γHV68 v-bcl-2 mutant viruses grow like wt γHV68 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 determined 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-γ^−/− (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 statistically significant (*) as follows. For B6 mice, day 9 spleen; wt γHV68 versus v-cyclin. MR, 1.8-fold difference, P = 0.03. For IFN-γ^−/− mice, day 4 spleen; v-cyclin.MR versus v-cyclin.Stop, 2.4-fold difference, P = 0.001.

v-bcl-2 and v-cyclin are critical for persistent replication of γHV68 in IFN-γ^−/− mice. IFN-γ^−/− mice were infected with 10⁶ PFU of virus and peritoneal cells were harvested at 16, 28, or 42 d after infection for quantitation of frequency of cells reactivating virus (A–C, left panels), amount of persistent virus (A–C, right panels), and frequency of latently infected cells (D). Data represent results of two to three experiments (mean ± SEM), with each experiment containing cells pooled from five mice. Since at day 42 time point, no significant difference between v-cyclin.MR and wt γHV68 was observed, only v-cyclin.MR was checked for day 16 and day 28 time points along with two independent isolates of v-bcl-2.Stop1 and v-cyclin.Stop. For day 42 experiments, v-bcl-2 mutant infection included two experiments with v-bcl-2.Stop1 and one experiment with v-bcl-2.Stop2. Similarly, v-cyclin mutant infection included two experiments with v-cyclin.Stop and one experiment with v-cyclin.LacZ at day 42 time point. The decreased frequencies of ex vivo reactivation and persistent productive replication of both v-bcl-2 mutant and v-cyclin mutant compared with wt γHV68 and v-cyclin.MR were statistically significant as follows: day 16 ex vivo reactivation, v-bcl-2.Stop1 versus v-cyclin.MR, P = 0.007; v-cyclin.Stop versus v-cyclin.MR, P = 0.003; for day 16 persistent productive replication, v-bcl-2.Stop1 versus v-cyclin.MR, P = 0.014; v-cyclin.Stop versus v-cyclin.MR, P = 0.011, for day 28 ex vivo reactivation, v-bcl-2.Stop1 versus v-cyclin.MR, P = 0.0009, v-cyclin.Stop versus v-cyclin.MR, P = 0.0001; for day 28 persistent productive replication, v-bcl-2.Stop1 versus v-cyclin.MR, P = 0.0326, v-cyclin.Stop versus v-cyclin.MR, P = 0.028, and for day 42 ex vivo reactivation, v-bcl-2 mutant versus wt γHV68, P = 0.0008, v-bcl-2 mutant versus v-cyclin.MR, P = 0.0003, v-cyclin mutant versus wt γHV68, P = 0.0001, v-cyclin mutant versus v-cyclin.MR, P = 0.0001; for day 42 persistent productive replication, v-bcl-2 mutant versus wt γHV68, P = 0.027, v-bcl-2 mutant versus v-cyclin.MR, P = 0.008, v-cyclin mutant versus wt γHV68, P = 0.032, v-cyclin mutant versus v-cyclin.MR, P = 0.011. The difference in frequency of genome positive cells at day 16 and day 42 were not statistically significant between the v-cyclin.MR versus v-bcl-2.Stop1 and v-cyclin.MR versus v-cyclin.Stop.

Genomic structure of v-bcl-2.Stop mutant viruses. (A) Schematic representation of the region of the γHV68 genome encoding the v-bcl-2 gene (M11 ORF; genomic coordinates 103,418–103,930) (reference 5), the structure of v-bcl-2 mutant viruses and the v-bcl-2 region Southern blot probe (genomic coordinates 101,654–105,377) (reference 5). The shaded region within ORF72 in the v-cyclin.LacZ virus represents a LacZ cassette insertion between 102,704 bp and 103,179 bp (reference 18). The crosshatched region in the M11 ORF in v-cyclin.LacZ signifies the part of the M11 ORF deleted in v-bcl-2.Stop1. (B) Schematic representation of the v-bcl-2 ORF showing the position of the conserved BH1 domain and the sites of Stop1 and Stop2 mutations in the v-bcl-2 ORF. Shaded regions are the portions of the ORF predicted to be translated. (C) Southern analysis of wt γHV68, v-bcl-2.Stop1 and Stop2 mutants, v-cyclin.LacZ, and v-cyclin.MR viruses. Purified genomic viral DNA (1 μg) from the indicated viruses was digested with XhoI and BamHI (lanes 1–5) or NheI and BamHI (lanes 6–10) and Southern analysis performed with the v- bcl-2 region probe (A; references 18 and 29). Expected fragment sizes for XhoI and BamHI digests: lane 1/wt γHV68, 5.2 kb; lane 2/v-cyclin.LacZ mutant, 3.8 kb and 1.1 kb; lane 3/v-bcl-2.Stop1A mutant, 3.3 and 1.9 kb; lane 4/v-bcl-2.Stop1B mutant, 3.3 and 1.9 kb; lane 5/v-cyclin.MR, 5.2 kb. Expected fragment sizes for NheI and BamHI digests: lane 6/wt γHV68, 5.2 kb; lane 7/v-cyclin-LacZ mutant, 3.8 kb and 1.5 kb; lane 8/v-bcl-2.Stop2A mutant, 3.5 and 1.7 kb; lane 9/v-bcl-2.Stop2B mutant, 3.5 and 1.7 kb; lane 10/v-cyclin.MR; 5.2 kb. (D) Immunoblot analysis of wt γHV68, v-bcl-2.Stop1 and Stop2 mutants, v-cyclin.LacZ, or v-cyclin.MR virus infected NIH 3T12 cell lysates for v-cyclin and β-actin protein expression.

v-bcl-2 mutants establish latency normally but fail to reactivate efficiently during ex vivo culture. B6 mice were infected 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 experiments using two independent mutant isolates (mean ± SEM), with each experiment containing cells pooled from five mice. The difference in frequency of genome positive cells at day 16 and day 42 were not statistically significant between the wt γHV68 versus v-bcl-2.Stop1 and v-cyclin.MR versus v-bcl-2.Stop2. At 16 d (A and B, left panels), the decreased frequencies of ex vivo reactivation of both v-bcl-2.Stop mutants compared with wt γHV68 and v-cyclin.MR were statistically significant as follows: v-bcl-2.Stop1 versus wt γHV68, P = 0.006; v-bcl-2.Stop2 versus wt γHV68, 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 γHV68 and v-cyclin.MR were statistically significant as follows: v-bcl-2.Stop1 versus wt γHV68, P = 0.004; v-bcl-2.Stop2 versus wt γHV68, 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.

v-bcl-2 and v-cyclin are necessary for lethal chronic disease but not for lethal acute disease. (A) IFNγR^−/− 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-cyclin.Stop mutant virus compared with v-cyclin.MR virus was statistically 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) Survival 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) Survival 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 infection. Data represent 20–30 mice per point pooled from 2–3 independent experiments. P = NS indicates that there is no statistical difference between the indicated groups.