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. 1994 Dec;68(12):8413–8417. doi: 10.1128/jvi.68.12.8413-8417.1994

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

W Willenbrink 1, W J Neubert 1
PMCID: PMC237314  PMID: 7966637

Abstract

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

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Selected References

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  1. Brunette E., Stribling R., Debs R. Lipofection does not require the removal of serum. Nucleic Acids Res. 1992 Mar 11;20(5):1151–1151. doi: 10.1093/nar/20.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buchholz C. J., Spehner D., Drillien R., Neubert W. J., Homann H. E. The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. J Virol. 1993 Oct;67(10):5803–5812. doi: 10.1128/jvi.67.10.5803-5812.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Calain P., Curran J., Kolakofsky D., Roux L. Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology. 1992 Nov;191(1):62–71. doi: 10.1016/0042-6822(92)90166-m. [DOI] [PubMed] [Google Scholar]
  4. Calain P., Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 1993 Aug;67(8):4822–4830. doi: 10.1128/jvi.67.8.4822-4830.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins P. L., Mink M. A., Hill M. G., 3rd, Camargo E., Grosfeld H., Stec D. S. Rescue of a 7502-nucleotide (49.3% of full-length) synthetic analog of respiratory syncytial virus genomic RNA. Virology. 1993 Jul;195(1):252–256. doi: 10.1006/viro.1993.1368. [DOI] [PubMed] [Google Scholar]
  6. Collins P. L., Mink M. A., Stec D. S. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9663–9667. doi: 10.1073/pnas.88.21.9663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Conzelmann K. K., Schnell M. Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. J Virol. 1994 Feb;68(2):713–719. doi: 10.1128/jvi.68.2.713-719.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curran J., Boeck R., Kolakofsky D. The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing. EMBO J. 1991 Oct;10(10):3079–3085. doi: 10.1002/j.1460-2075.1991.tb07860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Curran J., Marq J. B., Kolakofsky D. The Sendai virus nonstructural C proteins specifically inhibit viral mRNA synthesis. Virology. 1992 Aug;189(2):647–656. doi: 10.1016/0042-6822(92)90588-g. [DOI] [PubMed] [Google Scholar]
  10. De B. P., Banerjee A. K. Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3. Virology. 1993 Sep;196(1):344–348. doi: 10.1006/viro.1993.1486. [DOI] [PubMed] [Google Scholar]
  11. Einberger H., Mertz R., Hofschneider P. H., Neubert W. J. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro. J Virol. 1990 Sep;64(9):4274–4280. doi: 10.1128/jvi.64.9.4274-4280.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Felgner P. L., Ringold G. M. Cationic liposome-mediated transfection. Nature. 1989 Jan 26;337(6205):387–388. doi: 10.1038/337387a0. [DOI] [PubMed] [Google Scholar]
  14. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giesecke H., Obermaier B., Domdey H., Neubert W. J. Rapid sequencing of the Sendai virus 6.8 kb large (L) gene through primer walking with an automated DNA sequencer. J Virol Methods. 1992 Jul;38(1):47–60. doi: 10.1016/0166-0934(92)90168-d. [DOI] [PubMed] [Google Scholar]
  16. Gorman C. M., Gies D., McCray G., Huang M. The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology. 1989 Aug;171(2):377–385. doi: 10.1016/0042-6822(89)90605-3. [DOI] [PubMed] [Google Scholar]
  17. Gotoh H., Shioda T., Sakai Y., Mizumoto K., Shibuta H. Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes. Virology. 1989 Aug;171(2):434–443. doi: 10.1016/0042-6822(89)90612-0. [DOI] [PubMed] [Google Scholar]
  18. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  19. Hidaka Y., Kanda T., Iwasaki K., Nomoto A., Shioda T., Shibuta H. Nucleotide sequence of a Sendai virus genome region covering the entire M gene and the 3' proximal 1013 nucleotides of the F gene. Nucleic Acids Res. 1984 Nov 12;12(21):7965–7973. doi: 10.1093/nar/12.21.7965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoeijmakers J. H., Odijk H., Westerveld A. Differences between rodent and human cell lines in the amount of integrated DNA after transfection. Exp Cell Res. 1987 Mar;169(1):111–119. doi: 10.1016/0014-4827(87)90230-8. [DOI] [PubMed] [Google Scholar]
  21. Homann H. E., Willenbrink W., Buchholz C. J., Neubert W. J. Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein. J Virol. 1991 Mar;65(3):1304–1309. doi: 10.1128/jvi.65.3.1304-1309.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horikami S. M., Curran J., Kolakofsky D., Moyer S. A. Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. J Virol. 1992 Aug;66(8):4901–4908. doi: 10.1128/jvi.66.8.4901-4908.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huber M., Cattaneo R., Spielhofer P., Orvell C., Norrby E., Messerli M., Perriard J. C., Billeter M. A. Measles virus phosphoprotein retains the nucleocapsid protein in the cytoplasm. Virology. 1991 Nov;185(1):299–308. doi: 10.1016/0042-6822(91)90777-9. [DOI] [PubMed] [Google Scholar]
  24. Lamb R. A., Choppin P. W. The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of polypeptides. Virology. 1977 Sep;81(2):382–397. doi: 10.1016/0042-6822(77)90154-4. [DOI] [PubMed] [Google Scholar]
  25. Leppert M., Kort L., Kolakofsky D. Further characterization of Sendai virus DI-RNAs: a model for their generation. Cell. 1977 Oct;12(2):539–552. doi: 10.1016/0092-8674(77)90130-1. [DOI] [PubMed] [Google Scholar]
  26. Luytjes W., Krystal M., Enami M., Parvin J. D., Palese P. Amplification, expression, and packaging of foreign gene by influenza virus. Cell. 1989 Dec 22;59(6):1107–1113. doi: 10.1016/0092-8674(89)90766-6. [DOI] [PubMed] [Google Scholar]
  27. Mayne L. V., Jones T., Dean S. W., Harcourt S. A., Lowe J. E., Priestley A., Steingrimsdottir H., Sykes H., Green M. H., Lehmann A. R. SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA. Gene. 1988 Jun 15;66(1):65–76. doi: 10.1016/0378-1119(88)90225-9. [DOI] [PubMed] [Google Scholar]
  28. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mottet G., Curran J., Roux L. Intracellular stability of nonreplicating paramyxovirus nucleocapsids. Virology. 1990 May;176(1):1–7. doi: 10.1016/0042-6822(90)90223-e. [DOI] [PubMed] [Google Scholar]
  30. Neubert W. J. Cloning and sequencing of the polymerase gene (P) of Sendai virus (strain Fushimi). Nucleic Acids Res. 1989 Dec 11;17(23):10101–10101. doi: 10.1093/nar/17.23.10101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Neubert W. J., Eckerskorn C., Homann H. E. Sendai virus NP gene codes for a 524 amino acid NP protein. Virus Genes. 1991 Jan;5(1):25–32. doi: 10.1007/BF00571728. [DOI] [PubMed] [Google Scholar]
  32. Park K. H., Huang T., Correia F. F., Krystal M. Rescue of a foreign gene by Sendai virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5537–5541. doi: 10.1073/pnas.88.13.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Park K. H., Krystal M. In vivo model for pseudo-templated transcription in Sendai virus. J Virol. 1992 Dec;66(12):7033–7039. doi: 10.1128/jvi.66.12.7033-7039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pattnaik A. K., Ball L. A., LeGrone A. W., Wertz G. W. Infectious defective interfering particles of VSV from transcripts of a cDNA clone. Cell. 1992 Jun 12;69(6):1011–1020. doi: 10.1016/0092-8674(92)90619-n. [DOI] [PubMed] [Google Scholar]
  35. Portner A., Murti K. G. Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling. Virology. 1986 Apr 30;150(2):469–478. doi: 10.1016/0042-6822(86)90311-9. [DOI] [PubMed] [Google Scholar]
  36. Portner A., Murti K. G., Morgan E. M., Kingsbury D. W. Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy. Virology. 1988 Mar;163(1):236–239. doi: 10.1016/0042-6822(88)90257-7. [DOI] [PubMed] [Google Scholar]
  37. Shioda T., Hidaka Y., Kanda T., Shibuta H., Nomoto A., Iwasaki K. Sequence of 3,687 nucleotides from the 3' end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P and C proteins. Nucleic Acids Res. 1983 Nov 11;11(21):7317–7330. doi: 10.1093/nar/11.21.7317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shioda T., Iwasaki K., Shibuta H. Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins. Nucleic Acids Res. 1986 Feb 25;14(4):1545–1563. doi: 10.1093/nar/14.4.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spehner D., Kirn A., Drillien R. Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein. J Virol. 1991 Nov;65(11):6296–6300. doi: 10.1128/jvi.65.11.6296-6300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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