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

<jats:title>ABSTRACT</jats:title> <jats:p> 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 <jats:italic>cre</jats:italic> recombinase gene for <jats:italic>egfp</jats:italic> . 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- <jats:italic>loxP</jats:italic> (r), was identical to that of wild-type RhCMV except for the residual <jats:italic>loxP</jats:italic> site. The presence of the <jats:italic>loxP</jats:italic> sequence did not alter the expression profiles of neighboring open reading frames. In addition, RhCMV- <jats:italic>loxP</jats:italic> (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. </jats:p>