FIG. 6.

The v-cyclin.LacZ mutant establishes latency but fails to reactivate efficiently in cells from B-cell-deficient mice. B-cell-deficient mice were infected with 10⁶ PFU of v-cyclin.LacZ (triangles), wt γHV68 (squares), or v-cyclin.MR (circles), and cells were harvested 42 days postinfection for quantitation of the frequency of viral-genome-positive cells (A and B) and the frequency of cells reactivating virus (C and D). (A and B) Latently infected splenocytes and PECs were analyzed for the frequency of viral-genome-positive cells by limiting-dilution nested PCR, as described in the legend to Fig. 5 and in Materials and Methods. (C and D) Cells were harvested 42 days postinfection for limiting-dilution ex vivo reactivation analysis on MEF indicator monolayers, as described in the legend to Fig. 5 and in Materials and Methods. The presence of preformed infectious virus was assessed by limiting-dilution analysis of disrupted cells, with a control being included in each experiment (open symbols). Curve fit lines were derived by nonlinear-regression analysis (standard errors of the means are shown [error bars]). The dashed line is at 63%, the value which was used to calculate the frequency of genome-positive cells (A and B) or the frequency of reactivating cells (C and D) as indicated by a Poisson distribution. Data represent independent experiments as indicated, with each experiment utilizing cells pooled from four to seven mice. ∗∗∗, differences in the frequencies of reactivation for v-cyclin.LacZ and wt γHV68 were statistically significant (PECs, P = 0.0005; splenocytes, P = 0.0162).