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. 1988 Jun;62(6):2124–2133. doi: 10.1128/jvi.62.6.2124-2133.1988

Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro.

J M Polo 1, N L Davis 1, C M Rice 1, H V Huang 1, R E Johnston 1
PMCID: PMC253309  PMID: 2835514

Abstract

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

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Selected References

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

  1. Ahlquist P., Dasgupta R., Kaesberg P. Near identity of 3- RNA secondary structure in bromoviruses and cucumber mosaic virus. Cell. 1981 Jan;23(1):183–189. doi: 10.1016/0092-8674(81)90283-x. [DOI] [PubMed] [Google Scholar]
  2. Ahlquist P., French R., Janda M., Loesch-Fries L. S. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7066–7070. doi: 10.1073/pnas.81.22.7066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baric R. S., Trent D. W., Johnston R. E. A Sindbis virus variant with a cell-determined latent period. Virology. 1981 Apr 15;110(1):237–242. doi: 10.1016/0042-6822(81)90029-5. [DOI] [PubMed] [Google Scholar]
  4. Barrett P. N., Sheahan B. J., Atkins G. J. Isolation and preliminary characterization of Semliki Forest virus mutants with altered virulence. J Gen Virol. 1980 Jul;49(1):141–147. doi: 10.1099/0022-1317-49-1-141. [DOI] [PubMed] [Google Scholar]
  5. Bose H. R., Sagik B. P. The virus envelope in cell attachment. J Gen Virol. 1970 Nov;9(2):159–161. doi: 10.1099/0022-1317-9-2-159. [DOI] [PubMed] [Google Scholar]
  6. Burge B. W., Pfefferkorn E. R. Isolation and characterization of conditional-lethal mutants of Sindbis virus. Virology. 1966 Oct;30(2):204–213. doi: 10.1016/0042-6822(66)90096-1. [DOI] [PubMed] [Google Scholar]
  7. Condreay L. D., Brown D. T. Exclusion of superinfecting homologous virus by Sindbis virus-infected Aedes albopictus (mosquito) cells. J Virol. 1986 Apr;58(1):81–86. doi: 10.1128/jvi.58.1.81-86.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cress D. E., Kiefer M. C., Owens R. A. Construction of infectious potato spindle tuber viroid cDNA clones. Nucleic Acids Res. 1983 Oct 11;11(19):6821–6835. doi: 10.1093/nar/11.19.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dalrymple J. M., Schlesinger S., Russell P. K. Antigenic characterization of two sindbis envelope glycoproteins separated by isoelectric focusing. Virology. 1976 Jan;69(1):93–103. doi: 10.1016/0042-6822(76)90197-5. [DOI] [PubMed] [Google Scholar]
  10. Davis N. L., Fuller F. J., Dougherty W. G., Olmsted R. A., Johnston R. E. A single nucleotide change in the E2 glycoprotein gene of Sindbis virus affects penetration rate in cell culture and virulence in neonatal mice. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6771–6775. doi: 10.1073/pnas.83.18.6771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis N. L., Pence D. F., Meyer W. J., Schmaljohn A. L., Johnston R. E. Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein. Virology. 1987 Nov;161(1):101–108. doi: 10.1016/0042-6822(87)90175-9. [DOI] [PubMed] [Google Scholar]
  12. Dietzschold B., Wunner W. H., Wiktor T. J., Lopes A. D., Lafon M., Smith C. L., Koprowski H. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc Natl Acad Sci U S A. 1983 Jan;80(1):70–74. doi: 10.1073/pnas.80.1.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emini E. A., Wiebe M. E. An attenuated mutant of Venezuelan encephalitis virus: biochemical alterations and their genetic association with attenuation. Virology. 1981 Apr 15;110(1):185–196. doi: 10.1016/0042-6822(81)90020-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
  16. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  17. Hahn C. S., Dalrymple J. M., Strauss J. H., Rice C. M. Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2019–2023. doi: 10.1073/pnas.84.7.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanahan D., Meselson M. Plasmid screening at high colony density. Gene. 1980 Jun;10(1):63–67. doi: 10.1016/0378-1119(80)90144-4. [DOI] [PubMed] [Google Scholar]
  19. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  20. Johnson B. J., Kinney R. M., Kost C. L., Trent D. W. Molecular determinants of alphavirus neurovirulence: nucleotide and deduced protein sequence changes during attenuation of Venezuelan equine encephalitis virus. J Gen Virol. 1986 Sep;67(Pt 9):1951–1960. doi: 10.1099/0022-1317-67-9-1951. [DOI] [PubMed] [Google Scholar]
  21. Johnson R. T., McFarland H. F., Levy S. E. Age-dependent resistance to viral encephalitis: studies of infections due to Sindbis virus in mice. J Infect Dis. 1972 Mar;125(3):257–262. doi: 10.1093/infdis/125.3.257. [DOI] [PubMed] [Google Scholar]
  22. Johnston R. E., Smith J. F. Selection for accelerated penetration in cell culture coselects for attenuated mutants of Venezuelan equine encephalitis virus. Virology. 1988 Feb;162(2):437–443. doi: 10.1016/0042-6822(88)90484-9. [DOI] [PubMed] [Google Scholar]
  23. Kaplan G., Lubinski J., Dasgupta A., Racaniello V. R. In vitro synthesis of infectious poliovirus RNA. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8424–8428. doi: 10.1073/pnas.82.24.8424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaye K. M., Spriggs D. R., Bassel-Duby R., Fields B. N., Tyler K. L. Genetic basis for altered pathogenesis of an immune-selected antigenic variant of reovirus type 3 (Dearing). J Virol. 1986 Jul;59(1):90–97. doi: 10.1128/jvi.59.1.90-97.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kinney R. M., Johnson B. J., Brown V. L., Trent D. W. Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins. Virology. 1986 Jul 30;152(2):400–413. doi: 10.1016/0042-6822(86)90142-x. [DOI] [PubMed] [Google Scholar]
  26. La Monica N., Almond J. W., Racaniello V. R. A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans. J Virol. 1987 Sep;61(9):2917–2920. doi: 10.1128/jvi.61.9.2917-2920.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  29. Mizutani S., Colonno R. J. In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14. J Virol. 1985 Nov;56(2):628–632. doi: 10.1128/jvi.56.2.628-632.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nomoto A., Omata T., Toyoda H., Kuge S., Horie H., Kataoka Y., Genba Y., Nakano Y., Imura N. Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5793–5797. doi: 10.1073/pnas.79.19.5793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Olmsted R. A., Baric R. S., Sawyer B. A., Johnston R. E. Sindbis virus mutants selected for rapid growth in cell culture display attenuated virulence in animals. Science. 1984 Jul 27;225(4660):424–427. doi: 10.1126/science.6204381. [DOI] [PubMed] [Google Scholar]
  32. Olmsted R. A., Meyer W. J., Johnston R. E. Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals. Virology. 1986 Jan 30;148(2):245–254. doi: 10.1016/0042-6822(86)90322-3. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Reinarz A. B., Broome M. G., Sagik B. P. Age-dependent resistance of mice to sindbis virus infection: viral replication as a function of host age. Infect Immun. 1971 Feb;3(2):268–273. doi: 10.1128/iai.3.2.268-273.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rice C. M., Strauss J. H. Association of sindbis virion glycoproteins and their precursors. J Mol Biol. 1982 Jan 15;154(2):325–348. doi: 10.1016/0022-2836(82)90067-5. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Schlesinger J. J., Brandriss M. W., Monath T. P. Monoclonal antibodies distinguish between wild and vaccine strains of yellow fever virus by neutralization, hemagglutination inhibition, and immune precipitation of the virus envelope protein. Virology. 1983 Feb;125(1):8–17. doi: 10.1016/0042-6822(83)90059-4. [DOI] [PubMed] [Google Scholar]
  40. Seif I., Coulon P., Rollin P. E., Flamand A. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J Virol. 1985 Mar;53(3):926–934. doi: 10.1128/jvi.53.3.926-934.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stanway G., Hughes P. J., Mountford R. C., Reeve P., Minor P. D., Schild G. C., Almond J. W. Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon 12a1b. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1539–1543. doi: 10.1073/pnas.81.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Strauss E. G., Rice C. M., Strauss J. H. Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology. 1984 Feb;133(1):92–110. doi: 10.1016/0042-6822(84)90428-8. [DOI] [PubMed] [Google Scholar]
  43. Taniguchi T., Palmieri M., Weissmann C. QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host. Nature. 1978 Jul 20;274(5668):223–228. doi: 10.1038/274223a0. [DOI] [PubMed] [Google Scholar]
  44. Vrati S., Faragher S. G., Weir R. C., Dalgarno L. Ross River virus mutant with a deletion in the E2 gene: properties of the virion, virus-specific macromolecule synthesis, and attenuation of virulence for mice. Virology. 1986 Jun;151(2):222–232. doi: 10.1016/0042-6822(86)90044-9. [DOI] [PubMed] [Google Scholar]
  45. Webster R. G., Rott R. Influenza virus A pathogenicity: the pivotal role of hemagglutinin. Cell. 1987 Aug 28;50(5):665–666. doi: 10.1016/0092-8674(87)90321-7. [DOI] [PubMed] [Google Scholar]
  46. Zimmern D., Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. doi: 10.1073/pnas.75.9.4257. [DOI] [PMC free article] [PubMed] [Google Scholar]

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