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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
-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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