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Cloning Of A Transmissible Gastroenteritis Coronavirus Full-Length cDNA

  • Jose M. Gonzalez
  • Fernando Almazan
  • Zoltan Penzes
  • Enrique Calvo
  • Luis Enjuanes
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 494)

Abstract

To understand gene function and expression in coronaviruses it would be of interest to obtain a cDNA encoding a full-length infectious RNA. This has not yet been possible due to the large size of the Coronavirus genome and the instability of plasmids carrying Coronavirus replicase sequences. In this report we describe the construction of a transmissible gastroenteritis virus (TGEV) full-length cDNA. For this purpose, we started from a cDNA encoding the defective RNA DI-C, that was stably and efficiently replicated by the helper virus (Izeta et al., 1999). During the completion of the cDNA an ORF 1a fragment that was toxic to the bacteria was identified. Advantage of this finding was taken by reintroducing the toxic fragment into the viral cDNA in the last cloning step. To enhance the stability of the viral sequence, the cDNA was cloned as a bacterial artificial chromosome (BAC), a system that has been useful to stably clone large DNA from a variety of complex genomic sources into bacteria. The cytomegalovirus (CMV) immediate-early promoter was placed upstream of the cDNA to make use of a two-step amplification system that couples RNA pol II-driven transcription in the nucleus with the amplification by the viral replicase in the cytoplasm.

Keywords

Bacterial Artificial Chromosome Hepatitis Delta Virus Helper Virus Transmissible Gastroenteritis Virus Viral Replicase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Almazán, F., González, J. M., Pénzes, Z., Izeta, A., Calvo, E., Plana-Durán, J. and Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad Sci. USA 97, 5516–5521.PubMedCrossRefGoogle Scholar
  2. González, J. M, Almazán, F., Pénzes, Z., Calvo, E. and Enjuanes, L. (2000). Construction of a stable transmissible gastroenteritis Coronavirus full-length cDNA. Submitted.Google Scholar
  3. Izeta, A., Smerdou, C., Alonso, S., Pénzes, Z., Méndez, A., Plana-Durán, J. y Enjuanes, L. (1999). Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J. Virol. 73, 1535–1545.PubMedGoogle Scholar
  4. Sánchez, C. M., Izeta, A., Sánchez-Morgado, J. M., Alonso, S., Sola, I., Balasch, M, Plana-Durán, J. y Enjuanes, L. (1999). Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis Coronavirus is a determinant of its enteric tropism and virulence. J. Virol. 73, 7607–7618.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Jose M. Gonzalez
    • 1
  • Fernando Almazan
    • 1
  • Zoltan Penzes
    • 1
  • Enrique Calvo
    • 1
  • Luis Enjuanes
    • 1
  1. 1.Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, CampusUniversidad AutónomaCantoblancoMadridSpain

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