Progress towards the construction of a transmissible gastroenteritis coronavirus self-replicating RNA using a two-layer expression system.
Three transmissible gastroenteritis coronavirus (TGEV) defective interfering RNAs of 21, 10.6 and 9.7 kb (DI-A, DI-B and DI-C, respectively) were isolated. Dilution experiments showed that the largest DI RNA, DI-A, is a self-replicating RNA (replicon), and thus codes for a functional RNA polymerase and all the necessary replication signals. In order to engineer a cDNA encoding the RNA replicon a strategy based on the cloning of DI-C cDNA, followed by the insertion of the sequences required to complete the DI-A sequence has been developed. A cDNA complementary to DI-C RNA was cloned under the control of the CMV promoter (pDI-C-CMV) and rescued with a helper virus. In the ORF 1a of polymerase gene pDI-C-CMV contained a 10 kb deletion and in ORF 1b a 1.1 kb deletion. The consensus sequence corresponding to the deleted regions was cloned, and the deletions in pDI-C-CMV were replaced to yield a complete cDNA clone of DI-A, pDI-A-21-CMV, containing a full-length TGEV polymerase, driven by a CMV promoter. Expression of a functional TGEV polymerase is being investigated.
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Madrid, Spain.
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Pénzes
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González
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Izeta
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Muntión
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Enjuanes
D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D016679:Genome, Viral; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000235:genetics*
D012367:RNA, Viral; D012319:RNA Polymerase II