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Journal of Virology, April 2003, p. 4357-4369, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4357-4369.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Engineering the Transmissible Gastroenteritis Virus Genome as an Expression Vector Inducing Lactogenic Immunity

Isabel Sola,1 Sara Alonso,1 Sonia Zúñiga,1 Mónica Balasch,2 Juan Plana-Durán,2 and Luis Enjuanes1*

Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, Madrid,1 Fort-Dodge Veterinaria, Department of Research and Development, Girona, Spain2

Received 4 October 2002/ Accepted 7 January 2003

The genome of the coronavirus transmissible gastroenteritis virus (TGEV) has been engineered as an expression vector with an infectious cDNA. The vector led to the efficient (>40 µg/106 cells) and stable (>20 passages) expression of a heterologous gene (green fluorescent protein [GFP]), driven by the transcription-regulating sequences (TRS) of open reading frame (ORF) 3a inserted in the site previously occupied by the nonessential ORFs 3a and 3b. Expression levels driven by this TRS were higher than those of an expression cassette under the control of regulating sequences engineered with the N gene TRS. The recombinant TGEV including the GFP gene was still enteropathogenic, albeit with a 10- to 102-fold reduction in enteric tissue growth. Interestingly, a specific lactogenic immune response against the heterologous protein has been elicited in sows and their progeny. The engineering of an additional insertion site for the heterologous gene between viral genes N and 7 led to instability and to a new genetic organization of the 3' end of the recombinant viruses. As a consequence, a major species of subgenomic mRNA was generated from a TRS with the noncanonical core sequence 5'-CUAAAA-3'. Extension of the complementarity between the TRS and sequences at the 3' end of the viral leader was associated with transcriptional activation of noncanonical core sequences. The engineered vector led to expression levels as high as those of well-established vectors and seems very promising for the development of vaccines and, possibly, for gene therapy.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4555. Fax: 34 91 585 4915. E-mail: L.Enjuanes{at}cnb.uam.es.

Journal of Virology, April 2003, p. 4357-4369, Vol. 77, No. 7
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.7.4357-4369.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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