Figure 3. GrB cleaves the essential HSV-1 IE protein ICP4.

(A&B) B6WT3 fibroblasts were infected with recombinant HSV-1 expressing EGFP from the ICP0 promoter for 1 hr, washed, and exposed to no, WT, Pfn^−/−, or GrB^−/− gB-CD8 for 5 hrs. (A) Histograms from flow analysis of recovered cells show total fibroblasts (CD8⁻) recovered from cultures (top left) and percent infected (EGFP⁺; top right). (B) Lysates of recovered cells were subjected to Western blot for ICP4 (Lane 1: Noninfected; Lanes 2–5: Infected with No, WT, Pfn^−/−, or GrB^−/− gB-CD8, respectively). Bar graphs show optical density (O.D.) readings that were not adjusted (middle) or adjusted for the number of infected fibroblasts recovered from each culture (right). (C) Lysates from 293T cells that were either non-transfected (lane 1) or transfected with an ICP4-expressing plasmid (lanes 2–5) and exposed to different concentrations of recombinant GrB (lanes 1–5: 0, 0, 25, 50, 100 nM GrB, respectively) for 1 hr at 37° C. Lysates from 293T cells transfected with an ICP4-expressing plasmid (D), lysates from B6WT3 cells infected with HSV-1 (E), or ICP4 immunoprecipitated from HSV-1-infected fibroblasts (F) were exposed to 0 (lanes 1–3, 5, 7) or 100 nM GrB (lanes 4, 6, 8) for varying times at 37° C (lanes 1–2: 0 hr; 3–4: 1 hr; 5–6: 2 hrs; 7–8: 3 hrs). Lane 1 contains non-transfected 293T cell lysate (D) or noninfected fibroblast lysate (E).