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. 1993 Jun;67(6):3691–3695. doi: 10.1128/jvi.67.6.3691-3695.1993

A three-nucleotide insertion in the H stem-loop of the 5' untranslated region of Theiler's virus attenuates neurovirulence.

P K Bandyopadhyay 1, A Pritchard 1, K Jensen 1, H L Lipton 1
PMCID: PMC237727  PMID: 7684472

Abstract

The highly structured 5' untranslated region (5' UTR) of Theiler's murine encephalomyelitis virus is involved in cap-independent translation of the viral RNA. Previously, we reported that the bicistronic mRNA chloramphenicol acetyltransferase-5' UTR-luciferase (Luc) efficiently expressed Luc both in a rabbit reticulocyte lysate and when transfected into BHK-21 cells. Insertion of 3 nucleotides at position 665 in the 5' UTR of this bicistronic mRNA resulted in greatly reduced Luc expression in BHK-21 cells but had little effect on expression of Luc in rabbit reticulocyte lysate. This mutation was also introduced into a virulent Theiler's murine encephalomyelitis virus chimera, Chi-VL. The kinetics of viral RNA and protein synthesis and virus production in BHK-21 cells were slower for the mutant chimera [Chi-VL(IN668)] than for Chi-VL; however, the final virus yields were comparable. Intracerebral inoculation of mice with the chimeras revealed that Chi-VL(IN668) was completely attenuated in neurovirulence. The reduced neurovirulence of Chi-VL(IN668) may be ascribed to its reduced growth in the central nervous system, most likely due to an impaired ability to synthesize viral proteins.

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Selected References

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  1. Bandyopadhyay P. K., Wang C., Lipton H. L. Cap-independent translation by the 5' untranslated region of Theiler's murine encephalomyelitis virus. J Virol. 1992 Nov;66(11):6249–6256. doi: 10.1128/jvi.66.11.6249-6256.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Duke G. M., Osorio J. E., Palmenberg A. C. Attenuation of Mengo virus through genetic engineering of the 5' noncoding poly(C) tract. Nature. 1990 Feb 1;343(6257):474–476. doi: 10.1038/343474a0. [DOI] [PubMed] [Google Scholar]
  4. Duke G. M., Palmenberg A. C. Cloning and synthesis of infectious cardiovirus RNAs containing short, discrete poly(C) tracts. J Virol. 1989 Apr;63(4):1822–1826. doi: 10.1128/jvi.63.4.1822-1826.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Lipton H. L., Calenoff M., Bandyopadhyay P., Miller S. D., Dal Canto M. C., Gerety S., Jensen K. The 5' noncoding sequences from a less virulent Theiler's virus dramatically attenuate GDVII neurovirulence. J Virol. 1991 Aug;65(8):4370–4377. doi: 10.1128/jvi.65.8.4370-4377.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lipton H. L. Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect Immun. 1975 May;11(5):1147–1155. doi: 10.1128/iai.11.5.1147-1155.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Pevear D. C., Borkowski J., Calenoff M., Oh C. K., Ostrowski B., Lipton H. L. Insights into Theiler's virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains. Virology. 1988 Jul;165(1):1–12. doi: 10.1016/0042-6822(88)90652-6. [DOI] [PubMed] [Google Scholar]
  10. Pevear D. C., Calenoff M., Rozhon E., Lipton H. L. Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses. J Virol. 1987 May;61(5):1507–1516. doi: 10.1128/jvi.61.5.1507-1516.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pilipenko E. V., Blinov V. M., Chernov B. K., Dmitrieva T. M., Agol V. I. Conservation of the secondary structure elements of the 5'-untranslated region of cardio- and aphthovirus RNAs. Nucleic Acids Res. 1989 Jul 25;17(14):5701–5711. doi: 10.1093/nar/17.14.5701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pritchard A. E., Calenoff M. A., Simpson S., Jensen K., Lipton H. L. A single base deletion in the 5' noncoding region of Theiler's virus attenuates neurovirulence. J Virol. 1992 Apr;66(4):1951–1958. doi: 10.1128/jvi.66.4.1951-1958.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Sarnow P., Jacobson S. J., Najita L. Poliovirus genetics. Curr Top Microbiol Immunol. 1990;161:155–188. doi: 10.1007/978-3-642-75602-3_6. [DOI] [PubMed] [Google Scholar]
  16. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Stein S. B., Zhang L., Roos R. P. Influence of Theiler's murine encephalomyelitis virus 5' untranslated region on translation and neurovirulence. J Virol. 1992 Jul;66(7):4508–4517. doi: 10.1128/jvi.66.7.4508-4517.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Svitkin Y. V., Cammack N., Minor P. D., Almond J. W. Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U. Virology. 1990 Mar;175(1):103–109. doi: 10.1016/0042-6822(90)90190-3. [DOI] [PubMed] [Google Scholar]
  20. Svitkin Y. V., Maslova S. V., Agol V. I. The genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies. Virology. 1985 Dec;147(2):243–252. doi: 10.1016/0042-6822(85)90127-8. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  23. Zurbriggen A., Yamada M., Thomas C., Fujinami R. S. Restricted virus replication in the spinal cords of nude mice infected with a Theiler's virus variant. J Virol. 1991 Feb;65(2):1023–1030. doi: 10.1128/jvi.65.2.1023-1030.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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