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. 1994 Sep 15;13(18):4195–4203. doi: 10.1002/j.1460-2075.1994.tb06739.x

Infectious rabies viruses from cloned cDNA.

M J Schnell 1, T Mebatsion 1, K K Conzelmann 1
PMCID: PMC395346  PMID: 7925265

Abstract

The generation of infectious rabies virus (RV), a non-segmented negative-stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full-length RV antigenome-like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non-translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non-essential genetic material might be present in the genomes of non-segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure--potentially applicable also for other negative-stranded viruses--greatly facilitates the investigation of RV genetics, virus-host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

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Selected References

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

  1. Ball L. A. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors. J Virol. 1992 Apr;66(4):2335–2345. doi: 10.1128/jvi.66.4.2335-2345.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer J. C., Haenni A. L. Infectious transcripts and cDNA clones of RNA viruses. Virology. 1994 Feb;198(2):415–426. doi: 10.1006/viro.1994.1053. [DOI] [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., Cox J. H., Schneider L. G., Thiel H. J. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology. 1990 Apr;175(2):485–499. doi: 10.1016/0042-6822(90)90433-r. [DOI] [PubMed] [Google Scholar]
  8. Conzelmann K. K., Cox J. H., Thiel H. J. An L (polymerase)-deficient rabies virus defective interfering particle RNA is replicated and transcribed by heterologous helper virus L proteins. Virology. 1991 Oct;184(2):655–663. doi: 10.1016/0042-6822(91)90435-e. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Coslett G. D., Holloway B. P., Obijeski J. F. The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species. J Gen Virol. 1980 Jul;49(1):161–180. doi: 10.1099/0022-1317-49-1-161. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Dimock K., Collins P. L. Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3. J Virol. 1993 May;67(5):2772–2778. doi: 10.1128/jvi.67.5.2772-2778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Enami M., Luytjes W., Krystal M., Palese P. Introduction of site-specific mutations into the genome of influenza virus. Proc Natl Acad Sci U S A. 1990 May;87(10):3802–3805. doi: 10.1073/pnas.87.10.3802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flamand A., Raux H., Gaudin Y., Ruigrok R. W. Mechanisms of rabies virus neutralization. Virology. 1993 May;194(1):302–313. doi: 10.1006/viro.1993.1261. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kurath G., Ahern K. G., Pearson G. D., Leong J. C. Molecular cloning of the six mRNA species of infectious hematopoietic necrosis virus, a fish rhabdovirus, and gene order determination by R-loop mapping. J Virol. 1985 Feb;53(2):469–476. doi: 10.1128/jvi.53.2.469-476.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kurilla M. G., Cabradilla C. D., Holloway B. P., Keene J. D. Nucleotide sequence and host La protein interactions of rabies virus leader RNA. J Virol. 1984 Jun;50(3):773–778. doi: 10.1128/jvi.50.3.773-778.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Pattnaik A. K., Wertz G. W. Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1379–1383. doi: 10.1073/pnas.88.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pattnaik A. K., Wertz G. W. Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. J Virol. 1990 Jun;64(6):2948–2957. doi: 10.1128/jvi.64.6.2948-2957.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Perrotta A. T., Been M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature. 1991 Apr 4;350(6317):434–436. doi: 10.1038/350434a0. [DOI] [PubMed] [Google Scholar]
  24. Schubert M., Harmison G. G., Meier E. Primary structure of the vesicular stomatitis virus polymerase (L) gene: evidence for a high frequency of mutations. J Virol. 1984 Aug;51(2):505–514. doi: 10.1128/jvi.51.2.505-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tordo N., Poch O., Ermine A., Keith G., Rougeon F. Completion of the rabies virus genome sequence determination: highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses. Virology. 1988 Aug;165(2):565–576. doi: 10.1016/0042-6822(88)90600-9. [DOI] [PubMed] [Google Scholar]
  26. Tordo N., Poch O., Ermine A., Keith G., Rougeon F. Walking along the rabies genome: is the large G-L intergenic region a remnant gene? Proc Natl Acad Sci U S A. 1986 Jun;83(11):3914–3918. doi: 10.1073/pnas.83.11.3914. [DOI] [PMC free article] [PubMed] [Google Scholar]

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