Figure 3
RNA exosome depletion increases both nuclear accumulation of viral intronless mRNAs and virion production. (a) Immunoblot: Western blotting analysis of HEK293EBV∆BMLF1 cells extracts showing protein depletion upon hRRP40 siRNA treatments. HEK293EBV∆BMLF1 cells were treated with specific (hRRP40) or control (Firefly) siRNAs and transfected with a control vector (lanes 1 and 4) or an EB1 expression vector in order to induce the viral productive cycle (lanes 2, 3, 4 and 5) and transcomplemented with an EB2 expression vector (lanes 3 and 5). Membranes were probed with the indicated antibodies (anti-hRRP40, -EB1, -EB2, -gp350 and -Tubulin). Anti-Tubulin antibody was used as a loading control. (b) and (c) Nuclear and cytoplasmic mRNA accumulation: Quantification by RT-qPCR of nuclear (b) and cytoplasmic (c) EBV-encoding early (E) mRNA (BMRF1) or late (L) mRNAs (BDLF1, BdRF1 and BFRF3) in HEK293EBV∆BMLF1 cells transfected as described in (a). Data are displayed as mean values normalized to the control siRNA and GAPDH mRNA as an internal control. Error bars represent the s.d. from three independent experiments (n = 3). * Indicates a P-value of <0.05 and ** indicates a P-value of <0.01. (d) Virus production: HEK293EBV∆BMLF1 cells supernatant were collected 48 h after the induction of the productive cycle, filtered and used to infect Raji cells. The number of EBV-infected Raji cells was evaluated by quantification of GFP-expressing cells 72 h later by FACS analysis. Error bars represent the s.d. from three independent experiments (n = 3). ** Indicates a P-value of <0.01.
