Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron

ABSTRACT The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.

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Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis

Journal of Virology ◽

10.1128/jvi.77.9.5073-5083.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5073-5083 ◽

Cited By ~ 66

Author(s):

W. L. William Chang ◽

Peter A. Barry

Keyword(s):

Bacterial Artificial Chromosome ◽

Viral Genome ◽

Mammalian Cells ◽

Expression Profiles ◽

Genomic Structure ◽

Genome Structure ◽

Artificial Chromosome ◽

Full Length ◽

Iterative Sequence ◽

Wild Type

ABSTRACT Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were recovered from rhesus fibroblasts transfected with pRhCMV/BAC-EGFP. However, peak virus yields of cells infected with reconstituted progeny were 10-fold lower than wild-type RhCMV, suggesting that inclusion of the 9-kb BAC sequence impeded viral replication. Accordingly, pRhCMV/BAC-EGFP was further modified to enable efficient excision of the BAC vector from the viral genome after transfection into mammalian cells. Allelic exchange was performed in bacteria to substitute the cre recombinase gene for egfp. Transfection of rhesus fibroblasts with pRhCMV/BAC-Cre resulted in a pure progeny population lacking the vector backbone without the need of further manipulation. The genomic structure of the BAC-reconstituted virus, RhCMV-loxP(r), was identical to that of wild-type RhCMV except for the residual loxP site. The presence of the loxP sequence did not alter the expression profiles of neighboring open reading frames. In addition, RhCMV-loxP(r) replicated with wild-type kinetics both in tissue culture and seronegative immunocompetent macaques. Restriction analysis of the viral genome present within individual BAC clones and virions revealed that (i) RhCMV exhibits a simple genome structure and that (ii) there is a variable number of a 750-bp iterative sequence present at the S terminus.

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