(A). The CPE on BCBL-1 cells infected by HSV-2 (original magnification, ×40). HSV-2 was inoculated into BCBL-1 cells for 0 to 120 hours and changes of cell morphology were observed under light microscope. (B). RT-PCR analysis for HSV-2 immediate early, early, and late genes mRNA in HSV-2-infected BCBL-1 cells. HSV-2 UL48, UL5 and US6 mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 6, 12, 24, 48, and 72 hours was detected by RT-PCR. β-Actin was readily detectable in all samples indicating the presence of amplifiable cDNA. (C). Western blot analysis for HSV-2 structural protein expression in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected with Mock or HSV-2 for 24, 48, 72 and 96 hours; or Vero cells were infected with HSV-2 for 48 hours (positive control). Whole cell lysate were subjected to Western blot with the indicated antibody. Results shown are from a representative experiment of three independent experiments with similar results.

(A). Rta mRNA expression in HSV-2-infected BCBL-1 cells. Rta mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 3, 6, 12, 24, 48, 72 and 96 hours was quantitated by RT-qPCR. Relative quantities of Rta expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. * p<0.05 and ** p<0.01 for Student's t-test versus mock group, respectively. (B). ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. ORF26 mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 3, 6, 12, 24, 48, 72 and 96 hours 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. * p<0.05 and ** p<0.01 for Student's t-test versus mock group, respectively. (C). RT-PCR analysis for ORF57 and vIL-6 mRNA in BCBL-1 cells infected with HSV-2. ORF57 and vIL-6 mRNA expression in BCBL-1 cells infected with Mock or HSV-2 for 24, 48, 72 and 96 hours was detected by RT-PCR. β-Actin was readily detectable in all samples indicating the presence of amplifiable cDNA. (D). Rta protein expression in BCBL-1 cells infected by HSV-2. BCBL-1 cells were infected with HSV-2 or mock for 24, 72 and 96 h and whole-cell lysates were subjected to Western blot with Rta antibody. The membrane was stripped and reprobed with anti-actin antibody as a loading control. Results shown are from a representative experiment of three independent experiments with similar results. (E). Immunofluorescence assay staining of BCBL-1 cells infected with HSV-2 (original magnification, 40×). KSHV lytic protein ORF59 expression in normal BCBL-1 cells (untreated) and BCBL-1 cells infected with mock or HSV-2 for 24 and 72 h was detected by immunofluorescence assay staining with ORF59 MAb. (F). Quantification of results in (E). Quantitative and statistic methods were described in the text. ** p<0.01 for Student's t-test versus mock group.

(A). Real-time DNA-PCR analysis for the viral copy number of KSHV. BCBL-1 cells were infected with HSV-2 or Mock. At 72 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. ** p<0.01 for Student's t-test versus mock group. (B). RT-PCR analysis for ORF26 mRNA in BCBL-1 or HEK293 cells treated with supernatant from HSV-2-infected BCBL-1 cells. ORF26 mRNA expression in BCBL-1 cells treated with TPA (Lane 1), HEK293 cells treated for 48 hours with supernatant from TPA-treated BCBL-1 cells for 72 hours (Lane 2), HEK293 cells cultured alone for 27 hours (Lane 3), HEK293 cells infected with HSV-2 for 27 hours (Lane 4), HEK293 cells treated for 27 hours with supernatant from Mock-treated BCBL-1 cells for 72 hours (Lane 5), and HEK293 cells treated for 27 hours with supernatant from HSV-2-infected BCBL-1 cells for 72 hours (Lane 6) were detected by RT-PCR. (C). Western blot for detection of vIL-6 expression in BCBL-1 cells or HEK293 cells treated with supernatant from HSV-2-infected BCBL-1 cells. vIL-6 expression in BCBL-1 cells treated with TPA (Lane 1), HEK293 cells treated for 48 hours with supernatant from TPA-treated BCBL-1 cells for 72 hours (Lane 2), HEK293 cells cultured alone for 27 hours (Lane 3), HEK293 cells infected with HSV-2 for 27 hours (Lane 4), HEK293 cells treated for 27 hours with supernatant from Mock-treated BCBL-1 cells for 72 hours (Lane 5), and HEK293 cells treated for 27 hours with supernatant from HSV-2-infected BCBL-1 cells for 72 hours (Lane 6) were detected by Western blot.

(A). HSV-2 infection promotes induction of KSHV Rta promoter activity. BCBL-1, BC-3, 293/Bac36, B95-8 and Vero cells were infected with Mock (Mock+p50-Luc) or HSV-2 (HSV-2+p50-Luc), or treated with TPA (TPA+p50-Luc), and then cotransfected with the firefly luciferase reporter construct p50-Luc (0.4 µg) and a Renilla luciferase construct pRL-TK (0.02 µg). Luciferase activities were measured, normalized to inner control, and presented as fold increase (n-fold). All data points were the averages of three independent experiments performed in triplicate. * p<0.05 and ** p<0.01 for Student's t-test versus Mock+p50-Luc group, respectively. (B). Western blot analysis for Rta protein in si-construct-transfected BCBL-1 cells infected with HSV-2. BCBL-1 cells were transfected with pRNAT-Rta or pRNAT-vector, and then infected with HSV-2 or Mock for 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody.

(A). HSV-2 infection elevated the P65 level of NF-κB pathway in BCBL-1 cells. 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. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock and HSV-2 groups, respectively. (B). HSV-2 infection increased NF-κB activity in BCBL-1 cells. 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. ** p<0.01 for Student's t-test versus Mock group. (C). 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. 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. ** p<0.01 for Student's t-test versus Mock+DMSO group; ^## p<0.01 and ^& p<0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (D). Bay 11-7082 inhibited HSV-2-induced activity of NF-κB in BCBL-1 cells. 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. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively. (E). Bay 11-7082 enhanced ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. 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. * p<0.05 and ** p<0.01 for Student's t-test versus Mock+DMSO group, respectively; ^# p<0.05 and ^## p<0.01 for Student's t-test versus HSV-2+DMSO group, respectively. (F). Bay 11-7082 enhanced vIL-6 expression in HSV-2-infected BCBL-1 cells. 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. (G). Real-time DNA-PCR analysis for the viral copy number of KSHV. 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. * p<0.05 and ** p<0.01 for Student's t-test versus Mock+DMSO and HSV-2+DMSO groups, respectively.

(A). Transfection of IKK₂EE promoted the binding activity of P65 of NF-κB pathway in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. 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 MIGRI-transfected and HSV-2-infected BCBL-1 cells for competition assays (HSV-2+MIGRI+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. * p<0.05 and ** p<0.01 for Student's t-test versus Mock+MIGRI group, respectively; ^## p<0.01 and ^& p<0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (B). Transfection of IKK₂EE promoted HSV-2-induced activity of NF-κB in BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were co-transfected with the plasmid IKK₂EE and 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. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively. (C). Transfection of IKK₂EE inhibited ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Cells were collected and 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. ** p<0.01 for Student's t-test versus Mock+MIGRI group; ^# p<0.05 and ^## p<0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (D). Transfection of IKK₂EE inhibited vIL-6 expression in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (E). Real-time DNA-PCR analysis for the viral copy number of KSHV. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI. 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. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively.

(A). Effect of Tat on ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. Real-time quantitative PCR was performed to detect KSHV ORF26 mRNA expression in Mock-treated and pcDNA3.1-transfected BCBL-1 cells (Mock+pcDNA), Mock-treated and pTat-transfected BCBL-1 cells (Mock+Tat), or HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells (HSV-2+pcDNA), HSV-2-infected and pTat-transfected BCBL-1 cells (HSV-2+Tat) for 6, 24, 48 and 72 hours. The results shown were from three independent experiments performed in triplicate. ** p<0.01 for Student's t-test versus HSV-2+pcDNA group. (B). Effect of Tat on vIL-6 protein expression in HSV-2-infected BCBL-1 cells. Cell lysates were collected from HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells, or HSV-2-infected and pTat-transfected BCBL-1 cells for 48 and 72 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (C). Effect of Tat on Rta promoter activities in HSV-2-infected BCBL-1 cells. BCBL-1, B95-8 and Vero cells were infected with Mock and then co-transfected with pcDNA3.1, p50-Luc and Renilla vector pRL-TK (Mock+pcDNA3.1), infected with HSV-2 and then co-transfected with pcDNA3.1, p50-Luc and Renilla vector pRL-TK (HSV-2+pcDNA3.1), or infected with HSV-2 and then co-transfected with pTat, p50-Luc and Renilla vector pRL-TK (HSV-2+Tat). Luciferase activities were measured, normalized to Rellina inner control, and presented as fold increase (n-fold). All data points were the averages of three independent experiments performed in triplicate. * p<0.05 and ** p<0.01 for Student's t-test versus HSV-2+pcDNA3.1 group, respectively. (D). Effect of Tat on HSV-2 replication in BCBL-1 cells. BCBL-1 cells were infected with Mock and then transfected with pcDNA3.1 (Mock+pcDNA), infected with HSV-2 and then transfected with pcDNA3.1 (HSV-2+pcDNA), or infected with HSV-2 and then transfected with pTat (HSV-2+pTat) for 24, 48 and 72 hours. Whole cell lysate were subjected to Western blot with the indicated antibody.