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. 1985 Oct;56(1):227–239. doi: 10.1128/jvi.56.1.227-239.1985

Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions.

C M Rice, C A Franke, J H Strauss, D E Hruby
PMCID: PMC252510  PMID: 4032536

Abstract

We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication.

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Selected References

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

  1. Aliperti G., Schlesinger M. J. Evidence for an autoprotease activity of sindbis virus capsid protein. Virology. 1978 Oct 15;90(2):366–369. doi: 10.1016/0042-6822(78)90321-5. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Callis A. H., Ritzi E. M. Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay. J Virol. 1980 Sep;35(3):876–887. doi: 10.1128/jvi.35.3.876-887.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. DeFilippes F. M. Restriction enzyme mapping of vaccinia virus DNA. J Virol. 1982 Jul;43(1):136–149. doi: 10.1128/jvi.43.1.136-149.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Garoff H., Kondor-Koch C., Pettersson R., Burke B. Expression of Semliki Forest virus proteins from cloned complementary DNA. II. The membrane-spanning glycoprotein E2 is transported to the cell surface without its normal cytoplasmic domain. J Cell Biol. 1983 Sep;97(3):652–658. doi: 10.1083/jcb.97.3.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Hahn C. S., Strauss E. G., Strauss J. H. Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid protein autoprotease. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4648–4652. doi: 10.1073/pnas.82.14.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hruby D. E., Ball L. A. Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase. Virology. 1981 Sep;113(2):594–601. doi: 10.1016/0042-6822(81)90187-2. [DOI] [PubMed] [Google Scholar]
  14. Hruby D. E., Guarino L. A., Kates J. R. Vaccinia virus replication. I. Requirement for the host-cell nucleus. J Virol. 1979 Feb;29(2):705–715. doi: 10.1128/jvi.29.2.705-715.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hruby D. E., Lynn D. L., Condit R. C., Kates J. R. Cellular differences in the molecular mechanisms of vaccinia virus host range restriction. J Gen Virol. 1980 Apr;47(2):485–488. doi: 10.1099/0022-1317-47-2-485. [DOI] [PubMed] [Google Scholar]
  16. Hruby D. E., Roberts W. K. Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity. J Virol. 1976 Aug;19(2):325–330. doi: 10.1128/jvi.19.2.325-330.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hutchison C. A., 3rd, Edgell M. H. Genetic assay for small fragments of bacteriophage phi X174 deoxyribonucleic acid. J Virol. 1971 Aug;8(2):181–189. doi: 10.1128/jvi.8.2.181-189.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huth A., Rapoport T. A., Käriäinen L. Envelope proteins of Semliki Forest virus synthesized in Xenopus oocytes are transported to the cell surface. EMBO J. 1984 Apr;3(4):767–771. doi: 10.1002/j.1460-2075.1984.tb01882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones K. J., Waite M. R., Bose H. R. Cleavage of a viral envelope precursor during the morphogenesis of Sindbis virus. J Virol. 1974 Apr;13(4):809–817. doi: 10.1128/jvi.13.4.809-817.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kieny M. P., Lathe R., Drillien R., Spehner D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocq J. P. Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature. 1984 Nov 8;312(5990):163–166. doi: 10.1038/312163a0. [DOI] [PubMed] [Google Scholar]
  21. Kondor-Koch C., Burke B., Garoff H. Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein. J Cell Biol. 1983 Sep;97(3):644–651. doi: 10.1083/jcb.97.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kondor-Koch C., Riedel H., Söderberg K., Garoff H. Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4525–4529. doi: 10.1073/pnas.79.15.4525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mackett M., Smith G. L., Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7415–7419. doi: 10.1073/pnas.79.23.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mackett M., Yilma T., Rose J. K., Moss B. Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle. Science. 1985 Jan 25;227(4685):433–435. doi: 10.1126/science.2981435. [DOI] [PubMed] [Google Scholar]
  27. Mayne J. T., Rice C. M., Strauss E. G., Hunkapiller M. W., Strauss J. H. Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3. Virology. 1984 Apr 30;134(2):338–357. doi: 10.1016/0042-6822(84)90302-7. [DOI] [PubMed] [Google Scholar]
  28. Moss B., Smith G. L., Gerin J. L., Purcell R. H. Live recombinant vaccinia virus protects chimpanzees against hepatitis B. Nature. 1984 Sep 6;311(5981):67–69. doi: 10.1038/311067a0. [DOI] [PubMed] [Google Scholar]
  29. Nakano E., Panicali D., Paoletti E. Molecular genetics of vaccinia virus: demonstration of marker rescue. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1593–1596. doi: 10.1073/pnas.79.5.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Panicali D., Davis S. W., Weinberg R. L., Paoletti E. Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5364–5368. doi: 10.1073/pnas.80.17.5364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Panicali D., Paoletti E. Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4927–4931. doi: 10.1073/pnas.79.16.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Paoletti E., Lipinskas B. R., Samsonoff C., Mercer S., Panicali D. Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proc Natl Acad Sci U S A. 1984 Jan;81(1):193–197. doi: 10.1073/pnas.81.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pierce J. S., Strauss E. G., Strauss J. H. Effect of ionic strength on the binding of Sindbis virus to chick cells. J Virol. 1974 May;13(5):1030–1036. doi: 10.1128/jvi.13.5.1030-1036.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Rice C. M., Strauss J. H. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2062–2066. doi: 10.1073/pnas.78.4.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schmaljohn A. L., Kokubun K. M., Cole G. A. Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein. Virology. 1983 Oct 15;130(1):144–154. doi: 10.1016/0042-6822(83)90124-1. [DOI] [PubMed] [Google Scholar]
  37. Schupham R. K., Jones K. J., Sagik B. P., Bose H. R., Jr Virus-directed post-translational cleavage in Sindbus virus-infected cells. J Virol. 1977 May;22(2):568–571. doi: 10.1128/jvi.22.2.568-571.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  39. Simmons D. T., Strauss J. H. Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes. J Mol Biol. 1974 Jun 25;86(2):397–409. doi: 10.1016/0022-2836(74)90027-8. [DOI] [PubMed] [Google Scholar]
  40. Smith C., Wolcott J. A., Wust C. J., Brown A. Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells. J Virol. 1985 Jan;53(1):198–204. doi: 10.1128/jvi.53.1.198-204.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smith G. L., Godson G. N., Nussenzweig V., Nussenzweig R. S., Barnwell J., Moss B. Plasmodium knowlesi sporozoite antigen: expression by infectious recombinant vaccinia virus. Science. 1984 Apr 27;224(4647):397–399. doi: 10.1126/science.6200932. [DOI] [PubMed] [Google Scholar]
  42. Smith G. L., Mackett M., Moss B. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen. Nature. 1983 Apr 7;302(5908):490–495. doi: 10.1038/302490a0. [DOI] [PubMed] [Google Scholar]
  43. Smith G. L., Moss B. Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA. Gene. 1983 Nov;25(1):21–28. doi: 10.1016/0378-1119(83)90163-4. [DOI] [PubMed] [Google Scholar]
  44. Smith G. L., Murphy B. R., Moss B. Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza virus infection in hamsters. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7155–7159. doi: 10.1073/pnas.80.23.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  46. Strauss E. G., Lenches E. M., Strauss J. H. Mutants of sindbis virus. I. Isolation and partial characterization of 89 new temperature-sensitive mutants. Virology. 1976 Oct 1;74(1):154–168. doi: 10.1016/0042-6822(76)90137-9. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  49. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  50. Venkatesan S., Moss B. In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites. J Virol. 1981 Feb;37(2):738–747. doi: 10.1128/jvi.37.2.738-747.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Villarreal L. P., Berg P. Hybridization in situ of SV40 plaques: detection of recombinant SV40 virus carrying specific sequences of nonviral DNA. Science. 1977 Apr 8;196(4286):183–185. doi: 10.1126/science.191907. [DOI] [PubMed] [Google Scholar]
  52. Weir J. P., Bajszár G., Moss B. Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1210–1214. doi: 10.1073/pnas.79.4.1210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weir J. P., Moss B. Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations. J Virol. 1983 May;46(2):530–537. doi: 10.1128/jvi.46.2.530-537.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Welch W. J., Sefton B. M. Two small virus-specific polypeptides are produced during infection with Sindbis virus. J Virol. 1979 Mar;29(3):1186–1195. doi: 10.1128/jvi.29.3.1186-1195.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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