FIG 4.

Viral gene expression in HAP1 KO cells. (A to D) Western blotting. Control, RNase L KO, PKR KO, and DKO HAP1 cells were infected with 5 PFU/cell of purified vD10rev, vD9muD10mu, or vΔE3L and harvested at the indicated times (shown in hours). The lysates were analyzed by Western blotting using antibodies to VACV (A), the E3 early protein (B), the D13 intermediate protein (C), and the A3 late protein (D). Antibody to actin served as a loading control (not shown). One set of cells was infected in the presence of AraC to inhibit viral DNA replication and confirm the stage of expression of the viral proteins. (E) Northern blotting. HAP1 control, RNase L KO, PKR KO, and DKO cells were mock infected or infected with 5 PFU/cell of purified vD10rev, vD9muD10mu, or vΔE3L virus. At 13 h after infection, the cells were harvested, and the total RNAs were isolated and resolved on glyoxal gels. rRNAs were stained with ethidium bromide and detected by UV fluorescence; reverse images are shown. The RNAs were transferred to a nylon membrane, incubated with the digoxigenin-labeled probes to the viral early C11 mRNA, viral late F17 mRNA, and cellular GAPDH mRNA, detected with alkaline phosphatase-conjugated antibody to digoxigenin, and visualized with the chemiluminescence substrate and X-ray film. p.i., postinfection; Ab, antibody.