FIG 7.

Effects of K/SIM mutations on the degradation of PML II and PML II-associated proteins. (A) Half-life assays for wild-type PML II and PML II K/SIMmt in RHG101-infected cells as described above. (B) K/SIM mutations block PML II ubiquitination in RHG101-infected cells. HEp-2 TetOn cells expressing wild-type PML II or PML II K/SIMmt were induced, infected, and harvested for Ni-NTA pulldown under denaturing conditions. PML II precipitates were then probed with anti-Ub (upper) and anti-myc (lower) antibodies. (C and D) Effects of PML II K/SIM mutant on the degradation of Sp100 (C) and PML isoforms III-VII (D) in HEp-2 TetOn cells infected by HSV-1(F) at 5 PFU/cell. (E) HEp-2 TetOn cells expressing wild-type PML II (left) or PML II K/SIMmt (right) were infected by HSV-1(F) at 0.1 PFU/cell in the presence or absence of 1 μg/ml of doxycycline. At 2, 6, 10, 24, and 48 hpi, triplicate infected cells were harvested and titrated on U2OS cells. Titers with the standard error were plotted by Microsoft Excel.