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. 1996 May;70(5):2861–2868. doi: 10.1128/jvi.70.5.2861-2868.1996

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

X Y Jia 1, M Tesar 1, D F Summers 1, E Ehrenfeld 1
PMCID: PMC190143  PMID: 8627760

Abstract

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

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Selected References

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  1. Agol V. I. The 5'-untranslated region of picornaviral genomes. Adv Virus Res. 1991;40:103–180. doi: 10.1016/S0065-3527(08)60278-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander L., Lu H. H., Wimmer E. Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1406–1410. doi: 10.1073/pnas.91.4.1406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson D. A., Ross B. C. Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles. J Virol. 1990 Nov;64(11):5284–5289. doi: 10.1128/jvi.64.11.5284-5289.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Borman A. M., Deliat F. G., Kean K. M. Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis. EMBO J. 1994 Jul 1;13(13):3149–3157. doi: 10.1002/j.1460-2075.1994.tb06613.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown E. A., Day S. P., Jansen R. W., Lemon S. M. The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro. J Virol. 1991 Nov;65(11):5828–5838. doi: 10.1128/jvi.65.11.5828-5838.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown E. A., Zajac A. J., Lemon S. M. In vitro characterization of an internal ribosomal entry site (IRES) present within the 5' nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J Virol. 1994 Feb;68(2):1066–1074. doi: 10.1128/jvi.68.2.1066-1074.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cho M. W., Ehrenfeld E. Rapid completion of the replication cycle of hepatitis A virus subsequent to reversal of guanidine inhibition. Virology. 1991 Feb;180(2):770–780. doi: 10.1016/0042-6822(91)90090-x. [DOI] [PubMed] [Google Scholar]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Cohen J. I., Ticehurst J. R., Feinstone S. M., Rosenblum B., Purcell R. H. Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. J Virol. 1987 Oct;61(10):3035–3039. doi: 10.1128/jvi.61.10.3035-3039.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duke G. M., Hoffman M. A., Palmenberg A. C. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 Mar;66(3):1602–1609. doi: 10.1128/jvi.66.3.1602-1609.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Glass M. J., Jia X. Y., Summers D. F. Identification of the hepatitis A virus internal ribosome entry site: in vivo and in vitro analysis of bicistronic RNAs containing the HAV 5' noncoding region. Virology. 1993 Apr;193(2):842–852. doi: 10.1006/viro.1993.1193. [DOI] [PubMed] [Google Scholar]
  14. Glass M. J., Summers D. F. A cis-acting element within the hepatitis A virus 5'-non-coding region required for in vitro translation. Virus Res. 1992 Oct;26(1):15–31. doi: 10.1016/0168-1702(92)90143-w. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Harmon S. A., Emerson S. U., Huang Y. K., Summers D. F., Ehrenfeld E. Hepatitis A viruses with deletions in the 2A gene are infectious in cultured cells and marmosets. J Virol. 1995 Sep;69(9):5576–5581. doi: 10.1128/jvi.69.9.5576-5581.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hyypiä T., Horsnell C., Maaronen M., Khan M., Kalkkinen N., Auvinen P., Kinnunen L., Stanway G. A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8847–8851. doi: 10.1073/pnas.89.18.8847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Jia X. Y., Ehrenfeld E., Summers D. F. Proteolytic activity of hepatitis A virus 3C protein. J Virol. 1991 May;65(5):2595–2600. doi: 10.1128/jvi.65.5.2595-2600.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jia X. Y., Scheper G., Brown D., Updike W., Harmon S., Richards O., Summers D., Ehrenfeld E. Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5' noncoding region. Virology. 1991 Jun;182(2):712–722. doi: 10.1016/0042-6822(91)90612-f. [DOI] [PubMed] [Google Scholar]
  21. Jia X. Y., Summers D. F., Ehrenfeld E. Primary cleavage of the HAV capsid protein precursor in the middle of the proposed 2A coding region. Virology. 1993 Mar;193(1):515–519. doi: 10.1006/viro.1993.1157. [DOI] [PubMed] [Google Scholar]
  22. Johnston J. M., Harmon S. A., Binn L. N., Richards O. C., Ehrenfeld E., Summers D. F. Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli. J Infect Dis. 1988 Jun;157(6):1203–1211. doi: 10.1093/infdis/157.6.1203. [DOI] [PubMed] [Google Scholar]
  23. Kuge S., Nomoto A. Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5' noncoding sequence in viral replication. J Virol. 1987 May;61(5):1478–1487. doi: 10.1128/jvi.61.5.1478-1487.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Le S. Y., Chen J. H., Sonenberg N., Maizel J. V., Jr Conserved tertiary structural elements in the 5' nontranslated region of cardiovirus, aphthovirus and hepatitis A virus RNAs. Nucleic Acids Res. 1993 May 25;21(10):2445–2451. doi: 10.1093/nar/21.10.2445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lemon S. M., Whetter L., Chang K. H., Brown E. A. Why do human hepatitis viruses replicate so poorly in cell cultures? FEMS Microbiol Lett. 1992 Dec 15;100(1-3):455–459. doi: 10.1111/j.1574-6968.1992.tb14076.x. [DOI] [PubMed] [Google Scholar]
  26. Pal-Ghosh R., Morrow C. D. A poliovirus minireplicon containing an inactive 2A proteinase is expressed in vaccinia virus-infected cells. J Virol. 1993 Aug;67(8):4621–4629. doi: 10.1128/jvi.67.8.4621-4629.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parks G. D., Duke G. M., Palmenberg A. C. Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region. J Virol. 1986 Nov;60(2):376–384. doi: 10.1128/jvi.60.2.376-384.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pelletier J., Sonenberg N. Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts. J Virol. 1989 Jan;63(1):441–444. doi: 10.1128/jvi.63.1.441-444.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Racaniello V. R., Baltimore D. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science. 1981 Nov 20;214(4523):916–919. doi: 10.1126/science.6272391. [DOI] [PubMed] [Google Scholar]
  30. Racaniello V. R., Meriam C. Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA. Virology. 1986 Dec;155(2):498–507. doi: 10.1016/0042-6822(86)90211-4. [DOI] [PubMed] [Google Scholar]
  31. Rivera V. M., Welsh J. D., Maizel J. V., Jr Comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses. Virology. 1988 Jul;165(1):42–50. doi: 10.1016/0042-6822(88)90656-3. [DOI] [PubMed] [Google Scholar]
  32. Rohll J. B., Percy N., Ley R., Evans D. J., Almond J. W., Barclay W. S. The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation. J Virol. 1994 Jul;68(7):4384–4391. doi: 10.1128/jvi.68.7.4384-4391.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shaffer D. R., Brown E. A., Lemon S. M. Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes. J Virol. 1994 Sep;68(9):5568–5578. doi: 10.1128/jvi.68.9.5568-5578.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simoes E. A., Sarnow P. An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells. J Virol. 1991 Feb;65(2):913–921. doi: 10.1128/jvi.65.2.913-921.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Sonenberg N. Poliovirus translation. Curr Top Microbiol Immunol. 1990;161:23–47. doi: 10.1007/978-3-642-75602-3_2. [DOI] [PubMed] [Google Scholar]
  37. Stanway G., Kalkkinen N., Roivainen M., Ghazi F., Khan M., Smyth M., Meurman O., Hyypiä T. Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group. J Virol. 1994 Dec;68(12):8232–8238. doi: 10.1128/jvi.68.12.8232-8238.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tesar M., Harmon S. A., Summers D. F., Ehrenfeld E. Hepatitis A virus polyprotein synthesis initiates from two alternative AUG codons. Virology. 1992 Feb;186(2):609–618. doi: 10.1016/0042-6822(92)90027-m. [DOI] [PubMed] [Google Scholar]
  39. Trono D., Andino R., Baltimore D. An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis. J Virol. 1988 Jul;62(7):2291–2299. doi: 10.1128/jvi.62.7.2291-2299.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Usdin T. B., Brownstein M. J., Moss B., Isaacs S. N. SP6 RNA polymerase containing vaccinia virus for rapid expression of cloned genes in tissue culture. Biotechniques. 1993 Feb;14(2):222–224. [PubMed] [Google Scholar]
  41. Whetter L. E., Day S. P., Elroy-Stein O., Brown E. A., Lemon S. M. Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells. J Virol. 1994 Aug;68(8):5253–5263. doi: 10.1128/jvi.68.8.5253-5263.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]

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