Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2004 Apr 14;50(2):153–162. doi: 10.1016/0092-8674(87)90211-X

Vaccinia virus produces late mRNAs by discontinuous synthesis

Christine Bertholet 1, Erwin Van Meir 1, Béatrice ten Heggeler-Bordier 1, Riccardo Wittek 1
PMCID: PMC7133321  PMID: 3036368

Abstract

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3′ end of other RNAs.

References

  1. Bajszar G., Wittek R., Weir J.P., Moss B. Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants. J. Virol. 1983;45:62–72. doi: 10.1128/jvi.45.1.62-72.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baric R.S., Stohlman S.A., Razavi M.K., Lai M.M.C. Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription. Virus Res. 1985;3:19–33. doi: 10.1016/0168-1702(85)90038-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnes W.M., Bevan M. Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition. Nucl. Acids Res. 1983;11:349–368. doi: 10.1093/nar/11.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berk A.J., Sharp P.A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977;12:721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Bertholet C., Drillien R., Wittek R. Vol. 82. 1985. One hundred base pairs of 5′ flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription; pp. 2096–2100. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bertholet C., Stocco P., Van Meir E., Wittek R. Functional analysis of the 5′ flanking sequence of a vaccinia virus late gene. EMBO J. 1986;5:1951–1957. doi: 10.1002/j.1460-2075.1986.tb04449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boone R.F., Parr R.P., Moss B. Intermolecular duplexes formed from polyadenylated vaccinia virus RNA. J. Virol. 1979;30:365–374. doi: 10.1128/jvi.30.1.365-374.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Ann. Rev. Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  9. Brack C., Hirama M., Lenhard-Schuller R., Tonegawa S. A complete immunoglobulin gene is created by somatic recombination. Cell. 1978;15:1–14. doi: 10.1016/0092-8674(78)90078-8. [DOI] [PubMed] [Google Scholar]
  10. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Ann. Rev. Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  11. Bünemann H. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions. Nucl. Acids Res. 1982;10:7181–7196. doi: 10.1093/nar/10.22.7181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bünemann H., Westhoff P., Herrmann R.G. Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures. Nucl. Acids Res. 1982;10:7163–7180. doi: 10.1093/nar/10.22.7163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cochran M.A., Puckett C., Moss B. In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J. Virol. 1985;54:30–37. doi: 10.1128/jvi.54.1.30-37.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Colby C., Duesberg P.H. Double-stranded RNA in vaccinia virus infected cells. Nature. 1969;222:940–944. doi: 10.1038/222940a0. [DOI] [PubMed] [Google Scholar]
  15. Colby C., Jurale C., Kates J.R. Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro. J. Virol. 1971;7:71–76. doi: 10.1128/jvi.7.1.71-76.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cooper J., Wittek R., Moss B. Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs. J. Virol. 1981;39:733–745. doi: 10.1128/jvi.39.3.733-745.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Denhardt A.T. A member filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Comm. 1966;23:641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  18. Duesberg P.H., Colby C. Vol. 64. 1969. On the biosynthesis and structure of double-stranded RNA in vaccinia virus-infected cells; pp. 393–403. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Feinberg A.P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 1983;132:6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  20. Hänggi M., Bannwarth W., Stunnenberg H.G. Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. EMBO J. 1986;5:1071–1076. doi: 10.1002/j.1460-2075.1986.tb04324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herz C., Stavnezer E., Krug R.M., Gurney T., Jr. Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell. 1981;26:391–400. doi: 10.1016/0092-8674(81)90208-7. [DOI] [PubMed] [Google Scholar]
  22. Hirt P., Hiller G., Wittek R. Localization and fine structure of a vaccinia virus gene encoding an envelope antigen. J. Virol. 1986;58:757–764. doi: 10.1128/jvi.58.3.757-764.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hruby D.E., Ball L.A. control of expression of the vaccinia virus thymidine kinase gene. J. Virol. 1981;40:456–464. doi: 10.1128/jvi.40.2.456-464.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jungwirth C., Joklik W.K. Studies on “early” enzymes in HeLa cells infected with vaccinia virus. Virology. 1965;27:80–93. doi: 10.1016/0042-6822(65)90145-5. [DOI] [PubMed] [Google Scholar]
  25. Kates J., Beeson J. Ribonucleic acid synthesis in vaccinia virus. II. Synthesis of polyriboadenylic acid. J. Mol. Biol. 1970;50:19–23. doi: 10.1016/0022-2836(70)90101-4. [DOI] [PubMed] [Google Scholar]
  26. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978;15:1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  27. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986;47:481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  28. Krug R.M. The role of RNA priming in viral and trypanosomal mRNA synthesis. Cell. 1985;41:651–652. doi: 10.1016/S0092-8674(85)80041-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lai M.M.C., Baric R.S., Brayton P.R., Stohlman S.A. Vol. 81. 1984. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus; pp. 3626–3630. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mahr A., Roberts B. Arrangement of late mRNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment. J. Virol. 1984;49:510–520. doi: 10.1128/jvi.49.2.510-520.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maniatis T., Fritsch E.F., Sambrook J. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1982. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  32. McAuslan B.R. Control of induced thymidine kinase activity in the poxvirus infected cell. Virology. 1963;20:162–168. doi: 10.1016/0042-6822(63)90199-5. [DOI] [PubMed] [Google Scholar]
  33. McAuslan B.R. The induction and repression of thymidine kinase in the poxvirus-infected HeLa cell. Virology. 1963;21:383–389. doi: 10.1016/0042-6822(63)90199-5. [DOI] [PubMed] [Google Scholar]
  34. McMaster G.K., Carmichael G.G. Vol. 74. 1977. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange; pp. 4835–4838. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moss B. Reproduction of poxviruses. In: Fraenkel-Conrat H., Wagner R.R., editors. vol. 3. Plenum Publishing Corp; New York: 1974. pp. 405–473. (Comprehensive Virology). [Google Scholar]
  36. Moss B. Replication of poxviruses. In: Fields B.N., Chanock R.M., Roizman B., editors. Virology. Raven Press; New York: 1985. pp. 685–703. [Google Scholar]
  37. Murphy W.J., Watkins K.P., Agabian N. Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell. 1986;47:517–525. doi: 10.1016/0092-8674(86)90616-1. [DOI] [PubMed] [Google Scholar]
  38. Oda K., Joklik W.K. Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA. J. Mol. Biol. 1967;27:395–419. doi: 10.1016/0022-2836(67)90047-2. [DOI] [PubMed] [Google Scholar]
  39. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol. Cell. Biol. 1983;3:280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Paoletti E., Lipinskas B.R. Soluble endoribonuclease activity from vaccinia virus: specific cleavage of virion-associated high-molecular weight RNA. J. Virol. 1978;26:822–824. doi: 10.1128/jvi.26.3.822-824.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Patterson J.L., Holloway B., Kolakofsky D. La crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease. J. Virol. 1984;52:215–222. doi: 10.1128/jvi.52.1.215-222.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Plotch S.J., Bouloy M., Ulmanen I., Krug R.M. A unique cap (m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell. 1981;23:847–858. doi: 10.1016/0092-8674(81)90449-9. [DOI] [PubMed] [Google Scholar]
  43. Rigby P.W.J., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 1977;113:237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  44. Sahli R., McMaster G.K., Hirt B. DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice. Nucl. Acids Res. 1985;13:3617–3633. doi: 10.1093/nar/13.10.3617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A.R. Vol. 74. 1977. DNA sequencing with chain-terminating inhibitors; pp. 5463–5467. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sarov I., Joklik W.K. Studies on the nature and location of the capsid polypeptides of vaccinia virions. Virology. 1972;50:579–592. doi: 10.1016/0042-6822(72)90409-6. [DOI] [PubMed] [Google Scholar]
  47. Seed B. Diazotizable arylamine cellulose papers for the couplling and hybridization of nucleic acids. Nucl. Acids Res. 1982;10:1799–1810. doi: 10.1093/nar/10.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Smith A.J.H. DNA sequence analysis by primed synthesis. Methods Enzymol. 1980;65:560–580. doi: 10.1016/s0076-6879(80)65060-5. [DOI] [PubMed] [Google Scholar]
  49. Sutton R.E., Boothroyd J.C. Evidence for trans splicing in trypanosomes. Cell. 1986;47:527–535. doi: 10.1016/0092-8674(86)90617-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thomas P.S. Vol. 77. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose; pp. 5201–5205. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Van der Ploeg L.H.T. Discontinuous transcription and splicing in trypanosomes. Cell. 1986;47:479–480. doi: 10.1016/0092-8674(86)90608-2. [DOI] [PubMed] [Google Scholar]
  52. 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;37:738–747. doi: 10.1128/jvi.37.2.738-747.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weaver R.F., Weissman C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5′ termini of β-globin mRNA have identical map coordinates. Nucl. Acids Res. 1979;7:1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wei C.M., Moss B. Vol. 72. 1975. Methylated nucleotides block 5′-terminus of vaccinia virus mRNA; pp. 318–322. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weinrich S.L., Hruby D.E. A tandemly-oriented late gene cluster within the vaccinia virus genome. Nucl. Acids Res. 1986;14:3003–3016. doi: 10.1093/nar/14.7.3003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weir J.P., Moss B. Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations. J. Virol. 1983;46:530–537. doi: 10.1128/jvi.46.2.530-537.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weir J.P., Moss B. Regulation of expression and nucleotide sequence of a late vaccinia virus gene. J. Virol. 1984;51:662–669. doi: 10.1128/jvi.51.3.662-669.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wittek R., Cooper J., Barbosa E., Moss B. Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition. Cell. 1980;21:487–493. doi: 10.1016/0092-8674(80)90485-7. [DOI] [PubMed] [Google Scholar]
  59. Wittek R., Hänggi M., Hiller G. Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J. Virol. 1984;49:371–378. doi: 10.1128/jvi.49.2.371-378.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zaslavsky V., Yakobson E. Control of thymidine kinase synthesis in IHD vaccinia virus-infected thymidine kinase-deficient LM cells. J. Virol. 1975;16:210–213. doi: 10.1128/jvi.16.1.210-213.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cell are provided here courtesy of Elsevier

RESOURCES