Inhibition of NF-κB signaling enhances HSV-2-induced KSHV replication.

<p>(<b>A</b>). <b>HSV-2 infection elevated the P65 level of NF-κB pathway in BCBL-1 cells.</b> BCBL-1 cells were infected by HSV-2 or Mock for 12, 24 and 48 hours. Then cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of HSV-2-infected BCBL-1 cells for competition assays (HSV-2+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock and HSV-2 groups, respectively. (<b>B</b>). <b>HSV-2 infection increased NF-κB activity in BCBL-1 cells.</b> BCBL-1 cells adsorbed with HSV-2 or Mock for 1 hour, then were transfected with an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** <i>p</i><0.01 for Student's t-test versus Mock group. (<b>C</b>). <b>A pharmacologic inhibitor of IKK–NF-κB activation, Bay 11-7082, inhibited the binding activity of P65 of NF-κB pathway in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. Then cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of DMSO-pre-treated and HSV-2-infected BCBL-1 cells for competition assays (HSV-2+DMSO+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO group; ^##<i>p</i><0.01 and ^&<i>p</i><0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (<b>D</b>). <b>Bay 11-7082 inhibited HSV-2-induced activity of NF-κB in BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 1 hour and then were transfected with an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** <i>p</i><0.01 and ^##<i>p</i><0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively. (<b>E</b>). <b>Bay 11-7082 enhanced ORF26 mRNA expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. ORF26 mRNA expression in BCBL-1 cells was measured by RT-qPCR. Relative quantities of ORF26 expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO group, respectively; ^#<i>p</i><0.05 and ^##<i>p</i><0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (<b>F</b>). <b>Bay 11-7082 enhanced vIL-6 expression in HSV-2-infected BCBL-1 cells.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection for 12, 24 and 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (<b>G</b>). <b>Real-time DNA-PCR analysis for the viral copy number of KSHV.</b> BCBL-1 cells were pre-treated by Bay 11-7082 (5 µM) or DMSO for 1 hour followed by HSV-2 or Mock infection. At 24 and 48 hours postinfection, supernatant virus was collected, concentrated, and used for real-time DNA-PCR analysis. Results shown were from three independent experiments performed in triplicate. * <i>p</i><0.05 and ** <i>p</i><0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively.