Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Jun;66(6):3522–3530. doi: 10.1128/jvi.66.6.3522-3530.1992

Identification and characterization of a coronavirus packaging signal.

J A Fosmire 1, K Hwang 1, S Makino 1
PMCID: PMC241133  PMID: 1316465

Abstract

Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.

Full text

PDF
3522

Images in this article

3525Image
on p.3525
3527Image
on p.3527
3527Image
on p.3527

Selected References

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

  1. Adam M. A., Miller A. D. Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions. J Virol. 1988 Oct;62(10):3802–3806. doi: 10.1128/jvi.62.10.3802-3806.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alford R. L., Honda S., Lawrence C. B., Belmont J. W. RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus. Virology. 1991 Aug;183(2):611–619. doi: 10.1016/0042-6822(91)90990-s. [DOI] [PubMed] [Google Scholar]
  3. Baric R. S., Nelson G. W., Fleming J. O., Deans R. J., Keck J. G., Casteel N., Stohlman S. A. Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. J Virol. 1988 Nov;62(11):4280–4287. doi: 10.1128/jvi.62.11.4280-4287.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barton D. J., Sawicki S. G., Sawicki D. L. Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6. J Virol. 1988 Oct;62(10):3597–3602. doi: 10.1128/jvi.62.10.3597-3602.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernardi A., Spahr P. F. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3033–3037. doi: 10.1073/pnas.69.10.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berzin V., Borisova G. P., Cielens I., Gribanov V. A., Jansone I., Rosenthal G., Gren E. J. The regulatory region of MS2 phage RNA replicase cistron. Functional activity of individual MS2 RNA fragments. J Mol Biol. 1978 Feb 15;119(1):101–131. doi: 10.1016/0022-2836(78)90272-3. [DOI] [PubMed] [Google Scholar]
  7. Boursnell M. E., Brown T. D., Foulds I. J., Green P. F., Tomley F. M., Binns M. M. Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J Gen Virol. 1987 Jan;68(Pt 1):57–77. doi: 10.1099/0022-1317-68-1-57. [DOI] [PubMed] [Google Scholar]
  8. Bredenbeek P. J., Pachuk C. J., Noten A. F., Charité J., Luytjes W., Weiss S. R., Spaan W. J. The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. Nucleic Acids Res. 1990 Apr 11;18(7):1825–1832. doi: 10.1093/nar/18.7.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Compton S. R., Rogers D. B., Holmes K. V., Fertsch D., Remenick J., McGowan J. J. In vitro replication of mouse hepatitis virus strain A59. J Virol. 1987 Jun;61(6):1814–1820. doi: 10.1128/jvi.61.6.1814-1820.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denison M. R., Zoltick P. W., Leibowitz J. L., Pachuk C. J., Weiss S. R. Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59. J Virol. 1991 Jun;65(6):3076–3082. doi: 10.1128/jvi.65.6.3076-3082.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fujimura T., Esteban R., Esteban L. M., Wickner R. B. Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae. Cell. 1990 Aug 24;62(4):819–828. doi: 10.1016/0092-8674(90)90125-x. [DOI] [PubMed] [Google Scholar]
  13. Hahn Y. S., Grakoui A., Rice C. M., Strauss E. G., Strauss J. H. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4. J Virol. 1989 Mar;63(3):1194–1202. doi: 10.1128/jvi.63.3.1194-1202.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kamer G., Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984 Sep 25;12(18):7269–7282. doi: 10.1093/nar/12.18.7269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keck J. G., Matsushima G. K., Makino S., Fleming J. O., Vannier D. M., Stohlman S. A., Lai M. M. In vivo RNA-RNA recombination of coronavirus in mouse brain. J Virol. 1988 May;62(5):1810–1813. doi: 10.1128/jvi.62.5.1810-1813.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keck J. G., Stohlman S. A., Soe L. H., Makino S., Lai M. M. Multiple recombination sites at the 5'-end of murine coronavirus RNA. Virology. 1987 Feb;156(2):331–341. doi: 10.1016/0042-6822(87)90413-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kumanishi T. Brain tumors induced with Rous sarcoma virus, Schmidt-Ruppin strain. I. Induction of brain tumors in adult mice with Rous chicken sarcoma cells. Jpn J Exp Med. 1967 Oct;37(5):461–474. [PubMed] [Google Scholar]
  18. Lai M. M., Baric R. S., Brayton P. R., Stohlman S. A. Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3626–3630. doi: 10.1073/pnas.81.12.3626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lai M. M., Brayton P. R., Armen R. C., Patton C. D., Pugh C., Stohlman S. A. Mouse hepatitis virus A59: mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3. J Virol. 1981 Sep;39(3):823–834. doi: 10.1128/jvi.39.3.823-834.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lai M. M. Coronavirus: organization, replication and expression of genome. Annu Rev Microbiol. 1990;44:303–333. doi: 10.1146/annurev.mi.44.100190.001511. [DOI] [PubMed] [Google Scholar]
  21. Lai M. M., Patton C. D., Baric R. S., Stohlman S. A. Presence of leader sequences in the mRNA of mouse hepatitis virus. J Virol. 1983 Jun;46(3):1027–1033. doi: 10.1128/jvi.46.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lai M. M., Stohlman S. A. RNA of mouse hepatitis virus. J Virol. 1978 May;26(2):236–242. doi: 10.1128/jvi.26.2.236-242.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee H. J., Shieh C. K., Gorbalenya A. E., Koonin E. V., La Monica N., Tuler J., Bagdzhadzhyan A., Lai M. M. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology. 1991 Feb;180(2):567–582. doi: 10.1016/0042-6822(91)90071-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leibowitz J. L., Wilhelmsen K. C., Bond C. W. The virus-specific intracellular RNA species of two murine coronaviruses: MHV-a59 and MHV-JHM. Virology. 1981 Oct 15;114(1):39–51. doi: 10.1016/0042-6822(81)90250-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Makino S., Fleming J. O., Keck J. G., Stohlman S. A., Lai M. M. RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6567–6571. doi: 10.1073/pnas.84.18.6567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Makino S., Fujioka N., Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol. 1985 May;54(2):329–336. doi: 10.1128/jvi.54.2.329-336.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Makino S., Joo M., Makino J. K. A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J Virol. 1991 Nov;65(11):6031–6041. doi: 10.1128/jvi.65.11.6031-6041.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Makino S., Keck J. G., Stohlman S. A., Lai M. M. High-frequency RNA recombination of murine coronaviruses. J Virol. 1986 Mar;57(3):729–737. doi: 10.1128/jvi.57.3.729-737.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Makino S., Lai M. M. High-frequency leader sequence switching during coronavirus defective interfering RNA replication. J Virol. 1989 Dec;63(12):5285–5292. doi: 10.1128/jvi.63.12.5285-5292.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Makino S., Shieh C. K., Keck J. G., Lai M. M. Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs. Virology. 1988 Mar;163(1):104–111. doi: 10.1016/0042-6822(88)90237-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Makino S., Shieh C. K., Soe L. H., Baker S. C., Lai M. M. Primary structure and translation of a defective interfering RNA of murine coronavirus. Virology. 1988 Oct;166(2):550–560. doi: 10.1016/0042-6822(88)90526-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Makino S., Taguchi F., Fujiwara K. Defective interfering particles of mouse hepatitis virus. Virology. 1984 Feb;133(1):9–17. doi: 10.1016/0042-6822(84)90420-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Makino S., Taguchi F., Hirano N., Fujiwara K. Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain. Virology. 1984 Nov;139(1):138–151. doi: 10.1016/0042-6822(84)90335-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Makino S., Yokomori K., Lai M. M. Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal. J Virol. 1990 Dec;64(12):6045–6053. doi: 10.1128/jvi.64.12.6045-6053.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mann R., Baltimore D. Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. J Virol. 1985 May;54(2):401–407. doi: 10.1128/jvi.54.2.401-407.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  37. Pugatsch T., Stacey D. W. Identification of a sequence likely to be required for avian retroviral packaging. Virology. 1983 Jul 30;128(2):505–511. doi: 10.1016/0042-6822(83)90279-9. [DOI] [PubMed] [Google Scholar]
  38. Sawicki S. G., Sawicki D. L. Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis. J Virol. 1986 Jan;57(1):328–334. doi: 10.1128/jvi.57.1.328-334.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sethna P. B., Hofmann M. A., Brian D. A. Minus-strand copies of replicating coronavirus mRNAs contain antileaders. J Virol. 1991 Jan;65(1):320–325. doi: 10.1128/jvi.65.1.320-325.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sorge J., Ricci W., Hughes S. H. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol. 1983 Dec;48(3):667–675. doi: 10.1128/jvi.48.3.667-675.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stohlman S. A., Baric R. S., Nelson G. N., Soe L. H., Welter L. M., Deans R. J. Specific interaction between coronavirus leader RNA and nucleocapsid protein. J Virol. 1988 Nov;62(11):4288–4295. doi: 10.1128/jvi.62.11.4288-4295.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stohlman S. A., Lai M. M. Phosphoproteins of murine hepatitis viruses. J Virol. 1979 Nov;32(2):672–675. doi: 10.1128/jvi.32.2.672-675.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sturman L. S., Holmes K. V., Behnke J. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J Virol. 1980 Jan;33(1):449–462. doi: 10.1128/jvi.33.1.449-462.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wei N., Heaton L. A., Morris T. J., Harrison S. C. Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA. J Mol Biol. 1990 Jul 5;214(1):85–95. doi: 10.1016/0022-2836(90)90148-F. [DOI] [PubMed] [Google Scholar]
  45. Weiss B., Nitschko H., Ghattas I., Wright R., Schlesinger S. Evidence for specificity in the encapsidation of Sindbis virus RNAs. J Virol. 1989 Dec;63(12):5310–5318. doi: 10.1128/jvi.63.12.5310-5318.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. van der Most R. G., Bredenbeek P. J., Spaan W. J. A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol. 1991 Jun;65(6):3219–3226. doi: 10.1128/jvi.65.6.3219-3226.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES