FIG. 1.
Genome structure of recombinant MCMVs. The HindIII cleavage map of the MCMV (Smith strain) genome is shown at the top. Deletion and insertion of the m157 gene, respectively, were achieved by homologous recombination between the parental MCMV BACs and a linear DNA fragment containing the desired sequence, a kanamycin resistance gene (Kn), and flanking homologies to the target site in the viral genome. The deletion genome pΔm157 was generated by homologous recombination of the linear DNA fragment and w.t. MCMV BAC pSM3fr. An independent Δm157 genome was constructed as follows. First, a 48-bp FRT site (grey box) was inserted into the intergenic region between genes m16 and m17, generating pSM3fr-FRT, to prove that insertion of sequences at this positions does not interfere with virus replication. In a second mutagenesis step, the m157 gene was deleted from pSM3fr-FRT, generating the second independent m157 deletion genome, pΔm157-FRT. Finally, the m157 gene, including its native promoter, was reinserted into pΔm157-FRT at the ectopic positions between genes m16 and m17, thereby removing the FRT site. The names of the MCMV BACs are indicated on the left. Recombinant viruses were reconstituted by transfection of the recombinant MCMV BACs into permissive MEFs. The names of the corresponding reconstituted viruses are given on the right.