FIG. 4.

Generation of a marker rescue mutant of ORF49^null. (A) Schematic diagram of construction of a marker rescue mutant of ORF49^null. Shown are the genomic organization of ORF49 with a transposon insertion and neighboring genes (pORF49^null) (top), a homologous region used to construct marker rescue mutant of ORF49^null (middle), and DNA fragments from EcoRI digestion of ORF49^null (bottom). The sizes of the diagrams are proportional to their actual genomic sizes. (B) Southern analysis of ORF49^null and ORF49^nullMR. The BAC DNAs of pMHV-68, pORF49^null, and pORF49^nullMR were digested with EcoRI and analyzed with a ³²P-labeled Southern probe specific to the ORF49-containing fragment as indicated in panel A. (C to E) BHK21 cells were transfected with the BAC DNAs (50 ng) of ORF49^null and ORF49^nullMR. MHV-68-cloned BAC DNA (pMHV-68; 50 ng) was also transfected as a control. The transfected cells were harvested at the indicated time points and analyzed for viral DNA replication (C), viral gene expressions (D), and virion production (E) as described in the legend to Fig. 3. Western analysis was performed with rabbit serum against MHV-68-infected rabbit cell lysates (α-MHV-68) and with α-tubulin as a control. The asterisk indicates nonspecific bands.