Advertisement

Plant Molecular Biology

, Volume 6, Issue 6, pp 455–465 | Cite as

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

  • Nevin Dale Young
  • Milton Zaitlin
Article

Summary

The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3′-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5′-end of the plus strand.

Keywords

replicative form replicative intermediate molecular hybridization tobacco mosaic virus plant viral replication 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Baltimore D: Structure of the poliovirus replicative intermediate. J Mol Biol 32: 359–368, 1968.Google Scholar
  2. 2.
    Baltimore D, Girard M: An intermediate in the synthesis of poliovirus RNA. Proc Natl Acad Sci USA 56: 741–748, 1966.Google Scholar
  3. 3.
    Beachy R, Zaitlin M: Replication of tobacco mosaic virus. VI. Replicative intermediate and TMV-RNA related RNAs associated with polyribosomes. Virology 63: 84–97, 1975.Google Scholar
  4. 4.
    Bové JM, Bové C, Mocquot B: Turnip yellow mosaic virus RNA synthesis in vitro: Evidence for antive double stranded RNA. Biochem Biophys Res Commun 32: 480–486, 1968.Google Scholar
  5. 5.
    Bradley DW, Zaitlin M: Replication of tobacco mosaic virus. II. The in vitro synthesis of high molecular weight virus-specific RNAs. Virology 45: 192–199, 1971.Google Scholar
  6. 6.
    Dorsseers L, Van der Meer J, Van Kammen A, Zabel P: The cowpea mosaic virus RNA replication complex and the host-encoded RNA-dependent RNA polymerase-template complex are functionally different. Virology 125: 155–174, 1983.Google Scholar
  7. 7.
    Dreher TW, Bujarski J, Hall TC: Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. Nature 311: 171–175, 1984.Google Scholar
  8. 8.
    Duda CT, Zaitlin M, Siegel A: In vitro synthesis of double-stranded RNA by an enzyme system isolated from tobacco leaves. Biochem Biophys Acta 319: 62–71, 1973.Google Scholar
  9. 9.
    Erikson RL, Erikson E, Gordon JA: Structure and function of bacteriophage R17 replicative RNA. I. Studies on sedimentation and infectivity. J Mol Biol 22: 257–268, 1966.Google Scholar
  10. 10.
    Garnier M, Mamoun R, Bové JM: TYMV RNA replication in vivo: replicative intermediate is mainly single stranded. Virology 104: 357–374, 1980.Google Scholar
  11. 11.
    Goelet P, Lomanossoff GP, Butler PJG, Akam ME, Gait MJ, Karn J: Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci USA 79: 5818–5822, 1982.Google Scholar
  12. 12.
    Hunter TR, Hunt T, Knowland J, Zimmern D: Messenger RNA for the coat protein of tobacco mosaic virus. Nature 260: 759–764, 1976.Google Scholar
  13. 13.
    Jackson AO, Zaitlin M, Siegel A, Francki RIB: Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48: 655–665, 1972.Google Scholar
  14. 14.
    Jaspars EMJ, Gill DS, Symons RH: Viral RNA synthesis by a particulate fraction from cucumber seedlings infected with cucumber mosaic virus. Virology 144: 410–425, 1985.Google Scholar
  15. 15.
    Kafatos FC, Jones CW, Efstradidis A: Determination of nucleic acid sequence homologies and relative concentrations by a dot blot hybridization procedure. Nucl Acid Res 7: 1541–1552, 1979.Google Scholar
  16. 16.
    Kielland-Brandt MC: Studies on the 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 87: 489–503, 1974.Google Scholar
  17. 17.
    Loening UE: The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J 102: 251–257, 1967.Google Scholar
  18. 18.
    Maule AJ, Hull R, Donson J: The application of spot hybridization to the detection of DNA and RNA viruses in plant tissues. J Virol Meth 6: 215–224, 1983.Google Scholar
  19. 19.
    May JT, Gillian JM, Symons RH: Properties of a plant virus-induced RNA polymerase in particulate fractions of cucumbers infected with cucumber mosaic virus. Virology 41: 653–664, 1970.Google Scholar
  20. 20.
    Messing J, Crea R, Seeburg PH: A system for shotgun DNA sequencing. Nucl Acid Res 9: 309–321, 1981.Google Scholar
  21. 21.
    Messing J: New M13 vectors for cloning. Meth Enzymol 101: 20–79, 1983.Google Scholar
  22. 22.
    Miller WA, Hall TC: Use of micrococcal nuclease in the purification of highly template dependent RNA-dependent RNA polymerase from brome mosaic virus-infected barley. Virology 125: 236–241, 1983.Google Scholar
  23. 23.
    Miller WA, Dreher TW, Hall TC: Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (−)-sense genomic RNA. Nature 313: 68–70, 1985.Google Scholar
  24. 24.
    Nilsson-Tillgren T: Studies on the biosynthesis of TMV. II. On the RNA synthesis of infected cells. Molec Gen Genet 105: 191–202, 1969.Google Scholar
  25. 25.
    Nilsson-Tillgren T: Studies on the biosynthesis of TMV. III. Isolation and characterization of the replicative form and the replicative intermediate RNA Molec Gen Genet 109: 246–256, 1970.Google Scholar
  26. 26.
    Ralph RK, Wojcik SJ: Double-stranded tobacco mosaic virus RNA. Virology 37: 276–282, 1969.Google Scholar
  27. 27.
    Ralph RK, Matthews REF, Matus AI, Mandel HG: Isolation and properties of double-stranded viral RNA from virus-infected plants. J Mol Biol 11: 202–212, 1965.Google Scholar
  28. 28.
    Robertson HD, Webster RE, Zinder ND: Purification and properties of ribonuclease III from Escherichia coli. J Biol Chem 243: 82–91, 1968.Google Scholar
  29. 29.
    Romane CP, Zaitlin M: RNA-dependent RNA-polymerases in uninfected and tobacco mosaic virus-infected tobacco leaves: Viral-induced stimulation of a host polymerase activity. Virology 86: 241–253, 1978.Google Scholar
  30. 30.
    Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977.Google Scholar
  31. 31.
    Seed B: Diazotizable arylamine cellulose papers for the coupling and hybridization of nucleic acid. Nucl Acid Res 10: 1799–1810, 1982.Google Scholar
  32. 32.
    Siegel A, Wildman SG: Some natural relationships among strains of tobacco virus. Phytopath 44: 277–282, 1954.Google Scholar
  33. 33.
    Siegel A, Hari V, Montgomery I, Kolacz K: A messenger RNA for capsid protein isolated from tobacco-mosaic-virus infected tissue. Virology 73: 363–371, 1976.Google Scholar
  34. 34.
    Sulzinski MA, Gabard KA, Palukaitis P, Zaitlin M: Replication of tobacco mosaic virus. VIII. Characterization of a third subgenomic TMV RNA. Virology 145: 132–140, 1985.Google Scholar
  35. 35.
    Thatch SS, Dobbertin D, Lawarence C, Golini F, Thatch RE: The mechanism of viral replication. Structure of replication complexes of encephalomyocarditis virus. Proc Natl Acad Sci USA 71: 2549–2553, 1974.Google Scholar
  36. 36.
    Watanabe Y, Okada Y: In vitro viral RNA synthesis by a subcellular fraction of TMV-inoculated tobacco protoplasts. Virology 149: 64–73, 1986.Google Scholar
  37. 37.
    Weissman C, Burdon RH, Billeter MA, Ochoa S: Replication of viral RNA. III. Double-stranded replicative form of MS2 phage RNA. Proc Natl Acad Sci USA 51: 682–690, 1964.Google Scholar
  38. 38.
    Zabel P, Weenen-Swaans H, Van Kammen A: In vitro replication of cowpea mosaic virus RNA. I. Isolation and properties of the membrane-bound replicase. J Virol 14: 1049–1055, 1974.Google Scholar
  39. 39.
    Zabel P, Dorsseers 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. Nucl Acid Res 9: 2433–2453, 1981.Google Scholar
  40. 40.
    Zaitlin M, Duda CT, Petti MA: Replication of tobacco mosaic virus. V. Properties of the bound and solubilized replicase. Virology 53: 300–311, 1972.Google Scholar
  41. 41.
    Zelcer A, Weaber KF, Balazs E, Zaitlin M: The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants. Virology 113: 417–427, 1981.Google Scholar

Copyright information

© Martinus Nijhoff Publishers 1986

Authors and Affiliations

  • Nevin Dale Young
    • 1
  • Milton Zaitlin
    • 1
  1. 1.Department of Plant PathologyCornell UniversityIthacaU.S.A.

Personalised recommendations