FIG 2.
Requirement of all four RNF4 SIMs when replacing ICP0 SIM362-364 in PML II degradation. (A) Experimental design of the PML II half-life assay. HEp-2 TetOn cells stably expressing PML II were seeded and induced by doxycycline for 24 h. Cells were then infected with recombinant viruses at 10 PFU/cell. The time of virus addition was marked as −2 h. The time of cycloheximide addition was marked as 0 h. (B to E) Effects of ICP0 central SIM on the PML II half-life. Half-life assays for PML II were conducted with indicated viruses and myc-tagged PML II and mCherry-tagged ICP0 at different time points after cycloheximide treatment were detected by Western blotting (B and D). PML II band density from three (C) or two (E) independent experiments was quantified by Image J and plotted with the standard error to show the PML II half-life in different infections. (F and G) HEp2 TetOn cells expressing PML II were infected by the indicated viruses at 0.1 PFU/cell to measure growth properties. Viral DNA fold increase from 2 hpi to 24 hpi was measured with qPCR targeting ICP27 and 18S rDNA for viral DNA and total cell DNA, respectively, and calculated by 2−ΔΔCt using 2 hpi as the reference (F), whereas viral titer increase from 2 hpi to 24 hpi was measured in U2OS cells (G).