title: Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.
abstract: 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 micro g/10(6) 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 10(2)-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.
authors.affiliation: Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Sola
authors.forename: Sara
authors.initials: S
authors.lastname: Alonso
authors.forename: Sonia
authors.initials: S
authors.lastname: Zúñiga
authors.forename: Mónica
authors.initials: M
authors.lastname: Balasch
authors.forename: Juan
authors.initials: J
authors.lastname: Plana-Durán
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003126:Colostrum / Q000276:immunology; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D005761:Gastroenteritis, Transmissible, of Swine / Q000276:immunology / Q000517:prevention & control; D005818:Genetic Engineering; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D034421:Sus scrofa; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*
chemical_list: D004279:DNA, Viral; D008164:Luminescent Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins