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. 2006 Sep 11;184(1):277–289. doi: 10.1016/0042-6822(91)90844-2

Construction of tobacco mosaic virus subgenomic replicons that are replicated and spread systemically in tobacco plants

Anthony J Raffo 1, William O Dawson 1,1
PMCID: PMC7131223  PMID: 1871972

Abstract

Two tobacco mosaic virus (TMV)derived replicons, created by deletion of most of the 126/183-kDa open reading frame (ORF), replicated and systemically invaded tobacco plants when supported by wild type TMV. One RNA replicon contained an internal direct repeat of 476 nucleotides from the 3′ end of the 30-kDa ORF. Although this RNA was replicated, most of the progeny were heterogeneous in size and smaller than the original transcript. A second TMV-derived RNA replicon, without any internally repeated sequences and containing a deletion of the 5′ portion of the 30-kDa ORF as well as most of the 126/183-kDa ORF, was created and coinoculated with wild type TMV as helper. This RNA also was replicated efficiently and systemically invaded tobacco plants. An examination of the sequences of cDNA clones obtained after PCR amplification of the progeny population of this RNA replicon demonstrated that the observed size heterogeneity was due to deletions and insertions adjacent to the artificially created deletion junction. These data demonstrate that a TMV infection is capable of supporting an artificially created RNA replicon, similar to defective interfering RNAs or satellites. However, these dependent RNAs were replicated without noticeably interfering with wild type TMV symptoms or replication.

Reference

  1. Ahlquist P., Janda M. cDNA cloning andin vitro transcription of the complete brome mosaic virus genome. Mol. Cell. Biol. 1984;4:2876–2882. doi: 10.1128/mcb.4.12.2876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Smith J.A. Wiley; New York: 1987. Current Protocols in Molecular Biology; pp. 4.9.1–4.9.8. [Google Scholar]; Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Smith J.A. Wiley; New York: 1987. Current Protocols in Molecular Biology; p. 3.5.9. [Google Scholar]; Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Smith J.A. Wiley; New York: 1987. Current Protocols in Molecular Biology; pp. 3.10.2–3.10.5. [Google Scholar]
  3. Barrett A.D.T., Crouch C.F., Dimmock N.J. Defective interfering Semliki forest virus populations are biologically and physically heterogeneous. J. Gen. Virol. 1984;65:1273–1283. doi: 10.1099/0022-1317-65-8-1273. [DOI] [PubMed] [Google Scholar]
  4. Beachy R.N., Zaitlin M. Characterization andin vitro translation of the RNAs from less-than-full-length, virus-related, nucleoprotein rods present in tobacco mosaic virus preparations. Virology. 1977;81:160–169. doi: 10.1016/0042-6822(77)90068-x. [DOI] [PubMed] [Google Scholar]
  5. Beck D.L., Dawson W.O. Deletion of repeated sequences from tobacco mosaic virus mutants with two coat protein genes. Virology. 1990;177:462–469. doi: 10.1016/0042-6822(90)90510-x. [DOI] [PubMed] [Google Scholar]
  6. Burgyan J., Grieco F., Russo M. A defective interfering RNA molecule in cymbidium ringspot virus infections. J. Gen. Virol. 1989;70:235–239. [Google Scholar]
  7. Chirgwin J.J., Przybyla A.E., MacDonald R.J., Rutter W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry. 1979;18:5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Dawson W.O., Beck D.L., Knorr D.A., Grantham G.L. Vol. 83. 1986. cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts; pp. 1832–1836. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dawson W.O., Bubrick P., Grantham G.L. Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomology. Phytopathology. 1988;78:783–789. [Google Scholar]
  10. Dawson W.O., Lewandowski D.J., Hilf M.E., Bubrick P., Raffo A.J., Shaw J.J., Grantham G.L., Desjardins P.R. A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology. 1989;172:285–292. doi: 10.1016/0042-6822(89)90130-x. [DOI] [PubMed] [Google Scholar]
  11. Francki R.I.B. Plant virus satellites. Annu. Rev. Microbiol. 1985;39:151–174. doi: 10.1146/annurev.mi.39.100185.001055. [DOI] [PubMed] [Google Scholar]
  12. French R., Ahlquist P. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J. Virol. 1987;61:1457–1465. doi: 10.1128/jvi.61.5.1457-1465.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goelet P., Lomonossoff G.P., Butler P.J.G., Akam M.E., Gait M.J., Karn J. Vol. 79. 1982. Nucleotide sequence of tobacco mosaic virus RNA; pp. 5818–5822. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldbach R., Eggen R., de Jager C., van Kammen A., van Lent J., Rezelman G., Wellink J. Genetic organization, evolution and expression of plant viral RNA genomes. In: Fraser R.S.S., editor. Recognition and Response in Plant Virus Interactions. Vol. 41. Springer-Verlag; Berlin: 1990. pp. 147–162. (NATO ASI Series). [Google Scholar]
  15. Hagino-Yamagishi K., Nomoto A. In vitro construction of poliovirus defective interfering particles. J. Virol. 1989;63:5386–5392. doi: 10.1128/jvi.63.12.5386-5392.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hanahan D. Studies on the transformation ofE. coli with plasmids. J. Mol. Biol. 1983;166:557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  17. Hillman B.I., Carrington J.C., Morris T.J. A defective interfering RNA that contains a mosaic of a plant virus genome. Cell. 1987;51:427–433. doi: 10.1016/0092-8674(87)90638-6. [DOI] [PubMed] [Google Scholar]
  18. Hunter T., Hunt T., Knowland J., Zimmern D. Messenger RNA for the coat protein of tobacco mosaic virus. Nature (London) 1976;260:759–764. doi: 10.1038/260759a0. [DOI] [PubMed] [Google Scholar]
  19. Jennings P.A., Finch J.T., Winter G., Robertson J.S. Does the higher order structure of the influenza virus ribonucleoprotein guide sequence rearrangements in influenza viral RNA? Cell. 1983;34:619–627. doi: 10.1016/0092-8674(83)90394-x. [DOI] [PubMed] [Google Scholar]
  20. Kaariainen L., Petterson R.F., Keranen S., Lehtovaara P., Sodelund H., Ukkonen P. Multiple structurally related defective-interfering RNAs formed during undiluted passages of Semliki forest virus. Virology. 1981;113:686–697. doi: 10.1016/0042-6822(81)90197-5. [DOI] [PubMed] [Google Scholar]
  21. Kaplan G., Racaniello V.R. Construction and characterization of poliovirus subgenomic replicons. J. Virol. 1988;62:1687–1696. doi: 10.1128/jvi.62.5.1687-1696.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King A.M.Q. Genetic recombination in positive strand RNA viruses. In: Domingo E., Holland J.J., Ahlquist P., editors. Vol. II. CRC Press; Boca Raton, FL: 1988. pp. 149–165. (RNA Genetics). [Google Scholar]
  23. Kuge S., Saito I., Nomoto A. Primary structure of poliovirus defective interfering particle genomes and possible generation mechanisms of the particles. J. Mol. Biol. 1986;192:473–487. doi: 10.1016/0022-2836(86)90270-6. [DOI] [PubMed] [Google Scholar]
  24. Lazzarini R.A., Keene J.D., Schubert M. The origins of defective interfering particles of the negative-strand RNA viruses. Cell. 1981;26:145–154. doi: 10.1016/0092-8674(81)90298-1. [DOI] [PubMed] [Google Scholar]
  25. Lehto K., Dawson W.O. Replication, stability and gene expression of tobacco mosaic virus mutants with a second 30K ORF. Virology. 1990;175:30–40. doi: 10.1016/0042-6822(90)90183-r. [DOI] [PubMed] [Google Scholar]
  26. Lehtovaara P., Soderlund H., Keranen S., Petterson R.F., Kaariainen L. Extreme ends of the genome are conserved and rearranged in the defective-interfering RNAs of Semliki forest virus. J. Mol. Biol. 1982;156:731–748. doi: 10.1016/0022-2836(82)90139-5. [DOI] [PubMed] [Google Scholar]
  27. Levis R., Weiss B.G., Tsaing M., Huang H., Schlesinger S. Deletion mapping of sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging. Cell. 1986;44:137–145. doi: 10.1016/0092-8674(86)90492-7. [DOI] [PubMed] [Google Scholar]
  28. Li X.H., Heaton L.A., Morris T.J., Simon A.E. Vol. 86. 1989. Turnip crinkle virus defective interfering RNAs intensifying viral symptoms and are generatedde novo; pp. 9173–9177. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Making S., Fujioka N., Fujiwara K. Structure of the intercellular defective viral RNAs of defective interfering particles of mouse hepatites virus. J. Virol. 1985;54:329–336. doi: 10.1128/jvi.54.2.329-336.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Making S., Lai M.M.C. High-frequency leader sequence switching during caronavirus defective interfering RNA replication. J. Virol. 1989;63:5285–5292. doi: 10.1128/jvi.63.12.5285-5292.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maniatis T., Fritsch E.F., Sambrook J. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY: 1982. pp. 230–232. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  32. Meshi T., Watanabe Y., Saito T., Sugimoto A., Maeda T., Okada Y. Function of the 30K protein of tobacco mosaic virus: Involvement in cell-to-cell movement and dispensability for replication. EMBO J. 1987;6:2557–2563. doi: 10.1002/j.1460-2075.1987.tb02544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mirkov T.E., Mathews D.M., Du Plessis D.H., Dodds J.A. Nucleotide sequence and translation of satellite tobacco mosaic virus RNA. Virology. 1989;170:139–146. doi: 10.1016/0042-6822(89)90361-9. [DOI] [PubMed] [Google Scholar]
  34. Okada Y., Meshi T., Watanabe Y. Structure and function of tobacco mosaic virus RNA. In: Pirone T.P., Shaw J.G., editors. Viral Genes and Plant Pathogenesis. Springer-Verlag; New York: 1990. pp. 23–38. [Google Scholar]
  35. Pacha F., Allison R.F., Ahlquist P. Cis-acting sequences required forin vivo amplification of genome RNA3 are organized differently in related bromoviruses. Virology. 1990;174:436–443. doi: 10.1016/0042-6822(90)90097-b. [DOI] [PubMed] [Google Scholar]
  36. Pelham H.R.B. Leaky LAG termination codon in tobacco mosaic virus RNA. Nature (London) 1978;272:469–471. doi: 10.1038/272469a0. [DOI] [PubMed] [Google Scholar]
  37. Pogue G.P., Marsh L.E., Hall T.C. Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate (+)-strand replication. Virology. 1990;178:152–160. doi: 10.1016/0042-6822(90)90388-8. [DOI] [PubMed] [Google Scholar]
  38. Rao A.L.N., Hall T.C. Requirements for a viral transacting factor encoded by brome mosaic virus RNAs provides strong selectionin vivo for functional recombinants. J. Virol. 1990;64:2437–2441. doi: 10.1128/jvi.64.5.2437-2441.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sanger H.L. Characteristics of tobacco rattle virus: Evidence that its two particles are functionally defective and mutually complementing. Mol. Gen. Genet. 1968;101:346–367. doi: 10.1007/BF00436232. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A.R. Vol. 74. 1977. DNA sequencing with chain-terminating inhibitors; pp. 5463–5467. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlesinger S. The generation and amplification of defective interfering RNAs. In: Domingo E., Holland J.J., Ahlquist P., editors. Vol. II. CRC Press; Boca Raton, FL: 1988. pp. 167–185. (RNA Genetics). [Google Scholar]
  42. Siegel A., Montgomery V.H.I., Kolacz K. A messenger RNA for coat protein isolated from tobacco mosaic virus infected tissue. Virology. 1976;73:363–371. doi: 10.1016/0042-6822(76)90397-4. [DOI] [PubMed] [Google Scholar]
  43. Siegel A., Zaitlin M., Sehgal O.M. Vol. 48. 1962. The isolation of defective tobacco mosaic virus strains; pp. 1845–1851. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Takamatsu N., Ishikawa M., Meshi T., Okada Y. Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO J. 1987;6:307–311. doi: 10.1002/j.1460-2075.1987.tb04755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Takamatsu N., Watanabe Y., Meshi T., Okada Y. Mutational analysis of the pseudoknot region in the 3′ noncoding region of tobacco mosaic virus RNA. J. Virol. 1990;64:3686–3693. doi: 10.1128/jvi.64.8.3686-3693.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tsiang M., Weiss B.G., Schlesinger S. Effects of 5′-terminal modifications on the biological activity of defective interfering RNAs of sindbis virus. J. Virol. 1988;62:47–53. doi: 10.1128/jvi.62.1.47-53.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Turner D.R., Joyce L.E., Butler P.J.G. The tobacco mosaic virus assembly origin RNA: Functional characteristics defined by directed mutagenesis. J. Mol. Biol. 1988;203:531–547. doi: 10.1016/0022-2836(88)90190-8. [DOI] [PubMed] [Google Scholar]
  48. Valverde R.A., Heick J.A., Dodds J.A. Interactions between satellite tobacco mosaic virus, helper tobamoviruses and their hosts. Phytopathology. 1991;81:99–104. [Google Scholar]
  49. Van Belkum A., Abrahams J.P., Pled C.W.A., Bosch L. 5 Pseudoknots are present at the 204 nucleotides long 3′ noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res. 1985;13:7673–7686. doi: 10.1093/nar/13.21.7673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Van Der Kuyl A.C., Langereis K., Houwing C.J., Jaspers E.M.J., Bol J.F. Cis-acting elements involved in replication of alfalfa mosaic virus RNAsin vitro. Virology. 1990;176:346–354. doi: 10.1016/0042-6822(90)90004-b. [DOI] [PubMed] [Google Scholar]

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