Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1992 Aug;66(8):5075–5086. doi: 10.1128/jvi.66.8.5075-5086.1992

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

A A Haller 1, B L Semler 1
PMCID: PMC241370  PMID: 1321289

Abstract

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.

Full text

PDF
5075

Images in this article

5081Image
on p.5081
5082Image
on p.5082
5083Image
on p.5083

Selected References

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

  1. Belsham G. J., Brangwyn J. K. A region of the 5' noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control. J Virol. 1990 Nov;64(11):5389–5395. doi: 10.1128/jvi.64.11.5389-5395.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bienkowska-Szewczyk K., Ehrenfeld E. An internal 5'-noncoding region required for translation of poliovirus RNA in vitro. J Virol. 1988 Aug;62(8):3068–3072. doi: 10.1128/jvi.62.8.3068-3072.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown B. A., Ehrenfeld E. Translation of poliovirus RNA in vitro: changes in cleavage pattern and initiation sites by ribosomal salt wash. Virology. 1979 Sep;97(2):396–405. doi: 10.1016/0042-6822(79)90350-7. [DOI] [PubMed] [Google Scholar]
  4. Campos R., Villarreal L. P. An SV40 deletion mutant accumulates late transcripts in a paranuclear extract. Virology. 1982 May;119(1):1–11. doi: 10.1016/0042-6822(82)90059-9. [DOI] [PubMed] [Google Scholar]
  5. Dildine S. L., Semler B. L. Conservation of RNA-protein interactions among picornaviruses. J Virol. 1992 Jul;66(7):4364–4376. doi: 10.1128/jvi.66.7.4364-4376.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dildine S. L., Stark K. R., Haller A. A., Semler B. L. Poliovirus translation initiation: differential effects of directed and selected mutations in the 5' noncoding region of viral RNAs. Virology. 1991 Jun;182(2):742–752. doi: 10.1016/0042-6822(91)90615-i. [DOI] [PubMed] [Google Scholar]
  7. Evans D. M., Dunn G., Minor P. D., Schild G. C., Cann A. J., Stanway G., Almond J. W., Currey K., Maizel J. V., Jr Increased neurovirulence associated with a single nucleotide change in a noncoding region of the Sabin type 3 poliovaccine genome. Nature. 1985 Apr 11;314(6011):548–550. doi: 10.1038/314548a0. [DOI] [PubMed] [Google Scholar]
  8. Garcia A. D., O'Connell A. M., Sharp S. J. Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region. Mol Cell Biol. 1987 Jun;7(6):2046–2051. doi: 10.1128/mcb.7.6.2046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hamlyn P. H., Browniee G. G., Cheng C. C., Gait M. J., Milstein C. Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing. Cell. 1978 Nov;15(3):1067–1075. doi: 10.1016/0092-8674(78)90290-8. [DOI] [PubMed] [Google Scholar]
  10. Helentjaris T., Ehrenfeld E., Brown-Luedi M. L., Hershey J. W. Alterations in initiation factor activity from poliovirus-infected HeLa cells. J Biol Chem. 1979 Nov 10;254(21):10973–10978. [PubMed] [Google Scholar]
  11. Iizuka N., Kohara M., Hagino-Yamagishi K., Abe S., Komatsu T., Tago K., Arita M., Nomoto A. Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome. J Virol. 1989 Dec;63(12):5354–5363. doi: 10.1128/jvi.63.12.5354-5363.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson R. J., Howell M. T., Kaminski A. The novel mechanism of initiation of picornavirus RNA translation. Trends Biochem Sci. 1990 Dec;15(12):477–483. doi: 10.1016/0968-0004(90)90302-r. [DOI] [PubMed] [Google Scholar]
  13. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jang S. K., Pestova T. V., Hellen C. U., Witherell G. W., Wimmer E. Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site. Enzyme. 1990;44(1-4):292–309. doi: 10.1159/000468766. [DOI] [PubMed] [Google Scholar]
  16. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [DOI] [PubMed] [Google Scholar]
  17. Kaminski A., Howell M. T., Jackson R. J. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO J. 1990 Nov;9(11):3753–3759. doi: 10.1002/j.1460-2075.1990.tb07588.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Konarska M. M., Sharp P. A. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. doi: 10.1016/0092-8674(86)90066-8. [DOI] [PubMed] [Google Scholar]
  19. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuge S., Kawamura N., Nomoto A. Genetic variation occurring on the genome of an in vitro insertion mutant of poliovirus type 1. J Virol. 1989 Mar;63(3):1069–1075. doi: 10.1128/jvi.63.3.1069-1075.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuhn R. J., Wimmer E., Semler B. L. Expression of the poliovirus genome from infectious cDNA is dependent upon arrangements of eukaryotic and prokaryotic sequences in recombinant plasmids. Virology. 1987 Apr;157(2):560–564. doi: 10.1016/0042-6822(87)90302-3. [DOI] [PubMed] [Google Scholar]
  22. Kühn R., Luz N., Beck E. Functional analysis of the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1990 Oct;64(10):4625–4631. doi: 10.1128/jvi.64.10.4625-4631.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. La Monica N., Meriam C., Racaniello V. R. Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. J Virol. 1986 Feb;57(2):515–525. doi: 10.1128/jvi.57.2.515-525.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lawson M. A., Dasmahapatra B., Semler B. L. Species-specific substrate interaction of picornavirus 3C proteinase suballelic exchange mutants. J Biol Chem. 1990 Sep 15;265(26):15920–15931. [PubMed] [Google Scholar]
  26. Macejak D. G., Sarnow P. Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature. 1991 Sep 5;353(6339):90–94. doi: 10.1038/353090a0. [DOI] [PubMed] [Google Scholar]
  27. Manzella J. M., Blackshear P. J. Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region. J Biol Chem. 1990 Jul 15;265(20):11817–11822. [PubMed] [Google Scholar]
  28. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  29. Meerovitch K., Nicholson R., Sonenberg N. In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5' untranslated region. J Virol. 1991 Nov;65(11):5895–5901. doi: 10.1128/jvi.65.11.5895-5901.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meerovitch K., Pelletier J., Sonenberg N. A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation. Genes Dev. 1989 Jul;3(7):1026–1034. doi: 10.1101/gad.3.7.1026. [DOI] [PubMed] [Google Scholar]
  31. Minor P. D., Dunn G. The effect of sequences in the 5' non-coding region on the replication of polioviruses in the human gut. J Gen Virol. 1988 May;69(Pt 5):1091–1096. doi: 10.1099/0022-1317-69-5-1091. [DOI] [PubMed] [Google Scholar]
  32. Muller A. J., Witte O. N. The 5' noncoding region of the human leukemia-associated oncogene BCR/ABL is a potent inhibitor of in vitro translation. Mol Cell Biol. 1989 Nov;9(11):5234–5238. doi: 10.1128/mcb.9.11.5234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Najita L., Sarnow P. Oxidation-reduction sensitive interaction of a cellular 50-kDa protein with an RNA hairpin in the 5' noncoding region of the poliovirus genome. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5846–5850. doi: 10.1073/pnas.87.15.5846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Nomoto A., Detjen B., Pozzatti R., Wimmer E. The location of the polio genome protein in viral RNAs and its implication for RNA synthesis. Nature. 1977 Jul 21;268(5617):208–213. doi: 10.1038/268208a0. [DOI] [PubMed] [Google Scholar]
  36. Pelletier J., Flynn M. E., Kaplan G., Racaniello V., Sonenberg N. Mutational analysis of upstream AUG codons of poliovirus RNA. J Virol. 1988 Dec;62(12):4486–4492. doi: 10.1128/jvi.62.12.4486-4492.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988 Mar;8(3):1103–1112. doi: 10.1128/mcb.8.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  39. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  40. Pestova T. V., Hellen C. U., Wimmer E. Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein. J Virol. 1991 Nov;65(11):6194–6204. doi: 10.1128/jvi.65.11.6194-6204.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pilipenko E. V., Blinov V. M., Romanova L. I., Sinyakov A. N., Maslova S. V., Agol V. I. Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence. Virology. 1989 Feb;168(2):201–209. doi: 10.1016/0042-6822(89)90259-6. [DOI] [PubMed] [Google Scholar]
  42. Pilipenko E. V., Gmyl A. P., Maslova S. V., Svitkin Y. V., Sinyakov A. N., Agol V. I. Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA. Cell. 1992 Jan 10;68(1):119–131. doi: 10.1016/0092-8674(92)90211-t. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Skinner M. A., Racaniello V. R., Dunn G., Cooper J., Minor P. D., Almond J. W. New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence. J Mol Biol. 1989 May 20;207(2):379–392. doi: 10.1016/0022-2836(89)90261-1. [DOI] [PubMed] [Google Scholar]
  45. Vaheri A., Pagano J. S. Infectious poliovirus RNA: a sensitive method of assay. Virology. 1965 Nov;27(3):434–436. doi: 10.1016/0042-6822(65)90126-1. [DOI] [PubMed] [Google Scholar]
  46. Ypma-Wong M. F., Semler B. L. In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase. Nucleic Acids Res. 1987 Mar 11;15(5):2069–2088. doi: 10.1093/nar/15.5.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES