FIG. 5.

Antiapoptotic effects of gJ. (A) Activated caspase 3 was measured by flow cytometry in RAW264.7 cells either mock infected or infected for 6 h with RAS116 (Us5 deletion), RAS137 (repair of RAS116), or the wild-type strain F. Shown are means ± standard deviations (SD) of triplicate determinations from one representative of four independent experiments. *, P < 0.01 versus mock infection; **, P < 0.001 versus mock infection. (B) Inhibition of fas-induced caspase 3 activation in EcR-293 cells expressing gJ. Activated caspase 3 was measured by flow cytometry in control cells (EcR-293), control cells under PonA induction (EcR-293/Pon A), EcR-293 cells transfected with Us5 but without PonA induction (EcR-293-gJ), or EcR-293 cells transfected with Us5 and expressing gJ under PonA induction (EcR-293-gJ/Pon A) either left untreated (white bars; no tx) or treated with anti-fas (100 ng/ml) for 16 h (black bars; anti-fas). PonA (5 μM) was used to induce gJ expression. The values are the averages of eight or nine independent experiments (mean ± SD). *, P < 0.001. (C and D) Protection of Jurkat cells from HSV-1-induced apoptosis. (C) The percentage of apoptotic cells was evaluated as described in Materials and Methods for Jurkat cells mock infected or infected with ICP27 deletion virus (vBSΔ27) and wild-type parental virus (KOS1.1) at an MOI of 5 for 24 h when gJ expression was induced (black bars; Us5 ON) or repressed (white bars; Us5 OFF). *, P < 0.05. (D) Immunoblotting for caspase 3 and 9 cleavage in cell lysates obtained after infection at an MOI of 5 for 24 h of Jurkat cells expressing gJ (Us5 ON) or Jurkat cells with gJ expression repressed (Us5 OFF). The anti-caspase 3 or 9 antibodies detect full-length caspase 3 or 9, and the absence of signal reflects the cleavage of those proteins. Actin was used as a loading control.