Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Jul 1;13(13):3149–3157. doi: 10.1002/j.1460-2075.1994.tb06613.x

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

A M Borman 1, F G Deliat 1, K M Kean 1
PMCID: PMC395206  PMID: 8039507

Abstract

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.

Full text

PDF
3149

Images in this article

3150Image
on p.3150
3151Image
on p.3151
3151Image
on p.3151
3152Image
on p.3152
3153Image
on p.3153
3154Image
on p.3154
3155Image
on p.3155

Selected References

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

  1. 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]
  2. Andino R., Rieckhof G. E., Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. doi: 10.1016/0092-8674(90)90170-j. [DOI] [PubMed] [Google Scholar]
  3. Bernstein H. D., Sarnow P., Baltimore D. Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. J Virol. 1986 Dec;60(3):1040–1049. doi: 10.1128/jvi.60.3.1040-1049.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borman A., Howell M. T., Patton J. G., Jackson R. J. The involvement of a spliceosome component in internal initiation of human rhinovirus RNA translation. J Gen Virol. 1993 Sep;74(Pt 9):1775–1788. doi: 10.1099/0022-1317-74-9-1775. [DOI] [PubMed] [Google Scholar]
  5. Borman A., Jackson R. J. Initiation of translation of human rhinovirus RNA: mapping the internal ribosome entry site. Virology. 1992 Jun;188(2):685–696. doi: 10.1016/0042-6822(92)90523-r. [DOI] [PubMed] [Google Scholar]
  6. Collis P. S., O'Donnell B. J., Barton D. J., Rogers J. A., Flanegan J. B. Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells. J Virol. 1992 Nov;66(11):6480–6488. doi: 10.1128/jvi.66.11.6480-6488.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dasso M. C., Jackson R. J. On the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system. Nucleic Acids Res. 1989 Apr 25;17(8):3129–3144. doi: 10.1093/nar/17.8.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Dorner A. J., Semler B. L., Jackson R. J., Hanecak R., Duprey E., Wimmer E. In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate. J Virol. 1984 May;50(2):507–514. doi: 10.1128/jvi.50.2.507-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hagino-Yamagishi K., Nomoto A. In vitro construction of poliovirus defective interfering particles. J Virol. 1989 Dec;63(12):5386–5392. doi: 10.1128/jvi.63.12.5386-5392.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haller A. A., Nguyen J. H., Semler B. L. Minimum internal ribosome entry site required for poliovirus infectivity. J Virol. 1993 Dec;67(12):7461–7471. doi: 10.1128/jvi.67.12.7461-7471.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hambidge S. J., Sarnow P. Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10272–10276. doi: 10.1073/pnas.89.21.10272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  16. Kaplan G., Racaniello V. R. Construction and characterization of poliovirus subgenomic replicons. J Virol. 1988 May;62(5):1687–1696. doi: 10.1128/jvi.62.5.1687-1696.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kean K. M., Teterina N. L., Marc D., Girard M. Analysis of putative active site residues of the poliovirus 3C protease. Virology. 1991 Apr;181(2):609–619. doi: 10.1016/0042-6822(91)90894-h. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kräusslich H. G., Hölscher C., Reuer Q., Harber J., Wimmer E. Myristoylation of the poliovirus polyprotein is required for proteolytic processing of the capsid and for viral infectivity. J Virol. 1990 May;64(5):2433–2436. doi: 10.1128/jvi.64.5.2433-2436.1990. [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. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leppert M., Kort L., Kolakofsky D. Further characterization of Sendai virus DI-RNAs: a model for their generation. Cell. 1977 Oct;12(2):539–552. doi: 10.1016/0092-8674(77)90130-1. [DOI] [PubMed] [Google Scholar]
  23. Liebig H. D., Ziegler E., Yan R., Hartmuth K., Klump H., Kowalski H., Blaas D., Sommergruber W., Frasel L., Lamphear B. Purification of two picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on in vitro translation. Biochemistry. 1993 Jul 27;32(29):7581–7588. doi: 10.1021/bi00080a033. [DOI] [PubMed] [Google Scholar]
  24. Marc D., Drugeon G., Haenni A. L., Girard M., van der Werf S. Role of myristoylation of poliovirus capsid protein VP4 as determined by site-directed mutagenesis of its N-terminal sequence. EMBO J. 1989 Sep;8(9):2661–2668. doi: 10.1002/j.1460-2075.1989.tb08406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marc D., Girard M., van der Werf S. A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly. J Gen Virol. 1991 May;72(Pt 5):1151–1157. doi: 10.1099/0022-1317-72-5-1151. [DOI] [PubMed] [Google Scholar]
  26. Meerovitch K., Svitkin Y. V., Lee H. S., Lejbkowicz F., Kenan D. J., Chan E. K., Agol V. I., Keene J. D., Sonenberg N. La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol. 1993 Jul;67(7):3798–3807. doi: 10.1128/jvi.67.7.3798-3807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Molla A., Jang S. K., Paul A. V., Reuer Q., Wimmer E. Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus. Nature. 1992 Mar 19;356(6366):255–257. doi: 10.1038/356255a0. [DOI] [PubMed] [Google Scholar]
  28. Morley S. J., Buhl W. J., Jackson R. J. A rabbit reticulocyte factor which stimulates protein synthesis in several mammalian cell-free systems. Biochim Biophys Acta. 1985 May 24;825(1):57–69. doi: 10.1016/0167-4781(85)90079-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Omata T., Kohara M., Kuge S., Komatsu T., Abe S., Semler B. L., Kameda A., Itoh H., Arita M., Wimmer E. Genetic analysis of the attenuation phenotype of poliovirus type 1. J Virol. 1986 May;58(2):348–358. doi: 10.1128/jvi.58.2.348-358.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pallansch M. A., Kew O. M., Semler B. L., Omilianowski D. R., Anderson C. W., Wimmer E., Rueckert R. R. Protein processing map of poliovirus. J Virol. 1984 Mar;49(3):873–880. doi: 10.1128/jvi.49.3.873-880.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Pöyry T., Kinnunen L., Hovi T. Genetic variation in vivo and proposed functional domains of the 5' noncoding region of poliovirus RNA. J Virol. 1992 Sep;66(9):5313–5319. doi: 10.1128/jvi.66.9.5313-5319.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Teterina N. L., Kean K. M., Gorbalenya A. E., Agol V. I., Girard M. Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein. J Gen Virol. 1992 Aug;73(Pt 8):1977–1986. doi: 10.1099/0022-1317-73-8-1977. [DOI] [PubMed] [Google Scholar]
  40. Toyoda H., Kohara M., Kataoka Y., Suganuma T., Omata T., Imura N., Nomoto A. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. J Mol Biol. 1984 Apr 25;174(4):561–585. doi: 10.1016/0022-2836(84)90084-6. [DOI] [PubMed] [Google Scholar]
  41. Wimmer E., Hellen C. U., Cao X. Genetics of poliovirus. Annu Rev Genet. 1993;27:353–436. doi: 10.1146/annurev.ge.27.120193.002033. [DOI] [PubMed] [Google Scholar]
  42. Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES