Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome.
Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E(+) packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Delta3abDeltaE) was successfully rescued in these cell lines. rTGEV-Delta3abDeltaE reached high titers (10(7) PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 10(5) PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Delta3abDeltaE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E(+) packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.
Ortego
Javier
J
Centro Nacional de Biotecnología, CSIC, Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Escors
David
D
Laude
Hubert
H
Enjuanes
Luis
L
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005822:Genetic Vectors; D012093:Replicon; D012845:Sindbis Virus / Q000235:genetics; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
D014759:Viral Envelope Proteins