As v-bcl-2.Stop viruses were derived from the HV68 mutant v-cyclin.LacZ, we compared the mutant viruses to both wild-type HV68 (wt HV68) and v-cyclin.MR, a virus in which the v-cyclin.LacZ mutation has been res- cued using wt viral sequences (18). To definitively map the phenotypes we report to the v-bcl-2 gene, we character- ized two independently generated viral mutants containing each of two different mutations in the v-bcl-2 gene. The use of two independent isolates to map a phenotype to a herpesvirus gene is an accepted approach (35–37). No Role for v-bcl-2 in Acute Replication. We first tested whether the HV68 v-bcl-2 was required for viral replica- tion in cultured cells or in acutely infected wt B6 or immu- nocompromised IFN- / mice on the B6 background. As predicted from studies of an EBV v-bcl-2 mutant (17), HV68 v-bcl-2.Stop1 and v-bcl-2.Stop2 grew normally in cultured cells under both single step and multi-step growth conditions (Fig. 2, A and B). v-bcl-2 mutants also replicated as well as wt HV68 in the spleen and liver of wt B6 mice 4 or 9 d after infection as measured by plaque assay (Fig. 2 C, left panel). Similarly, growth of v-bcl-2 mutants during acute infection of IFN- / was indistinguishable from growth of wt HV68 (Fig. 2 C, right panel). These data show that v-bcl-2 is not essential for replication during acute infection in either wt B6 or immunocompromised IFN- / mice. We have previously demonstrated that the v-cyclin is not required for replication during acute infec- tion of wild type mice (18). We confirmed this finding and additionally showed that the v-cyclin, similar to the v-bcl-2, is not essential for replication in IFN- / mice (Fig. 2 C, right panel). While a 1.8-fold difference in titer between the wt HV68 and v-cyclin.MR at day 9 in B6 mice, and a 2.4-fold difference between v-cyclin.MR and v-cyclin. Stop at day 4 in IFN- / mice reached statistical differ- ence for spleen, the biological significance of such small differences in titer are unclear and do not influence our conclusions regarding v-bcl-2 mutants. v-bcl-2 Is Important for Ex Vivo Reactivation from Latency, but Not Establishment of Latency. Given the lack of a role for v-bcl-2 during acute infection, we turned our attention to parameters of chronic infection. We examined the role of v-bcl-2 in establishment of latency (stable carriage of the viral genome in a cell without active replication) and ex vivo reactivation from latency. By 16 d after infection, in- fectious HV68 is cleared from the spleen and peritoneal cells of wt B6 mice and latency is established (18, 28, 29, 32, 34). The HV68 v-bcl-2 is not required for establish- ment of latency since splenocytes (data not shown) and peritoneal cells harvested 16 or 42 d after infection of B6 mice with wt HV68, v-cyclin.MR, or v-bcl-2.Stop mu- tants contained equivalent frequencies of viral genome bearing cells (Fig. 3 A). Despite normal establishment of latency and absence of productive infection as shown by the disrupted samples in Fig. 3 B, v-bcl-2.Stop mutants re- activated 4–5-fold less efficiently than either wt HV68 or v-cyclin.MR virus in the peritoneal exudate cells (PECs) but not in the spleen (data not shown) both day 16 and day 42 after infection (Fig. 3 B). This viral phenotype is similar to v-cyclin mutants which also fail to efficiently reactivate from latency (18, 27), suggesting that regulation of both apoptosis and cell cycle is key to ex vivo reactivation from the latent state. The HV68 v-bcl-2 and v-cyclin Are Necessary for Persistent Replication of Virus in Immunocompromised Hosts. Persis- tent replication of -herpesviruses likely contributes to both disease in immunocompromised hosts and spread of 934 v-bcl-2 and v-cyclin Are Chronic Disease Herpesvirus Genes Figure 2. HV68 v-bcl-2 mutant viruses grow like wt HV68 in cultured cells and during acute infection of mice. NIH 3T12 cells were infected with 5.0 (A) or 0.05 (B) PFU of virus per cell and viral titer deter- mined at the times indicated. Data is pooled from two experiments (mean SEM). (C) Viral titers in spleen and liver of B6 (left panel) and IFN- / (right panel) mice 4 and 9 d after intraperitoneal infection with 106 PFU of virus. Data is pooled from two separate experiments with five mice total per time point/experiment (mean SEM). The virus titer in the spleen were statisti- cally significant (*) as follows. For B6 mice, day 9 spleen; wt HV68 versus v-cyclin. MR, 1.8-fold difference, P 0.03. For IFN- / mice, day 4 spleen; v-cyclin.MR versus v-cyclin.Stop, 2.4-fold difference, P 0.001.