10.1016/0042-6822(88)90675-7. [DOI] [PubMed] [Google Scholar]
  7. Dawson W. O., Lehto K. M. Regulation of tobamovirus gene expression. Adv Virus Res. 1990;38:307–342. doi: 10.1016/S0065-3527(08)60865-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dawson W. O. Tobamovirus-plant interactions. Virology. 1992 Feb;186(2):359–367. doi: 10.1016/0042-6822(92)90001-6. [DOI] [PubMed] [Google Scholar]
  9. Dunigan D. D., Zaitlin M. Capping of tobacco mosaic virus RNA. Analysis of viral-coded guanylyltransferase-like activity. J Biol Chem. 1990 May 15;265(14):7779–7786. [PubMed] [Google Scholar]
  10. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hayes R. J., Buck K. W. Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell. 1990 Oct 19;63(2):363–368. doi: 10.1016/0092-8674(90)90169-f. [DOI] [PubMed] [Google Scholar]
  12. Hayes R. J., Buck K. W. Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction. J Gen Virol. 1990 Nov;71(Pt 11):2503–2508. doi: 10.1099/0022-1317-71-11-2503. [DOI] [PubMed] [Google Scholar]
  13. Kielland-Brandt M. C. Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves. J Mol Biol. 1974 Aug 15;87(3):489–503. doi: 10.1016/0022-2836(74)90099-0. [DOI] [PubMed] [Google Scholar]
  14. Kitamura K., Namazue J., Campo-Vera H., Ogino T., Yamanishi K. Induction of neutralizing antibody against varicella-zoster virus (VZV) by VZV gp3 and cross-reactivity between VZV gp3 and herpes simplex viruses gB. Virology. 1986 Feb;149(1):74–82. doi: 10.1016/0042-6822(86)90088-7. [DOI] [PubMed] [Google Scholar]
  15. Koonin E. V., Dolja V. V. Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol. 1993;28(5):375–430. doi: 10.3109/10409239309078440. [DOI] [PubMed] [Google Scholar]
  16. Lehto K., Bubrick P., Dawson W. O. Time course of TMV 30K protein accumulation in intact leaves. Virology. 1990 Jan;174(1):290–293. doi: 10.1016/0042-6822(90)90077-5. [DOI] [PubMed] [Google Scholar]
  17. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  18. Meshi T., Ishikawa M., Motoyoshi F., Semba K., Okada Y. In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5043–5047. doi: 10.1073/pnas.83.14.5043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller W. A., Dreher T. W., Hall T. C. Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA. Nature. 1985 Jan 3;313(5997):68–70. doi: 10.1038/313068a0. [DOI] [PubMed] [Google Scholar]
  20. Molla A., Paul A. V., Wimmer E. Cell-free, de novo synthesis of poliovirus. Science. 1991 Dec 13;254(5038):1647–1651. doi: 10.1126/science.1661029. [DOI] [PubMed] [Google Scholar]
  21. Ohno T., Aoyagi M., Yamanashi Y., Saito H., Ikawa S., Meshi T., Okada Y. Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome. J Biochem. 1984 Dec;96(6):1915–1923. doi: 10.1093/oxfordjournals.jbchem.a135026. [DOI] [PubMed] [Google Scholar]
  22. Osman T. A., Buck K. W. Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants. J Gen Virol. 1991 Nov;72(Pt 11):2853–2856. doi: 10.1099/0022-1317-72-11-2853. [DOI] [PubMed] [Google Scholar]
  23. Rezaian M. A., Williams R. H., Gordon K. H., Gould A. R., Symons R. H. Nucleotide sequence of cucumber-mosaic-virus RNA 2 reveals a translation product significantly homologous to corresponding proteins of other viruses. Eur J Biochem. 1984 Sep 3;143(2):277–284. doi: 10.1111/j.1432-1033.1984.tb08370.x. [DOI] [PubMed] [Google Scholar]
  24. Rezaian M. A., Williams R. H., Symons R. H. Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs. Eur J Biochem. 1985 Jul 15;150(2):331–339. doi: 10.1111/j.1432-1033.1985.tb09025.x. [DOI] [PubMed] [Google Scholar]
  25. Schiebel W., Haas B., Marinković S., Klanner A., Sänger H. L. RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties. J Biol Chem. 1993 Jun 5;268(16):11851–11857. [PubMed] [Google Scholar]
  26. Song C., Simon A. E. RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+)- and (-)-strands of virus-associated RNAs. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8792–8796. doi: 10.1073/pnas.91.19.8792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu G., Kaper J. M. Requirement of 3'-terminal guanosine in (-)-stranded RNA for in vitro replication of cucumber mosaic virus satellite RNA by viral RNA-dependent RNA polymerase. J Mol Biol. 1994 May 20;238(5):655–657. doi: 10.1006/jmbi.1994.1326. [DOI] [PubMed] [Google Scholar]
  28. Wu S. X., Ahlquist P., Kaesberg P. Active complete in vitro replication of nodavirus RNA requires glycerophospholipid. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11136–11140. doi: 10.1073/pnas.89.23.11136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu S. X., Kaesberg P. Synthesis of template-sense, single-strand Flockhouse virus RNA in a cell-free replication system. Virology. 1991 Jul;183(1):392–396. doi: 10.1016/0042-6822(91)90153-3. [DOI] [PubMed] [Google Scholar]
  30. Xiong Z. G., Lommel S. A. Red clover necrotic mosaic virus infectious transcripts synthesized in vitro. Virology. 1991 May;182(1):388–392. doi: 10.1016/0042-6822(91)90687-7. [DOI] [PubMed] [Google Scholar]
  31. Young N., Forney J., Zaitlin M. Tobacco mosaic virus replicase and replicative structures. J Cell Sci Suppl. 1987;7:277–285. doi: 10.1242/jcs.1987.supplement_7.19. [DOI] [PubMed] [Google Scholar]
  32. Zabel P., Dorssers L., Wernars K., Van Kammen A. Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3'-end of an RNA primer. Nucleic Acids Res. 1981 Jun 11;9(11):2433–2453. doi: 10.1093/nar/9.11.2433. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES