Figure 5. v-bcl-2 and v-cyclin are necessary for lethal chronic disease but not for lethal acute disease. (A) IFN R / mice were infected intraperitoneally with v-cyclin.MR, v-bcl-2.Stop, or v-cyclin.Stop mutant virus and observed for lethality over 90 d after infection. The increase in percent survival of mice infected with v-bcl-2 mutant virus or v-cy- clin.Stop mutant virus compared with v-cyclin.MR virus was sta- tistically significant as follows: v-bcl-2.Stop versus v-cyclin.MR, P 0.0003; v-cyclin.Stop versus v-cylin.MR, P 0.012. (B) Sur- vival of RAG-1 / mice infected intraperitoneally with 106, 103, or 10 PFU of virus. Data are pooled from two experiments (n number of mice). (C and D) Sur- vival of CD1 mice (3–4 wk old) infected intracerebrally with v-cyclin.MR, v-bcl-2.Stop 1, or v-cyclin.Stop virus and observed for lethality over 21 d after infec- tion. Data represent 20–30 mice per point pooled from 2–3 in- dependent experiments. P NS indicates that there is no sta- tistical difference between the indicated groups. v-bcl-2 and v-cyclin mutant viruses was specific to chronic disease, we tested mutant viruses in two additional models of virulence during acute infection. The virulence of wt HV68 and v-bcl-2.Stop1 was comparable in RAG-1 / mice infected with 10, 103, or 106 PFU (Fig. 5 B). We pre- viously showed that v-cyclin mutant HV68 is not attenu- ated in this same model (18). HV68 also causes acute lethal meningitis when administered intracerebrally into weanling mice (39). The virulence of v-cyclin and v-bcl-2 mutants in this lethal meningitis model was indistinguish- able from that of wt HV68 (Fig. 5, C and D). Thus, the lack of virulence of v-bcl-2 and v-cyclin mutant viruses dur- ing chronic disease was not seen in two different models of acute disease, further supporting our conclusion that v-bcl-2 and v-cyclin are viral genes with a physiologically important and specific role during chronic infection. Discussion We show here for the first time that a -herpesvirus v-bcl-2, as previously shown for a v-cyclin, is necessary for efficient ex vivo reactivation from latency. The impor- tance of both anti-apoptotic and cell cycle regulatory pro- teins in ex vivo reactivation from latency suggests that the reactivation process requires cell cycle progression and triggers host or cellular apoptotic pathways that serve to prevent reactivation unless the virus counters with anti- apoptotic molecules. 937 Gangappa et al. Of special importance for understanding how herpesvi- ruses cause disease, we demonstrate for the first time that herpesviruses have genes (v-bcl-2 and v-cyclin) that are im- portant for persistent replication and chronic disease in an immunocompromised host, but not acute replication and acute disease. This identifies persistent replication as a pro- cess that is genetically distinct from replication during acute infection, and shows that genes important for persistent replication are key virulence determinants. v-bcl-2 Is Required for Efficient Ex Vivo Reactivation from Latency. HV68 establishes latency in a number of cells types including macrophages, dendritic cells, and B cells (28, 31, 40, 41). We found that establishment of latency does not require v-bcl-2. In this respect, v-bcl-2 is similar to other HV68 genes we have analyzed including the v-cyclin and M1 (18, 29). However, the v-bcl-2 mutation resulted in inefficient ex vivo reactivation of virus from la- tently infected cells. In this respect the v-bcl-2 gene is simi- lar to v-cyclin, but distinct from the M1 locus since mutations in the M1 locus enhance the efficiency of ex vivo reactivation (18, 29). Thus, HV68 contains genes that both enhance and inhibit ex vivo reactivation, sug- gesting that the balance between latency and ex vivo reac- tivation is under tight regulation by multiple viral genes. The importance of v-bcl-2 for ex vivo reactivation from la- tency and persistent replication is consistent with our prior demonstration that the v-bcl-2 gene is actively transcribed in latently infected tissues (42). It is possible that additional