Figure 2. Induction of replicon vector is dependent on the MCMV DNA replication.

(A) Induction of MCMV oriLyt is specific to MCMV infection. Cell line luc-ori cl.1 was infected with MCMV (beta herpesvirus; MOI = 0.5) or murine herpesvirus 68 (MHV68, gamma herpesvirus; MOI = 0.5) or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (B) NIH3T3 cells were stably transfected with pEpibo-luc (luc t1) or pEpibo-luc-ori (luc-ori t3). Depicted is the ratio of FL expression of the resulting cell pools before and after infection with MCMV at an MOI of 1. In the luc t1 cell pool, lacking the oriLyt sequence, FL expression is not enhanced by infection, in contrast to the luc-ori t3 cell pool, in which FL expression is induced about 40 fold at 36 h p.i. (C) NIH3T3 or luc-ori cell lines (luc-ori cl.1–cl.4) were infected with MCMV at an MOI of 0.5 (hatched bars) or left uninfected (plain bars). In addition, cell lines were either treated with phosponoacidic acid (PAA, 300 µg/ml, black bars) or left untreated (white bars). 36 h p.i. FL induction was measured via bioluminescence assay. While reporter expression is induced up to 1000-fold in MCMV infected cells lines cl.1–3, inhibition of viral DNA polymerase by PAA blocks the induction of FL expression. (D) Induction can be inhibited with PAA completely if it is added before replication has started and can be reduced if added after the onset of DNA replication. Cell line luc-ori cl.1 was infected with MCMV at an MOI = 0.5 or left uninfected. Arrows indicate time points when 300 µg/ml PAA was added to the cells. Cells were harvested at indicated time points and FL was measured via bioluminescence assay. Induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (p.i., post infection; BG = Background; ***: p<0.001, ns: p>0.05, Two-Way-ANOVA, depicted is mean+SD).