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. 1996 Aug;70(8):5266–5271. doi: 10.1128/jvi.70.8.5266-5271.1996

An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

V S Sriskanda 1, G Pruss 1, X Ge 1, V B Vance 1
PMCID: PMC190483  PMID: 8764036

Abstract

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.

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Selected References

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  1. An G. High efficiency transformation of cultured tobacco cells. Plant Physiol. 1985 Oct;79(2):568–570. doi: 10.1104/pp.79.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumenthal T., Carmichael G. G. RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem. 1979;48:525–548. doi: 10.1146/annurev.bi.48.070179.002521. [DOI] [PubMed] [Google Scholar]
  4. Bohjanen P. R., Petryniak B., June C. H., Thompson C. B., Lindsten T. An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA. Mol Cell Biol. 1991 Jun;11(6):3288–3295. doi: 10.1128/mcb.11.6.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brewer G. An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol Cell Biol. 1991 May;11(5):2460–2466. doi: 10.1128/mcb.11.5.2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C. Y., You Y., Shyu A. B. Two cellular proteins bind specifically to a purine-rich sequence necessary for the destabilization function of a c-fos protein-coding region determinant of mRNA instability. Mol Cell Biol. 1992 Dec;12(12):5748–5757. doi: 10.1128/mcb.12.12.5748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Czako M., An G. Expression of DNA coding for diphtheria toxin chain a is toxic to plant cells. Plant Physiol. 1991 Mar;95(3):687–692. doi: 10.1104/pp.95.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  10. Frankel A. D., Mattaj I. W., Rio D. C. RNA-protein interactions. Cell. 1991 Dec 20;67(6):1041–1046. doi: 10.1016/0092-8674(91)90282-4. [DOI] [PubMed] [Google Scholar]
  11. Hayes R. J., Buck K. W. Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell. 1990 Oct 19;63(2):363–368. doi: 10.1016/0092-8674(90)90169-f. [DOI] [PubMed] [Google Scholar]
  12. Horton R. M., Cai Z. L., Ho S. N., Pease L. R. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990 May;8(5):528–535. [PubMed] [Google Scholar]
  13. 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]
  14. Hunt A. G., MacDonald M. H. Deletion analysis of the polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase small-subunit gene. Plant Mol Biol. 1989 Aug;13(2):125–138. doi: 10.1007/BF00016132. [DOI] [PubMed] [Google Scholar]
  15. Kavanagh T., Goulden M., Santa Cruz S., Chapman S., Barker I., Baulcombe D. Molecular analysis of a resistance-breaking strain of potato virus X. Virology. 1992 Aug;189(2):609–617. doi: 10.1016/0042-6822(92)90584-c. [DOI] [PubMed] [Google Scholar]
  16. Levine T. D., Gao F., King P. H., Andrews L. G., Keene J. D. Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs. Mol Cell Biol. 1993 Jun;13(6):3494–3504. doi: 10.1128/mcb.13.6.3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  18. Nakhasi H. L., Rouault T. A., Haile D. J., Liu T. Y., Klausner R. D. Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA. New Biol. 1990 Mar;2(3):255–264. [PubMed] [Google Scholar]
  19. Nicholson R., Pelletier J., Le S. Y., Sonenberg N. Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies. J Virol. 1991 Nov;65(11):5886–5894. doi: 10.1128/jvi.65.11.5886-5894.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nüesch J. P., Weitz M., Siegl G. Proteins specifically binding to the 3' untranslated region of hepatitis A virus RNA in persistently infected cells. Arch Virol. 1993;128(1-2):65–79. doi: 10.1007/BF01309789. [DOI] [PubMed] [Google Scholar]
  21. Olsen H. S., Nelbock P., Cochrane A. W., Rosen C. A. Secondary structure is the major determinant for interaction of HIV rev protein with RNA. Science. 1990 Feb 16;247(4944):845–848. doi: 10.1126/science.2406903. [DOI] [PubMed] [Google Scholar]
  22. Pardigon N., Strauss J. H. Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA. J Virol. 1992 Feb;66(2):1007–1015. doi: 10.1128/jvi.66.2.1007-1015.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Quadt R., Kao C. C., Browning K. S., Hershberger R. P., Ahlquist P. Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1498–1502. doi: 10.1073/pnas.90.4.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roehl H. H., Semler B. L. Poliovirus infection enhances the formation of two ribonucleoprotein complexes at the 3' end of viral negative-strand RNA. J Virol. 1995 May;69(5):2954–2961. doi: 10.1128/jvi.69.5.2954-2961.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanfaçon H., Brodmann P., Hohn T. A dissection of the cauliflower mosaic virus polyadenylation signal. Genes Dev. 1991 Jan;5(1):141–149. doi: 10.1101/gad.5.1.141. [DOI] [PubMed] [Google Scholar]
  26. Sonenberg N., Shatkin A. J., Ricciardi R. P., Rubin M., Goodman R. M. Analysis of terminal structures of RNA from potato virus X. Nucleic Acids Res. 1978 Jul;5(7):2501–2512. doi: 10.1093/nar/5.7.2501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Todd S., Nguyen J. H., Semler B. L. RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA. J Virol. 1995 Jun;69(6):3605–3614. doi: 10.1128/jvi.69.6.3605-3614.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vakalopoulou E., Schaack J., Shenk T. A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. Mol Cell Biol. 1991 Jun;11(6):3355–3364. doi: 10.1128/mcb.11.6.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vance V. B. Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology. 1991 Jun;182(2):486–494. doi: 10.1016/0042-6822(91)90589-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Williams A. S., Marzluff W. F. The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein. Nucleic Acids Res. 1995 Feb 25;23(4):654–662. doi: 10.1093/nar/23.4.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. You Y., Chen C. Y., Shyu A. B. U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation. Mol Cell Biol. 1992 Jul;12(7):2931–2940. doi: 10.1128/mcb.12.7.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yu W., Leibowitz J. L. Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA. J Virol. 1995 Apr;69(4):2016–2023. doi: 10.1128/jvi.69.4.2016-2023.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhang S., Mehdy M. C. Binding of a 50-kD Protein to a U-Rich Sequence in an mRNA Encoding a Proline-Rich Protein That Is Destabilized by Fungal Elicitor. Plant Cell. 1994 Jan;6(1):135–145. doi: 10.1105/tpc.6.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zubiaga A. M., Belasco J. G., Greenberg M. E. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol Cell Biol. 1995 Apr;15(4):2219–2230. doi: 10.1128/mcb.15.4.2219. [DOI] [PMC free article] [PubMed] [Google Scholar]

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