Figure 1
EB2 stabilizes late viral mRNAs in the nucleus. (a) mRNA accumulation: Cytoplasmic and nuclear RNAs from HEK293EBV∆BMLF1 cells transiently transfected as indicated at the top of the figure were submitted to RT-PCR analysis using specific primers to detect cellular U6 snRNA and ß-actin mRNA, or EBV-encoding mRNAs (BDLF1, BdRF1, BFRF3 and BMRF1). The PCR products were loaded on a 2% agarose gel and visualized by ethidium bromide staining. The RT-PCR results were in the linear range of the PCR reaction. Expression of EB2, EB1 and Tubulin proteins expressed in HEK293EBV∆BMLF1 cells that have been transfected, or not (lane 1), with an EB1 expression plasmid (lane 2), or cotransfected with expression plasmids for both EB1 and EB2 (lane 3) were controlled by western-blotting. * Indicates an unspecific band recognized by the anti-EB2 serum. (b) Schematic representation of the pTRE2-BDLF1 construct which contains the viral gene BDLF1 under the control of the Tet-responsive promoter and the pCMV-RLuc construct which contains the Renilla Luciferase gene, RLuc, under the control of the CMV promoter. (c) (e) (g) and (i) Nuclear and cytoplasmic BDLF1and luciferase mRNAs accumulation: Quantification by RT-qPCR of the nuclear (c and g) and cytoplasmic (e and i) BDLF1 and luciferase mRNA expressed from HeLa cells co-transfected with the pTRE2-BDLF1 construct or the pCMV-RLuc construct without or with an EB2 expression vector (time 0 of the kinetic). Error bars represent the s.d. from three independent experiments (n = 3). ** Indicates P-value of <0.01 and *** indicates P-value of <0.001. (d) (f) (h) and (j) Nuclear and cytoplasmic BDLF1 or luciferase mRNA stability: Relative nuclear (d and h) and cytoplasmic (f and j) BDLF1 or luciferase mRNA level after transcription inhibition (expressed as a percentage of the amount at time point 0) was determined by RT-qPCR from HeLa cells co-transfected with the pTRE2-BDLF1 construct or the pCMV-RLuc construct without or with an EB2 expression vector. Numbers above the panel refer to hours after transcriptional shutoff of the BDLF1 or luciferase intronless gene. Half-lives (t1/2) and s.d were calculated from three independent experiments (n = 3).
