Figure 3. v-bcl-2 mutants es- tablish latency normally but fail to reactivate efficiently during ex vivo culture. B6 mice were in- fected with 106 PFU of virus and peritoneal cells were harvested at 16 or 42 d after infection for quantitation of the frequency of latently infected cells (A) and the frequency of cells reactivating from latency (B). Data represent results of two to five separate ex- periments using two indepen- dent mutant isolates (mean SEM), with each experiment containing cells pooled from five mice. The difference in fre- quency of genome positive cells at day 16 and day 42 were not sta- tistically significant between the wt HV68 versus v-bcl-2.Stop1 and v-cyclin.MR versus v-bcl- 2.Stop2. At 16 d (A and B, left panels), the decreased frequen- cies of ex vivo reactivation of both v-bcl-2.Stop mutants com- pared with wt HV68 and v-cyclin.MR were statistically significant as follows: v-bcl-2.Stop1 versus wt HV68, P 0.006; v-bcl-2.Stop2 versus wt HV68, P 0.005; v-bcl- 2.Stop1 versus v-cyclin.MR, P 0.013; v-bcl-2.Stop2 versus v-cyclin.MR, P 0.010. At 42 d (A and B, right panels), the decreased frequencies of ex vivo reactivation of both v-bcl-2.Stop mutants compared with wt HV68 and v-cyclin.MR were statistically significant as follows: v-bcl-2.Stop1 versus wt HV68, P 0.004; v-bcl-2.Stop2 versus wt HV68, P 0.005; v-bcl-2.Stop1 versus v-cyclin.MR, P 0.006; v-bcl-2.Stop2 versus v-cyclin.MR, P 0.009. the viruses between hosts. Consistent with prior reports (18, 29), we did not detect persistent replication of either wt HV68 or v-bcl-2.Stop viruses in the spleen (data not shown) or peritoneal cells of normal mice 16 and 42 d after infection (Fig. 3 B, open symbols). However, wt HV68 persistently replicates in peritoneal cells of IFN- / mice for at least 6 wk after infection (unpublished data), provid- ing a model for determining whether the v-bcl-2 has a role in persistent replication. One scenario is that persistent rep- lication is due to reactivation from latency. Because HV68 v-cyclin mutants share with v-bcl-2 mutants (above) the phenotype of decreased reactivation from latency de- spite normal establishment of latency (18), we analyzed v-bcl-2 and v-cyclin mutants for their capacity to persistently replicate in IFN- / mice. Consistent with findings in immunocompetent B6 mice (Fig. 3), v-bcl-2.Stop1 and v-bcl-2.Stop2 showed 4–5-fold decreased frequency of reactivation from peritoneal cells of IFN- / mice compared with wt HV68 and v-cyclin.MR virus (Fig. 4, left panels). This decrease in ex vivo reactiva- tion was not due to a decrease in the number of cells carry- ing the viral genome because PECs from IFN- / mice infected with v-cyclin.MR, v-bcl-2.Stop, or v-cyclin.Stop mutant contained comparable frequency of genome posi- tive cells (Fig. 4 D). The v-cyclin is also required for efficient ex vivo reactivation from latency in cells from IFN- / mice (Fig. 4, A–C, left panels). Interestingly, neither v-bcl-2.Stop nor v-cyclin.Stop mutants showed significant persistent replication in IFN- / mice (Fig. 4, A–C, right panels). Persistent replica- 935 Gangappa et al. tion was detected for both wt HV68 and control virus v-cyclin.MR. These results demonstrated the requirement for both v-bcl-2 and v-cyclin genes for persistent replication in immunocompromised hosts. This is in striking contrast to the lack of a role for these genes in acute replication in either wt or IFN- / mice (Fig. 2). This demonstrates that different viral genes are required for acute replication and persistent replication, strongly arguing that acute and persistent -herpesvirus replication are genetically distinct processes. Requirement for v-bcl-2 and v-cyclin for Lethal Chronic Dis- ease but Not Lethal Acute Disease. The data presented so far argues for a specific role of v-bcl-2 and v-cyclin in chronic but not acute infection. If this distinction is physiologically important, these genes should be required for virulence in models of chronic disease but not for virulence during acute infection. Chronically infected IFN- / and IFN R / mice develop lethal persistent infection and se- vere large vessel vasculitis (32). The vasculitis is due to per- sistent replication in smooth muscle cells of the immuno- privileged media of the great elastic arteries (33). Consistent with the data above demonstrating a critical role for v-cyclin and v-bcl-2 in persistent replication in IFN- / mice, v-bcl-2 mutant and v-cyclin mutant viruses killed IFN R / mice less efficiently than wt virus during chronic infection (Fig. 5 A). Evaluation of pathology of arteritic lesions revealed significant change in incidence of lesions (v-cyclin.MR [14/19 74%] versus v-bcl-2.Stop [10/35 28%], P 0.0002; v-cyclin.MR versus v-cyclin.Stop [7/20 35%], P 0.001) but not in the severity of aortic lesions between