FIG 7.

Production of infectious viruses is compromised by ORF45 mutation or deletion. (A) Growth curves for recombinant KSHV. Stable iSLK cells carrying wild-type BAC16, F66A, Stop45, or revertant viral genomes were induced with doxycycline and sodium butyrate. Supernatants of induced medium were collected at different time points as indicated. Viral genome copies in the medium were determined by real-time PCR and were plotted against the time since induction. (B) iSLK.BAC16 cells were pretreated for 1 h with the indicated kinase inhibitor, followed by induction of lytic reactivation in the presence of inhibitor. Viral genome copies were measured at 96 hpi as described for panel A. (C) Extracellular virions were collected and concentrated from the media of induced iSLK.BAC16 wild-type and ORF45 mutant cell lines as outlined in Materials and Methods. After normalizing by viral genome copy number, virion composition was analyzed by Western blotting with the indicated antibodies. (D to F) 293 cells were infected with 2-fold serial dilutions of wild-type and ORF45 mutant KSHV. Twenty-four h postinduction, GFP-positive cells were counted by fluorescence-activated cell sorting (FACS). The percentages of GFP-positive cells (D) and GFP median values (E) were calculated for each dilution. (F) Representative histograms of the FACS data.