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Journal of Virology, February 1999, p. 1535-1545, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Replication and Packaging of Transmissible Gastroenteritis Coronavirus-Derived Synthetic Minigenomes

Ander Izeta,1 Cristian Smerdou,1 Sara Alonso,1 Zoltan Penzes,1 Ana Mendez,1 Juan Plana-Durán,2 and Luis Enjuanes1,*

Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma, Canto Blanco, 28049 Madrid,1 and Fort Dodge Veterinaria, Vall de Bianya, 17813 Girona,2 Spain

Received 6 August 1998/Accepted 9 November 1998

The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes, Virology 217: 495-507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated in trans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene beta -glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 µg/106 cells of GUS.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas (CSIC), Campus Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. Phone and Fax: 34-91-585 4555. E-mail: L.Enjuanes{at}cnb.uam.es.

Journal of Virology, February 1999, p. 1535-1545, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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