Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jul;70(7):4370–4379. doi: 10.1128/jvi.70.7.4370-4379.1996

Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.

S Mahajan 1, V V Dolja 1, J C Carrington 1
PMCID: PMC190370  PMID: 8676460

Abstract

The roles of the capsid protein (CP) and the CP coding sequence of tobacco etch potyvirus (TEV) in genome amplification were analyzed. A series of frameshift-stop codon mutations that interrupted translation of the CP coding sequence at various positions were introduced into the TEV genome. A series of 3' deletion mutants that lacked the CP coding sequence beyond each of the frameshift-stop codon mutations were also produced. In addition, a series of 5' CP deletion mutants were generated. Amplification of genomes containing either frameshift-stop codon insertions after codons 1, 59, 103, and 138 or genomes containing the corresponding 3' deletions of the CP coding sequence was reduced by 100- to 1,000-fold relative to that of the parental genome in inoculated protoplasts. In contrast, a mutant containing a frameshift-stop codon after CP position 189 was amplified to 27% of the level of the parental virus, but the corresponding 3' deletion mutant lacking codons 190 to 261 was nonviable. Deletion mutants lacking CP codons 2 to 100, 2 to 150, 2 to 189, and 2 to 210 were amplified relatively efficiently in protoplasts, but a deletion mutant lacking codons 2 to 230 was nonviable. None of the amplification-defective frameshift-stop codon or deletion mutants was rescued in transgenic cells expressing TEV CP, although the transgenic CP was able to rescue intercellular movement defects of replication-competent CP mutants. Coupled with previous results, these data led to the conclusions that (i) TEV genome amplification requires translation to a position between CP codons 138 and 189 but does not require the CP product and (ii) the TEV CP coding sequence contains a cis-active RNA element between codons 211 and 246. The implications of these findings on mechanisms of RNA replication and genome evolution are discussed.

Full Text

The Full Text of this article is available as a PDF (628.7 KB).

Selected References

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

  1. Atreya C. D., Atreya P. L., Thornbury D. W., Pirone T. P. Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants. Virology. 1992 Nov;191(1):106–111. doi: 10.1016/0042-6822(92)90171-k. [DOI] [PubMed] [Google Scholar]
  2. Atreya C. D., Raccah B., Pirone T. P. A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology. 1990 Sep;178(1):161–165. doi: 10.1016/0042-6822(90)90389-9. [DOI] [PubMed] [Google Scholar]
  3. Atreya P. L., Atreya C. D., Pirone T. P. Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7887–7891. doi: 10.1073/pnas.88.17.7887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Atreya P. L., Lopez-Moya J. J., Chu M., Atreya C. D., Pirone T. P. Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids. J Gen Virol. 1995 Feb;76(Pt 2):265–270. doi: 10.1099/0022-1317-76-2-265. [DOI] [PubMed] [Google Scholar]
  5. Ball L. A., Li Y. cis-acting requirements for the replication of flock house virus RNA 2. J Virol. 1993 Jun;67(6):3544–3551. doi: 10.1128/jvi.67.6.3544-3551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berkhout B., Schmidt B. F., van Strien A., van Boom J., van Westrenen J., van Duin J. Lysis gene of bacteriophage MS2 is activated by translation termination at the overlapping coat gene. J Mol Biol. 1987 Jun 5;195(3):517–524. doi: 10.1016/0022-2836(87)90180-x. [DOI] [PubMed] [Google Scholar]
  7. Berkhout B., van Duin J. Mechanism of translational coupling between coat protein and replicase genes of RNA bacteriophage MS2. Nucleic Acids Res. 1985 Oct 11;13(19):6955–6967. doi: 10.1093/nar/13.19.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  9. Carrington J. C., Dougherty W. G. Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol. 1987 Aug;61(8):2540–2548. doi: 10.1128/jvi.61.8.2540-2548.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carrington J. C., Haldeman R., Dolja V. V., Restrepo-Hartwig M. A. Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo. J Virol. 1993 Dec;67(12):6995–7000. doi: 10.1128/jvi.67.12.6995-7000.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang Y. C., Borja M., Scholthof H. B., Jackson A. O., Morris T. J. Host effects and sequences essential for accumulation of defective interfering RNAs of cucumber necrosis and tomato bushy stunt tombusviruses. Virology. 1995 Jun 20;210(1):41–53. doi: 10.1006/viro.1995.1315. [DOI] [PubMed] [Google Scholar]
  12. Dolja V. V., Haldeman-Cahill R., Montgomery A. E., Vandenbosch K. A., Carrington J. C. Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology. 1995 Feb 1;206(2):1007–1016. doi: 10.1006/viro.1995.1023. [DOI] [PubMed] [Google Scholar]
  13. Dolja V. V., Haldeman R., Robertson N. L., Dougherty W. G., Carrington J. C. Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO J. 1994 Mar 15;13(6):1482–1491. doi: 10.1002/j.1460-2075.1994.tb06403.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dolja V. V., McBride H. J., Carrington J. C. Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10208–10212. doi: 10.1073/pnas.89.21.10208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dougherty W. G., Cary S. M., Parks T. D. Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology. 1989 Aug;171(2):356–364. doi: 10.1016/0042-6822(89)90603-x. [DOI] [PubMed] [Google Scholar]
  16. Dougherty W. G., Semler B. L. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes. Microbiol Rev. 1993 Dec;57(4):781–822. doi: 10.1128/mr.57.4.781-822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. French R., Ahlquist P. Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J Virol. 1987 May;61(5):1457–1465. doi: 10.1128/jvi.61.5.1457-1465.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodwin J., Chapman K., Swaney S., Parks T. D., Wernsman E. A., Dougherty W. G. Genetic and biochemical dissection of transgenic RNA-mediated virus resistance. Plant Cell. 1996 Jan;8(1):95–105. doi: 10.1105/tpc.8.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jaeger J. A., Turner D. H., Zuker M. Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706–7710. doi: 10.1073/pnas.86.20.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kasschau K. D., Carrington J. C. Requirement for HC-Pro processing during genome amplification of tobacco etch potyvirus. Virology. 1995 May 10;209(1):268–273. doi: 10.1006/viro.1995.1254. [DOI] [PubMed] [Google Scholar]
  21. Kim Y. N., Jeong Y. S., Makino S. Analysis of cis-acting sequences essential for coronavirus defective interfering RNA replication. Virology. 1993 Nov;197(1):53–63. doi: 10.1006/viro.1993.1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klein P. G., Klein R. R., Rodríguez-Cerezo E., Hunt A. G., Shaw J. G. Mutational analysis of the tobacco vein mottling virus genome. Virology. 1994 Nov 1;204(2):759–769. doi: 10.1006/viro.1994.1591. [DOI] [PubMed] [Google Scholar]
  23. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  24. Li X. H., Carrington J. C. Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):457–461. doi: 10.1073/pnas.92.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lindbo J. A., Dougherty W. G. Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol Plant Microbe Interact. 1992 Mar-Apr;5(2):144–153. doi: 10.1094/mpmi-5-144. [DOI] [PubMed] [Google Scholar]
  26. Lindbo J. A., Dougherty W. G. Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology. 1992 Aug;189(2):725–733. doi: 10.1016/0042-6822(92)90595-g. [DOI] [PubMed] [Google Scholar]
  27. Lindbo J. A., Silva-Rosales L., Proebsting W. M., Dougherty W. G. Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance. Plant Cell. 1993 Dec;5(12):1749–1759. doi: 10.1105/tpc.5.12.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Novak J. E., Kirkegaard K. Coupling between genome translation and replication in an RNA virus. Genes Dev. 1994 Jul 15;8(14):1726–1737. doi: 10.1101/gad.8.14.1726. [DOI] [PubMed] [Google Scholar]
  29. Oh C. S., Carrington J. C. Identification of essential residues in potyvirus proteinase HC-Pro by site-directed mutagenesis. Virology. 1989 Dec;173(2):692–699. doi: 10.1016/0042-6822(89)90582-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parks T. D., Leuther K. K., Howard E. D., Johnston S. A., Dougherty W. G. Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Anal Biochem. 1994 Feb 1;216(2):413–417. doi: 10.1006/abio.1994.1060. [DOI] [PubMed] [Google Scholar]
  31. Restrepo-Hartwig M. A., Carrington J. C. The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication. J Virol. 1994 Apr;68(4):2388–2397. doi: 10.1128/jvi.68.4.2388-2397.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Riechmann J. L., Laín S., García J. A. Highlights and prospects of potyvirus molecular biology. J Gen Virol. 1992 Jan;73(Pt 1):1–16. doi: 10.1099/0022-1317-73-1-1. [DOI] [PubMed] [Google Scholar]
  33. Shukla D. D., Ward C. W. Structure of potyvirus coat proteins and its application in the taxonomy of the potyvirus group. Adv Virus Res. 1989;36:273–314. doi: 10.1016/s0065-3527(08)60588-6. [DOI] [PubMed] [Google Scholar]
  34. Slade D. E., Johnston R. E., Dougherty W. G. Generation and characterization of monoclonal antibodies reactive with the 49-kDa proteinase of tobacco etch virus. Virology. 1989 Dec;173(2):499–508. doi: 10.1016/0042-6822(89)90562-x. [DOI] [PubMed] [Google Scholar]
  35. Smith H. A., Swaney S. L., Parks T. D., Wernsman E. A., Dougherty W. G. Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs. Plant Cell. 1994 Oct;6(10):1441–1453. doi: 10.1105/tpc.6.10.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Van Bokhoven H., Le Gall O., Kasteel D., Verver J., Wellink J., Van Kammen A. B. Cis- and trans-acting elements in cowpea mosaic virus RNA replication. Virology. 1993 Aug;195(2):377–386. doi: 10.1006/viro.1993.1387. [DOI] [PubMed] [Google Scholar]
  37. Verchot J., Carrington J. C. Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications. J Virol. 1995 Mar;69(3):1582–1590. doi: 10.1128/jvi.69.3.1582-1590.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verchot J., Carrington J. C. Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification. J Virol. 1995 Jun;69(6):3668–3674. doi: 10.1128/jvi.69.6.3668-3674.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weiland J. J., Dreher T. W. Cis-preferential replication of the turnip yellow mosaic virus RNA genome. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6095–6099. doi: 10.1073/pnas.90.13.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]

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

RESOURCES