1887

Journal of General Virology header logo

Volume 85, Issue 6

Characterization of regulatory elements within the coat protein (CP) coding region of affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter Free

Abstract

A replicon based on that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3′-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3′ terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem–loop structures, which are followed by an enhancer region.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.79838-0
2004-06-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/jgv/85/6/vir851727.html?itemId=/content/journal/jgv/10.1099/vir.0.79838-0&mimeType=html&fmt=ahah

References

  1. Balmori E., Gilmer D., Richards K., Guilley H., Jonard G. 1993; Mapping the promoter for subgenomic RNA synthesis on beet necrotic yellow vein virus RNA 3. Biochimie 75:517–521 [CrossRef]
     [Google Scholar]
  2. Blackwood E. M., Kadonaga J. T. 1998; Going the distance: a current view of enhancer action. Science 281:61–63
     [Google Scholar]
  3. Boccard F., Baucombe D. 1993; Mutational analysis of cis -acting sequences and gene function in RNA3 of cucumber mosaic virus. Virology 193:563–578 [CrossRef]
     [Google Scholar]
  4. Boyer J. C., Haenni A. L. 1994; Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415–426 [CrossRef]
     [Google Scholar]
  5. Buck K. W. 1996; Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47:159–251
     [Google Scholar]
  6. Buck K. W. 1999; Replication of tobacco mosaic virus RNA. Philos Trans R Soc Lond Ser B Biol Sci 354:613–627 [CrossRef]
     [Google Scholar]
  7. Carpenter C. D., Simon A. E. 1998; Analysis of sequences and predicted structures required for viral satellite RNA accumulation by in vivo genetic selection. Nucleic Acids Res 26:2426–2432 [CrossRef]
     [Google Scholar]
  8. Chiu W., Niwa Y., Zeng W., Hirano T., Kobayashi H., Sheen J. 1995; Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330
     [Google Scholar]
  9. Culver J. N., Lehto K., Close S. M., Hilf M. E., Dawson W. O. 1993; Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes. Proc Natl Acad Sci U S A 90:2055–2059 [CrossRef]
     [Google Scholar]
  10. Dawson W. O. 1992; Tobamovirus–plant interactions. Virology 186:359–367 [CrossRef]
     [Google Scholar]
  11. Dawson W. O., Lehto K. M. 1990; Regulation of tobamovirus gene expression. Adv Virus Res 38:307–342
     [Google Scholar]
  12. Dawson W. O., Lewandowski D. J., Hilf M. E., Bubrick P., Raffo A. J., Shaw J. J., Grantham G. L., Desjardins P. R. 1989; A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology 172:285–292 [CrossRef]
     [Google Scholar]
  13. De Graff M., Jaspars E. M. J. 1994; Plant viral RNA synthesis in cell-free systems. Annu Rev Phytopathol 32:311–335 [CrossRef]
     [Google Scholar]
  14. Deom C. M., Oliver M. J., Beachy R. N. 1987; The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science 237:384–389 [CrossRef]
     [Google Scholar]
  15. Donson J., Kearney C. M., Hilf M. E., Dawson W. O. 1991; Systemic expression of a bacterial gene by tobacco mosaic virus-based vector. Proc Natl Acad Sci U S A 88:7204–7208 [CrossRef]
     [Google Scholar]
  16. Epel B. L., Padgett H. S., Heinlein M., Beachy R. N. 1996; Plant virus movement protein dynamics probed with a GFP-protein fusion. Gene 173:75–79 [CrossRef]
     [Google Scholar]
  17. French R., Ahlquist P. 1998; Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA. J Virol 62:2411–2420
     [Google Scholar]
  18. Gafny R., Lapidot M., Berna A., Holt C. A., Deom C. M., Beachy R. N. 1992; Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus. Virology 187:499–507 [CrossRef]
     [Google Scholar]
  19. Gallie D. R., Feder J. N., Schimke R. T., Walbot V. 1991; Post-transcriptional regulation in higher eukaryotes: the role of reporter gene in controlling expression. Mol Gen Genet 228:258–264
     [Google Scholar]
  20. Goelet P., Lomonossoff G. P., Dutler P. J., Akam M. E., Gait M. J., Karn J. 1982; Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A 79:5818–5822 [CrossRef]
     [Google Scholar]
  21. Grdzelishvili V. Z., Chapman S. N., Dawson W. O., Lewandowski D. J. 2000; Mapping of the tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo . Virology 275:177–192 [CrossRef]
     [Google Scholar]
  22. Guilley H., Jonard G., Kukla B., Richards K. E. 1979; Sequence of 1000 nucleotides at the 3′ end of tobacco mosaic virus RNA. Nucleic Acids Res 6:1287–1308 [CrossRef]
     [Google Scholar]
  23. Haasnoot P. C., Brederode F. T., Olsthoorn R. C., Bol J. F. 2000; A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro . RNA 6:708–716 [CrossRef]
     [Google Scholar]
  24. Johnston J. C., Rochon D. M. 1995; Deletion analysis of the promoter for the cucumber necrosis virus 0·9-kb subgenomic RNA. Virology 214:100–109 [CrossRef]
     [Google Scholar]
  25. Koev G., Miller W. A. 2000; A positive-strand RNA virus with three very different subgenomic RNA promoters. J Virol 74:5988–5996 [CrossRef]
     [Google Scholar]
  26. Koev G., Mohan B. R., Miller W. A. 1999; Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1. J Virol 73:2876–2885
     [Google Scholar]
  27. Kumagai M. H., Turpen T. H., Weinzettl G. N. C. 9 other authors 1993; Rapid high-level expression of biologically active α -trichosanthin in transfected plants by an RNA viral vector. Proc Natl Acad Sci U S A 90:427–430 [CrossRef]
     [Google Scholar]
  28. Kumagai M. H., Donson J., Della-Cioppa G., Harvey D., Hanley K., Grill L. K. 1995; Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci U S A 92:1679–1683 [CrossRef]
     [Google Scholar]
  29. Maia I. G., Seron K., Haenni A. L., Bernardi F. 1996; Gene expression from viral RNA genomes. Plant Mol Biol 32:367–391 [CrossRef]
     [Google Scholar]
  30. Marsh L. E., Dreher T. W., Hall T. C. 1988; Mutational analysis of brome mosaic virus subgenomic promoter. Nucleic Acids Res 16:981–995 [CrossRef]
     [Google Scholar]
  31. Mushegian A. R., Shepherd R. J. 1995; Genetic elements of plant viruses as tools for genetic engineering. Microbiol Rev 59:548–578
     [Google Scholar]
  32. Nagy P. D., Pogany J., Simon A. E. 2001; In vivo and in vitro characterization of an RNA replication enhancer in satellite RNA associated with turnip crinkle virus. Virology 288:315–324 [CrossRef]
     [Google Scholar]
  33. Okada Y. 1999; Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation. Philos Trans R Soc Lond Ser B Biol Sci 354:569–582 [CrossRef]
     [Google Scholar]
  34. Pogue G. P., Lindbo J. A., Garger S. J., Fitzmaurice W. P. 2002; Making an ally from an enemy: plant virology and the new agriculture. Annu Rev Phytopathol 40:45–74 [CrossRef]
     [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  36. Shivprasad S., Pogue G. P., Lewandowski D. J., Hidalgo J., Donson J., Grill L. K., Dawson W. O. 1999; Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255:312–323 [CrossRef]
     [Google Scholar]
  37. Song C., Simon A. E. 1995; Requirement of a 3′-terminal stem–loop in in vitro transcription by an RNA-dependent RNA polymerase. J Mol Biol 254:6–14 [CrossRef]
     [Google Scholar]
  38. Stupina V., Simon A. E. 1997; Analysis in vivo of turnip crinkle virus satellite RNA C variants with mutations in the 3′-terminal minus strand promoter. Virology 238:470–477 [CrossRef]
     [Google Scholar]
  39. Szecsi J., Ding S. X., Lim C. O., Bendahmane M., Cho M. J., Nelson R. S., Beachy R. N. 1999; Development of tobacco mosaic virus infection sites in Nicotiana benthamiana . Mol Plant Microbe Interact 12:143–152 [CrossRef]
     [Google Scholar]
  40. Takamatsu N., Ishikawa M., Meshi T., Okada Y. 1987; Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO J 6:307–311
     [Google Scholar]
  41. Turpen T. H. 1999; Tobacco mosaic virus and virescence of biotechnology. Philos Trans R Soc Lond Ser B Biol Sci 354:665–673 [CrossRef]
     [Google Scholar]
  42. van der Kuyl A. C., Neelmeman L., Bol J. F. 1991; Deletion analysis of cis - and trans -acting elements involved in replication of alfalfa mosaic virus RNA3 in vivo . Virology 183:687–694 [CrossRef]
     [Google Scholar]
  43. van der Vossen E. A., Notenboom T., Bol J. F. 1995; Characterization of sequences controlling the synthesis of alfalfa mosaic virus subgenomic RNA in vivo . Virology 212:663–672 [CrossRef]
     [Google Scholar]
  44. Wang J., Simon A. E. 1997; Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro . Virology 232:174–186 [CrossRef]
     [Google Scholar]
  45. Wang J., Carpenter C. D., Simon A. E. 1999; Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus. Virology 253:327–336 [CrossRef]
     [Google Scholar]
  46. Yusibov V., Shivprasad S., Turpen T. H., Dawson W., Koprowski H. 1999; Plant viral vectors based on tobamoviruses. Curr Top Microbiol Immunol 240:81–94
     [Google Scholar]
  47. Zavriev S. K., Hickey C. M., Lommel S. A. 1996; Mapping of the red clover necrotic mosaic virus subgenomic RNA. Virology 216:407–410 [CrossRef]
     [Google Scholar]
  48. Zuker M. 1989; On finding all suboptimal foldings of an RNA molecule. Science 244:48–52 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.79838-0
Loading
Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter
J. Gen. Virol. 85, 1727 (2004); https://doi.org/10.1099/vir.0.79838-0
/content/journal/jgv/10.1099/vir.0.79838-0
/content/journal/jgv/10.1099/vir.0.79838-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error