Susceptibility to cytotoxic T lymphocyte-induced apoptosis is a function of the proliferative status of the target

Many DNA viruses induce quiescent cells to progress into a late G1/S-like phase to obtain cell-regulated components of the cell's DNA synthetic machinery (18) (19) (20) (21) . The mechanism of induction into the DNA synthetic state by DNA viruses differ, but the effects are the same, i.e., activation of the competence mechanism for cell cycle progression and the production of enzymes needed for DNA synthesis even if the DNA synthesized is preferentially viral (18) (19) (20) (21) . Numerous herpes viruses induce the activation of quiescent cells into the DNA synthetic phase and upregulate the activity of DNA topoisomerases I and/or II, which the viruses require for replication (20, 21) . When quiescent 3T3-A31 cells were infected with HSV-1, these cells became more susceptible to CTLinduced DNA fragmentation than 3T3-A31 ceils in log phase growth and markedly more susceptible than their quiescent counterparts (Fig. 5, A and B) . The level of CTL-induced membrane lysis was not significantly different between the three groups (Fig. 5 A) . A previous study (22) suggested that HSV-l-infected cells were highly sensitive to killing by TNF-ct, but inclusion of anti-TNF antisera in our assays did not block the DNA fragmentation nor the membrane lysis of the target cells. Pulse-labeling studies of the HSV-infected quiescent vs. uninfected quiescent targets showed, as expected, that DNA synthesis was initiated in the HSV-infected quiescent cells and remained high throughout the time of assay (data not shown). This DNA synthesis was probably viral, as HSV-1 infection inhibits cellular DNA synthesis while stimulating cellular DNA synthetic enzymes (23) . Thus, HSV-1 infection of the target cell induced a state of competence for extreme susceptibility to CTL-induced apoptosis. This virus-induced competence was not due to serum con- Figure 5 . Infection of quiescent 3T3-A31 cells with HSV-1 renders these targets, otherwise refractory to apoptosis, highly susceptible to CTbinduced apoptosis. (A) Lysis of targets either in log phase growth, quiescent, or quiescent+HSVol infection. (B) Sohbilized lZq-DNA rdease from targets as in A. Quiescent targets and targets in log phase growth were prepared as in Hg. 2.5 d before use (day -5), 3T3-A31 cells were subcultured into T150 era 2 flasks to obtain ~75% confluency. On day -4, they were labeled with 1zsI-IUDR at 1/~Ci/ml in a total volume of 10 ml for 18 h. On day -3 the medium was removed, fresh RPMI + 5% CPSK-2 was added, and the cells were allowed to quiesce for 72 h. For HSV-1 inf~ion, quiescent cells were infected with 5 ml of HSV-1 with a titer of 107 PFU/ml for 2 h at 37~ with rocking every 15 rain. Multiplicity of infection, determined after infection by counting the number of cells in the infected flask, was 20. Target cells were processed and assayed as in Fig. 1 in AIM-V media without or with 10% FBS as indicated. Cytotoxicity assays were performed as described (3) . Assay time for the data presented was 14 h. Time = 0 when the LCMV-CTLs were added to the target cells. Con A was at a final concentration of 5/~g/rrd. Total time from the HSV-1 infection of quiescent targets to harvest of the assay was 20 h. tamination of the virus inoculum, because FBS added back to quiescent targets or to HSV-l-infected quiescent targets, to a final concentration of 10%, did not significantly increase their susceptibility to CTL-induced apoptosis (Fig. 2, A and B) . The possibility that HSV-1 directly induced fragm