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. 1996 Aug;70(8):5533–5540. doi: 10.1128/jvi.70.8.5533-5540.1996

The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis.

K H Kim 1, C Hemenway 1
PMCID: PMC190512  PMID: 8764066

Abstract

A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs.

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Selected References

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  1. Allison R. F., Janda M., Ahlquist P. Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution. Virology. 1989 Sep;172(1):321–330. doi: 10.1016/0042-6822(89)90134-7. [DOI] [PubMed] [Google Scholar]
  2. Baulcombe D. C., Chapman S., Santa Cruz S. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 1995 Jun;7(6):1045–1053. doi: 10.1046/j.1365-313x.1995.07061045.x. [DOI] [PubMed] [Google Scholar]
  3. Beck D. L., Guilford P. J., Voot D. M., Andersen M. T., Forster R. L. Triple gene block proteins of white clover mosaic potexvirus are required for transport. Virology. 1991 Aug;183(2):695–702. doi: 10.1016/0042-6822(91)90998-q. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Boccard F., Baulcombe D. Mutational analysis of cis-acting sequences and gene function in RNA3 of cucumber mosaic virus. Virology. 1993 Apr;193(2):563–578. doi: 10.1006/viro.1993.1165. [DOI] [PubMed] [Google Scholar]
  6. Braun C. J., Hemenway C. L. Expression of Amino-Terminal Portions or Full-Length Viral Replicase Genes in Transgenic Plants Confers Resistance to Potato Virus X Infection. Plant Cell. 1992 Jun;4(6):735–744. doi: 10.1105/tpc.4.6.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bujarski J. J., Ahlquist P., Hall T. C., Dreher T. W., Kaesberg P. Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end. EMBO J. 1986 Aug;5(8):1769–1774. doi: 10.1002/j.1460-2075.1986.tb04425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chapman S., Hills G., Watts J., Baulcombe D. Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity. Virology. 1992 Nov;191(1):223–230. doi: 10.1016/0042-6822(92)90183-p. [DOI] [PubMed] [Google Scholar]
  9. De Graaff M., Man in't Veld M. R., Jaspars E. M. In vitro evidence that the coat protein of alfalfa mosaic virus plays a direct role in the regulation of plus and minus RNA synthesis: implications for the life cycle of alfalfa mosaic virus. Virology. 1995 Apr 20;208(2):583–589. doi: 10.1006/viro.1995.1189. [DOI] [PubMed] [Google Scholar]
  10. Doronin S. V., Hemenway C. Synthesis of potato virus X RNAs by membrane-containing extracts. J Virol. 1996 Jul;70(7):4795–4799. doi: 10.1128/jvi.70.7.4795-4799.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dreher T. W., Hall T. C. Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity. J Mol Biol. 1988 May 5;201(1):31–40. doi: 10.1016/0022-2836(88)90436-6. [DOI] [PubMed] [Google Scholar]
  12. Dreher T. W., Rao A. L., Hall T. C. Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation. J Mol Biol. 1989 Apr 5;206(3):425–438. doi: 10.1016/0022-2836(89)90491-9. [DOI] [PubMed] [Google Scholar]
  13. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  14. Eggen R., Verver J., Wellink J., Pleij K., van Kammen A., Goldbach R. Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants. Virology. 1989 Dec;173(2):456–464. doi: 10.1016/0042-6822(89)90558-8. [DOI] [PubMed] [Google Scholar]
  15. Fichot O., Girard M. An improved method for sequencing of RNA templates. Nucleic Acids Res. 1990 Oct 25;18(20):6162–6162. doi: 10.1093/nar/18.20.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Gargouri-Bouzid R., David C., Haenni A. L. The 3' promoter region involved in RNA synthesis directed by the turnip yellow mosaic virus genome in vitro. FEBS Lett. 1991 Dec 2;294(1-2):56–58. doi: 10.1016/0014-5793(91)81342-6. [DOI] [PubMed] [Google Scholar]
  18. Geliebter J., Zeff R. A., Melvold R. W., Nathenson S. G. Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: Kbm9 and Kbm6. Proc Natl Acad Sci U S A. 1986 May;83(10):3371–3375. doi: 10.1073/pnas.83.10.3371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gilmer D., Allmang C., Ehresmann C., Guilley H., Richards K., Jonard G., Ehresmann B. The secondary structure of the 5'-noncoding region of beet necrotic yellow vein virus RNA 3: evidence for a role in viral RNA replication. Nucleic Acids Res. 1993 Mar 25;21(6):1389–1395. doi: 10.1093/nar/21.6.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gilmer D., Richards K., Jonard G., Guilley H. cis-active sequences near the 5'-termini of beet necrotic yellow vein virus RNAs 3 and 4. Virology. 1992 Sep;190(1):55–67. doi: 10.1016/0042-6822(92)91192-w. [DOI] [PubMed] [Google Scholar]
  21. Hemenway C., Weiss J., O'Connell K., Tumer N. E. Characterization of infectious transcripts from a potato virus X cDNA clone. Virology. 1990 Apr;175(2):365–371. doi: 10.1016/0042-6822(90)90421-m. [DOI] [PubMed] [Google Scholar]
  22. Huisman M. J., Linthorst H. J., Bol J. F., Cornelissen J. C. The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses. J Gen Virol. 1988 Aug;69(Pt 8):1789–1798. doi: 10.1099/0022-1317-69-8-1789. [DOI] [PubMed] [Google Scholar]
  23. Huntley C. C., Hall T. C. Minus sense transcripts of brome mosaic virus RNA-3 intercistronic region interfere with viral replication. Virology. 1993 Jan;192(1):290–297. doi: 10.1006/viro.1993.1032. [DOI] [PubMed] [Google Scholar]
  24. Ishikawa M., Meshi T., Ohno T., Okada Y. Specific cessation of minus-strand RNA accumulation at an early stage of tobacco mosaic virus infection. J Virol. 1991 Feb;65(2):861–868. doi: 10.1128/jvi.65.2.861-868.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. King P., Goodbourn S. A method for sequence-specific deletion mutagenesis. Nucleic Acids Res. 1992 Mar 11;20(5):1039–1044. doi: 10.1093/nar/20.5.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lahser F. C., Marsh L. E., Hall T. C. Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation. J Virol. 1993 Jun;67(6):3295–3303. doi: 10.1128/jvi.67.6.3295-3303.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lai M. M. Coronavirus: organization, replication and expression of genome. Annu Rev Microbiol. 1990;44:303–333. doi: 10.1146/annurev.mi.44.100190.001511. [DOI] [PubMed] [Google Scholar]
  29. Lai M. M., Patton C. D., Baric R. S., Stohlman S. A. Presence of leader sequences in the mRNA of mouse hepatitis virus. J Virol. 1983 Jun;46(3):1027–1033. doi: 10.1128/jvi.46.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liao C. L., Lai M. M. Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription. J Virol. 1994 Aug;68(8):4727–4737. doi: 10.1128/jvi.68.8.4727-4737.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marsh L. E., Hall T. C. Evidence implicating a tRNA heritage for the promoters of positive-strand RNA synthesis in brome mosaic and related viruses. Cold Spring Harb Symp Quant Biol. 1987;52:331–341. doi: 10.1101/sqb.1987.052.01.038. [DOI] [PubMed] [Google Scholar]
  32. Marsh L. E., Huntley C. C., Pogue G. P., Connell J. P., Hall T. C. Regulation of (+):(-)-strand asymmetry in replication of brome mosaic virus RNA. Virology. 1991 May;182(1):76–83. doi: 10.1016/0042-6822(91)90650-z. [DOI] [PubMed] [Google Scholar]
  33. Marsh L. E., Pogue G. P., Hall T. C. Similarities among plant virus (+) and (-) RNA termini imply a common ancestry with promoters of eukaryotic tRNAs. Virology. 1989 Oct;172(2):415–427. doi: 10.1016/0042-6822(89)90184-0. [DOI] [PubMed] [Google Scholar]
  34. Meshi T., Ishikawa M., Takamatsu N., Ohno T., Okada Y. The 5'-terminal sequence of TMV RNA. Question on the polymorphism found in vulgare strain. FEBS Lett. 1983 Oct 17;162(2):282–285. doi: 10.1016/0014-5793(83)80772-8. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Niesters H. G., Strauss J. H. Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA. J Virol. 1990 Sep;64(9):4162–4168. doi: 10.1128/jvi.64.9.4162-4168.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pacha R. F., Allison R. F., Ahlquist P. cis-acting sequences required for in vivo amplification of genomic RNA3 are organized differently in related bromoviruses. Virology. 1990 Feb;174(2):436–443. doi: 10.1016/0042-6822(90)90097-b. [DOI] [PubMed] [Google Scholar]
  38. Pogue G. P., Hall T. C. The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation. J Virol. 1992 Feb;66(2):674–684. doi: 10.1128/jvi.66.2.674-684.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pogue G. P., Marsh L. E., Connell J. P., Hall T. C. Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA. Virology. 1992 Jun;188(2):742–753. doi: 10.1016/0042-6822(92)90529-x. [DOI] [PubMed] [Google Scholar]
  40. 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 Sep;178(1):152–160. doi: 10.1016/0042-6822(90)90388-8. [DOI] [PubMed] [Google Scholar]
  41. Pooggin M. M., Skryabin K. G. The 5'-untranslated leader sequence of potato virus X RNA enhances the expression of a heterologous gene in vivo. Mol Gen Genet. 1992 Aug;234(2):329–331. doi: 10.1007/BF00283854. [DOI] [PubMed] [Google Scholar]
  42. Price M. Examination of potato virus X proteins synthesized in infected tobacco plants. J Virol. 1992 Sep;66(9):5658–5661. doi: 10.1128/jvi.66.9.5658-5661.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rohll J. B., Holness C. L., Lomonossoff G. P., Maule A. J. 3'-terminal nucleotide sequences important for the accumulation of cowpea mosaic virus M-RNA. Virology. 1993 Apr;193(2):672–679. doi: 10.1006/viro.1993.1175. [DOI] [PubMed] [Google Scholar]
  44. Sit T. L., Leclerc D., AbouHaidar M. G. The minimal 5' sequence for in vitro initiation of papaya mosaic potexvirus assembly. Virology. 1994 Feb 15;199(1):238–242. doi: 10.1006/viro.1994.1118. [DOI] [PubMed] [Google Scholar]
  45. Skryabin K. G., Morozov SYu, Kraev A. S., Rozanov M. N., Chernov B. K., Lukasheva L. I., Atabekov J. G. Conserved and variable elements in RNA genomes of potexviruses. FEBS Lett. 1988 Nov 21;240(1-2):33–40. doi: 10.1016/0014-5793(88)80335-1. [DOI] [PubMed] [Google Scholar]
  46. Smirnyagina E. V., Morozov S. Y., Rodionova N. P., Miroshnichenko N. A., Solovev A. G., Fedorkin O. N., Atabekov J. G. Translational efficiency and competitive ability of mRNAs with 5'-untranslated alpha beta-leader of potato virus X RNA. Biochimie. 1991 May;73(5):587–598. doi: 10.1016/0300-9084(91)90027-x. [DOI] [PubMed] [Google Scholar]
  47. Spaan W., Delius H., Skinner M., Armstrong J., Rottier P., Smeekens S., van der Zeijst B. A., Siddell S. G. Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. EMBO J. 1983;2(10):1839–1844. doi: 10.1002/j.1460-2075.1983.tb01667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Takamatsu N., Watanabe Y., Iwasaki T., Shiba T., Meshi T., Okada Y. Deletion analysis of the 5' untranslated leader sequence of tobacco mosaic virus RNA. J Virol. 1991 Mar;65(3):1619–1622. doi: 10.1128/jvi.65.3.1619-1622.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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 Aug;64(8):3686–3693. doi: 10.1128/jvi.64.8.3686-3693.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tomashevskaya O. L., Solovyev A. G., Karpova O. V., Fedorkin O. N., Rodionova N. P., Morozov SYu, Atabekov J. G. Effects of sequence elements in the potato virus X RNA 5' non-translated alpha beta-leader on its translation enhancing activity. J Gen Virol. 1993 Dec;74(Pt 12):2717–2724. doi: 10.1099/0022-1317-74-12-2717. [DOI] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Traynor P., Young B. M., Ahlquist P. Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread. J Virol. 1991 Jun;65(6):2807–2815. doi: 10.1128/jvi.65.6.2807-2815.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tsai C. H., Dreher T. W. Second-site suppressor mutations assist in studying the function of the 3' noncoding region of turnip yellow mosaic virus RNA. J Virol. 1992 Sep;66(9):5190–5199. doi: 10.1128/jvi.66.9.5190-5199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsai C. H., Dreher T. W. Turnip yellow mosaic virus RNAs with anticodon loop substitutions that result in decreased valylation fail to replicate efficiently. J Virol. 1991 Jun;65(6):3060–3067. doi: 10.1128/jvi.65.6.3060-3067.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Van der Kuyl A. C., Neeleman L., Bol J. F. Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis. Virology. 1991 Nov;185(1):496–499. doi: 10.1016/0042-6822(91)90807-n. [DOI] [PubMed] [Google Scholar]
  57. White K. A., Bancroft J. B., Mackie G. A. Mutagenesis of a hexanucleotide sequence conserved in potexvirus RNAs. Virology. 1992 Aug;189(2):817–820. doi: 10.1016/0042-6822(92)90614-u. [DOI] [PubMed] [Google Scholar]
  58. White K. A., Mackie G. A. Control and expression of 3' open reading frames in clover yellow mosaic virus. Virology. 1990 Dec;179(2):576–584. doi: 10.1016/0042-6822(90)90124-a. [DOI] [PubMed] [Google Scholar]
  59. Zelenina D. A., Kulaeva O. I., Smirnyagina E. V., Solovyev A. G., Miroshnichenko N. A., Fedorkin O. N., Rodionova N. P., Morozov SYu, Atabekov J. G. Translation enhancing properties of the 5'-leader of potato virus X genomic RNA. FEBS Lett. 1992 Jan 27;296(3):267–270. doi: 10.1016/0014-5793(92)80301-v. [DOI] [PubMed] [Google Scholar]
  60. van der Kuyl A. C., Langereis K., Houwing C. J., Jaspars E. M., Bol J. F. cis-acting elements involved in replication of alfalfa mosaic virus RNAs in vitro. Virology. 1990 Jun;176(2):346–354. doi: 10.1016/0042-6822(90)90004-b. [DOI] [PubMed] [Google Scholar]
  61. van der Kuyl A. C., Neeleman L., Bol J. F. Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo. Virology. 1991 Aug;183(2):687–694. doi: 10.1016/0042-6822(91)90997-p. [DOI] [PubMed] [Google Scholar]
  62. van der Vossen E. A., Neeleman L., Bol J. F. Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA. Nucleic Acids Res. 1993 Mar 25;21(6):1361–1367. doi: 10.1093/nar/21.6.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]

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