genic when expressed in transgenic mice (26), and is re- quired for efficient ex vivo reactivation from viral latency, but not for viral replication in wild type mice during acute infection (18, 27). The role of the v-cyclin in persistent rep- lication and chronic disease has not been assessed. In this study we use loss of function mutagenesis to evaluate the role of the v-bcl-2 gene in HV68 infection in vivo. Because regulation of cell cycle and apoptosis is inti- mately intertwined during infection with papillomaviruses, polyomaviruses, and adenoviruses, we compared proper- ties of v-bcl-2 and v-cyclin mutants. We found that neither of these genes has an important role during acute infec- tion, but that both are important for ex vivo reactivation from latency, persistent replication, and disease during chronic infection. Materials and Methods Viruses and Tissue Culture. HV68 clone WUMS (ATCC VR1465) was passaged, grown, and titered in NIH 3T12 cells or BALB/c or C57Bl/6 murine embryonic fibroblasts (MEFs) as de- scribed (18, 28). Viral stocks were generated from NIH 3T12 cells infected at multiplicity of infection (MOI) 0.05 and har- vested at 50% cytopathic effect (CPE) (18). Generation of Mutant HV68 by Homologous Recombination. The parental genomic subclone for the targeting plasmids con- tains the 3,723 bp BamHI/BsrGI fragment of HV68 from ge- nomic coordinates 101,654 to 105,377 in Litmus 38 (pL3700; references 5 and 18). The pL3700-v-bcl-2.Stop1 mutant target- ing plasmid was generated using PCR and primers that excise 70 bp (from 103,641 to 103,711) and insert the 9 bp, 5 CTCGAG- TAG 3 , which includes an XhoI site (underlined) and an in frame stop codon (bold). For pL3700-v-bcl-2.Stop2, the primers insert 7 bp, 5 AGCTAGC 3 , which includes an NheI site (un- derlined) and a stop codon (bold) at genomic coordinate 103,450. The v-bcl-2 mutant viruses were generated by transfection of NIH 3T12 cells with v-cyclin.LacZ virus genomic DNA (1.5 g) and pL3700-v-bcl-2 mutant plasmid (1.5 g) (18, 29). Recombinant virus was purified by white plaque morphology after staining with X-Gal (5-bromo-4-chloro-3-indole- -D-galactoside) (18). The v-cyclin marker rescue virus (v-cyclin.MR) has been de- scribed previously (18). Plaque purified (three rounds) viral stocks were characterized by Southern blot and Western blot analyses (18, 29). Mice, Infections, and Organ Harvests. Recombination activat- ing gene (RAG)-1 / (The Jackson Laboratory; 002096) and IFN- / mice (The Jackson Laboratory; 002287) on a C57Bl/6 background (B6) were bred and maintained at Washington Uni- versity School of Medicine in accordance with all federal and university policies. C57Bl/6J (B6) mice were purchased from The Jackson Laboratory. interferon receptor-deficient (IFN R / ) mice on a 129 background were obtained from Michel Aguet, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland (30). Unless otherwise stated, mice were age and sex matched, used between 7 to 10 wk of age, and in- fected with 10, 103, or 106 PFU of HV68 by intraperitoneal in- jection in 0.5 ml of complete DMEM (18, 29). CD-1 outbred lactating mice with pups (12 d old) were obtained from Charles River Laboratories, and the pups were infected intracerebrally with virus diluted in 10 l of complete DMEM. Organs for titer were frozen at 80 C in 1 ml DMEM before plaque assay (18, 29). Peritoneal cells were harvested by peritoneal lavage with 10 ml DMEM (31). To assess inflammatory disease in the great elas- tic arteries, the heart and attached aortic base were harvested at the time of death or sacrifice from IFN R / mice and analyzed for incidence and severity of arteritic lesions (32, 33). Quantitation of Cells Harboring HV68 Genome. The fre- quency of cells harboring the HV68 genome was determined by a limiting dilution nested PCR assay that amplifies HV68 gene 50 sequences with single copy sensitivity (31, 34). Briefly, perito- neal cells and splenocytes were frozen in 10% DMSO at 80 C, thawed, counted, resuspended in isotonic buffer, and serially di- luted into 96-well PCR plates. Uninfected NIH 3T12 cells were added so that each well contained 104 cells. Cells were then lysed in proteinase K and nested PCR performed (31, 34). 10 PCR re- actions were analyzed for each cell dilution, with six dilutions per sample per experiment. Control reactions in each experiment in- cluded uninfected cells alone (6 reactions/plate) or cells with 10, 1, or 0.1 copies of plasmid DNA (pBam HIN) containing target sequence (six reactions/plate each). There were no false-positive PCR reactions in assays reported here, and all assays demon- strated approximately one-copy sensitivity for plasmid DNA, with reactions containing 10, 1, or 0.1 copies of plasmid DNA positive in 89, 29, and 3% of all reactions. Ex Vivo Reactivation from Latency. The frequency of cells re- activating from latency ex vivo was determined using a limiting dilution reactivation assay (28, 34). Briefly, peritoneal cells and splenocytes were harvested 16, 28, or 42 d after infection, and plated in serial twofold dilution (starting at 4 104 peritoneal cells and 105 splenocytes per well) onto MEF monolayers in 96- well plates. Wells were scored microscopically 21 d later for viral CPE. When CPE was difficult to discern at high cell numbers, wells were replated onto fresh MEFs and CPE assessed. 24 wells were plated per dilution and 12 dilutions were plated per sample. Preformed virus in tissues was detected by plating parallel cell samples that had been subjected to mechanical disruption. Me- chanical disruption does not inactivate virus but kills 99% of cells, and thus samples treated in this way detect preformed virus rather than virus reactivating from latency (28, 31, 34). Statistical Methods. All data was analyzed using GraphPad Prism software (GraphPad Software). Titer data were statistically analyzed with the nonparametric, Mann-Whitney t test. Fre- quencies were obtained from the cell number at which 63% of the wells scored positive for either reactivating virus or the pres- ence of viral genome based on Poisson distribution. Data were subjected to nonlinear regression analysis to obtain single-cell fre- quency for each limiting dilution analysis. The frequencies of re- activation and genome-positive cells were statistically analyzed by paired t test. Alterations in the incidence of arteritis were com- pared using -square test and Fisher’s exact test (which gave comparable results). Results Generation of Viruses Containing Mutations in the HV68 v-bcl-2 Gene. To determine the role of v-bcl-2 in viral in- fection, we constructed four HV68 v-bcl-2 mutants. The HV68 v-bcl-2 ORF (M11 original designation) spans 513 bp located between the v-cyclin gene (ORF72) and ORF73 (Fig. 1 A). Of the four recognized bcl-2 homology do- mains (BH domains; BH1-BH4) that are conserved be- tween different bcl-2 family members, the predicted HV68-v-bcl-2 protein has a recognizable BH1-like do- 932 v-bcl-2 and v-cyclin Are Chronic Disease Herpesvirus Genes