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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.

SUBMITTER: Sola I

PROVIDER: S-EPMC150661 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.

Sola Isabel I Alonso Sara S Zúñiga Sonia S Balasch Mónica M Plana-Durán Juan J Enjuanes Luis L

Journal of virology 20030401 7

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 ...[more]

PMID: 12634392

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Project description:We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600-10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected approximately 36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident approximately 18 h postinfection. Leader-containing GFP transcripts containing the ORF 3B and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines.

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Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.

Publications

Engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity.

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