Figure 1: vtRNA levels in response to expression of EBV-encoded latency III proteins.

(a) Northern blot analysis was used to assess the effects of overexpressing EBV-encoded proteins on the vtRNA1-1 levels in different BL2 cell lines. The 5S rRNA serves as internal loading control. (−) indicate untreated BL2 cells, whereas EVC indicates the empty vector control. EBV strain B95.8 was used as positive infection control (+ EBV). The mean and standard deviation of three different experiments are shown beneath the blot, whereas the vtRNA1-1 expression levels in untreated BL2 cells (−) was taken as 1.0. See also Supplementary Fig. 2. (b) Real-time qPCR was used to quantify vtRNA1-1 levels in the untreated BL2 control (−) or in BL2 cells overexpressing latency stage III proteins. Real-time qPCR data shown are the mean values from three individual assays; TBP served as housekeeping gene. ‘Fold induction’ was calculated using the comparative Δct method where BL2 served as calibrator. (c) Schematic representation of LMP1 domain organization. The different signalling pathways controlled by the signalling modules CTAR1 and CTAR2 are indicated. (d) Real-time qPCR to investigate the effect of LMP1 mutants (M_CTAR 1 and M_CTAR 2 carry detrimental amino-acid substitutions or a deletion in CTAR domains 1 or 2, respectively) on the vtRNA1-1 level in BL2 cells. Data shown are the mean values and standard deviations of three independent assays. (e) In the presence of an NF-κB inhibitor (inh.), northern blot analysis showed no LMP1-dependent upregulation of vtRNA1-1. 5.8 S rRNA serves as internal loading control. (f) ChIP efficiencies using an NF-κB p65 antibody were monitored using qPCR on vtRNA1-1 and vtRNA1-2 promoter regions from BL2 cells carrying the empty vector (evc), expressing LMP1 (+LMP1), or from EBV-infected BL2 cells. Data shown are mean values and standard deviations from three individual assays. Significant differences relative to the mock control (no antibody) were determined using the two-tailed unpaired Student’s t-test (***P<0.001, **P<0.01, *P<0.01). In b and d, significant differences in fold induction relative to the untreated BL2 cells (−) were determined using the two-tailed unpaired Student’s t-test (***P<0.001, **P<0.01).