Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Oct;66(10):6143–6154. doi: 10.1128/jvi.66.10.6143-6154.1992

Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.

D X Liu 1, S C Inglis 1
PMCID: PMC241492  PMID: 1527853

Abstract

mRNA3 specified by the coronavirus infectious bronchitis virus appears to be functionally tricistronic, having the capacity to encode three small proteins (3a, 3b, and 3c) from separate open reading frames (ORFs). The mechanism by which this can occur was investigated through in vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c ORFs, and the results suggest that translation of the most distal of the three ORFs, that for 3c, is mediated by an unconventional, cap-independent mechanism involving internal initiation. This conclusion is based on several observations. A synthetic mRNA whose peculiar 5' end structure prevents translation of the 5'-proximal ORFs (3a and 3b) directs the synthesis of 3c normally. Translation of 3c, unlike that of 3a and 3b, was insensitive to the presence of the 5' cap analog 7-methyl-GTP, and it was unaffected by alteration of the sequence contexts for initiation on the 3a and 3b ORFs. Finally, an mRNA in which the 3a/b/c infectious bronchitis virus coding region was placed downstream of the influenza A virus nucleocapsid protein gene directed the efficient synthesis of 3c as well as nucleocapsid protein, whereas initiation at 3a and 3b could not be detected. Expression of the 3c ORF from this mRNA, however, was abolished when the 3a and 3b coding region was deleted, indicating that 3c initiation is dependent on upstream sequence elements which together may serve as a ribosomal internal entry site similar to those described for picornaviruses.

Full text

PDF
6146

Images in this article

6146Image
on p.6146
6147Image
on p.6147
6148Image
on p.6148
6149Image
on p.6149
6150Image
on p.6150

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. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borovjagin A. V., Evstafieva A. G., Ugarova TYu, Shatsky I. N. A factor that specifically binds to the 5'-untranslated region of encephalomyocarditis virus RNA. FEBS Lett. 1990 Feb 26;261(2):237–240. doi: 10.1016/0014-5793(90)80561-v. [DOI] [PubMed] [Google Scholar]
  4. Boursnell M. E., Binns M. M., Brown T. D. Sequencing of coronavirus IBV genomic RNA: three open reading frames in the 5' 'unique' region of mRNA D. J Gen Virol. 1985 Oct;66(Pt 10):2253–2258. doi: 10.1099/0022-1317-66-10-2253. [DOI] [PubMed] [Google Scholar]
  5. Boursnell M. E., Brown T. D., Foulds I. J., Green P. F., Tomley F. M., Binns M. M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J Gen Virol. 1987 Jan;68(Pt 1):57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  6. Brierley I., Digard P., Inglis S. C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell. 1989 May 19;57(4):537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cavanagh D., Brian D. A., Enjuanes L., Holmes K. V., Lai M. M., Laude H., Siddell S. G., Spaan W., Taguchi F., Talbot P. J. Recommendations of the Coronavirus Study Group for the nomenclature of the structural proteins, mRNAs, and genes of coronaviruses. Virology. 1990 May;176(1):306–307. doi: 10.1016/0042-6822(90)90259-T. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Contreras R., Cheroutre H., Degrave W., Fiers W. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res. 1982 Oct 25;10(20):6353–6362. doi: 10.1093/nar/10.20.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Dotto G. P., Enea V., Zinder N. D. Functional analysis of bacteriophage f1 intergenic region. Virology. 1981 Oct 30;114(2):463–473. doi: 10.1016/0042-6822(81)90226-9. [DOI] [PubMed] [Google Scholar]
  11. Hassin D., Korn R., Horwitz M. S. A major internal initiation site for the in vitro translation of the adenovirus DNA polymerase. Virology. 1986 Nov;155(1):214–224. doi: 10.1016/0042-6822(86)90181-9. [DOI] [PubMed] [Google Scholar]
  12. Herman R. C. Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA. Biochemistry. 1987 Dec 15;26(25):8346–8350. doi: 10.1021/bi00399a048. [DOI] [PubMed] [Google Scholar]
  13. Herman R. C. Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein. J Virol. 1986 Jun;58(3):797–804. doi: 10.1128/jvi.58.3.797-804.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Inglis S. C., McGeoch D. J., Mahy B. W. Polypeptides specified by the influenza virus genoma. 2. Assignement of protein coding functions to individual genome segments by in vitro translation. Virology. 1977 May 15;78(2):522–536. doi: 10.1016/0042-6822(77)90128-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986 Nov 21;47(4):481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  22. Kozak M. Mechanism of mRNA recognition by eukaryotic ribosomes during initiation of protein synthesis. Curr Top Microbiol Immunol. 1981;93:81–123. doi: 10.1007/978-3-642-68123-3_5. [DOI] [PubMed] [Google Scholar]
  23. Kozak M. Regulation of protein synthesis in virus-infected animal cells. Adv Virus Res. 1986;31:229–292. doi: 10.1016/S0065-3527(08)60265-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  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., Stohlman S. A. Further characterization of mRNA's of mouse hepatitis virus: presence of common 5'-end nucleotides. J Virol. 1982 Feb;41(2):557–565. doi: 10.1128/jvi.41.2.557-565.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu D. X., Cavanagh D., Green P., Inglis S. C. A polycistronic mRNA specified by the coronavirus infectious bronchitis virus. Virology. 1991 Oct;184(2):531–544. doi: 10.1016/0042-6822(91)90423-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu D. X., Inglis S. C. Association of the infectious bronchitis virus 3c protein with the virion envelope. Virology. 1991 Dec;185(2):911–917. doi: 10.1016/0042-6822(91)90572-S. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Luz N., Beck E. Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1991 Dec;65(12):6486–6494. doi: 10.1128/jvi.65.12.6486-6494.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  39. Sarnow P. Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5795–5799. doi: 10.1073/pnas.86.15.5795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smith A. R., Boursnell M. E., Binns M. M., Brown T. D., Inglis S. C. Identification of a new gene product encoded by mRNA D of infectious bronchitis virus. Adv Exp Med Biol. 1987;218:47–54. doi: 10.1007/978-1-4684-1280-2_6. [DOI] [PubMed] [Google Scholar]
  41. Sonenberg N. Regulation of translation by poliovirus. Adv Virus Res. 1987;33:175–204. doi: 10.1016/s0065-3527(08)60318-8. [DOI] [PubMed] [Google Scholar]
  42. Stern D. F., Sefton B. M. Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome. J Virol. 1984 Apr;50(1):22–29. doi: 10.1128/jvi.50.1.22-29.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Verver J., Le Gall O., van Kammen A., Wellink J. The sequence between nucleotides 161 and 512 of cowpea mosaic virus M RNA is able to support internal initiation of translation in vitro. J Gen Virol. 1991 Oct;72(Pt 10):2339–2345. doi: 10.1099/0022-1317-72-10-2339. [DOI] [PubMed] [Google Scholar]
  44. del Angel R. M., Papavassiliou A. G., Fernández-Tomás C., Silverstein S. J., Racaniello V. R. Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8299–8303. doi: 10.1073/pnas.86.21.8299. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES