Journal: The Journal of Biological Chemistry

Article Title: IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3

doi: 10.1074/jbc.M117.802900

Figure Lengend Snippet: K-RTA transactivates the human IL-10 promoter. A, KSHV-infected PEL cell line JSC-1 and BC3 cells were co-transfected with the reporter plasmids IL-10 p-1000, ORF50, or PANp and pRL-TK vector (internal control). Two days after transfection, a luciferase assay was performed. Results are presented as the mean ± S.D. (n = 3). Student's t test; **, p < 0.05. B, KSHV-infected PEL cell line JSC-1 and BC3 cells were treated with or without TPA (20 ng/ml) for 24 h, and human IL-10 mRNA expression was determined by RT-qPCR. GAPDH mRNA was used to normalize data. Results are presented as the mean ± S.D. (n = 3). Student's t test; **, p < 0.05. C, human IL-10 promoter reporter plasmid p-265 and internal control pRL-TK were transfected into JSC-1 cells. One day later, cells were treated or not with TPA (20 ng/ml) for 24 h. Luciferase activities were determined 2 days after transfection. Results are presented as the mean ± S.D. (n = 3). The activation was statistically significant by Student's t test; p < 0.01. D, three human cell lines, JSC-1, BC3, and HCT 116 cells, were co-transfected with FLAG–K-RTA, IL-10 promoter reporter plasmid p-265, and the internal control pRL-TK plasmid (ratio 1:1:1). Two days after transfection, luciferase activity was examined. Results are presented as the mean ± S.D. (n = 3). The activation was statistically significant by Student's t test; *, p < 0.01. E, human IL-10 promoter reporter plasmid p-265 and internal control pRL-TK were co-transfected into cells with either an empty plasmid (i.e. without K-RTA) or with a plasmid carrying the wild-type K-RTA (WT) or the transactivation domain-deleted K-RTA (F490). Luciferase activity was determined 2 days after transfection. Results are presented as the mean ± S.D. (n = 3). Student's t test; *, p < 0.01. F, HCT 116 cells were transiently transfected with either K-RTA WT or K-RTA F490 expression plasmids. Two days after transfection, mRNA was prepared, and human IL-10 gene expression was measured by RT-qPCR. GAPDH mRNA was used to normalize data. Results are presented as the mean ± S.D. (n = 3). Student's t test. *, p < 0.01. G and H, binding efficiencies of K-RTA to PAN (PANp) or IL-10 (−265/−1 from p-265) promoter DNA probes were analyzed by EMSA. Five-fmol biotin-labeled promoter probes were incubated with increasing amounts of FLAG–K-RTA protein (aa 1–531). The amount of FLAG–K-RTA increased from 0 to 300 ng. A complex of FLAG–K-RTA–DNA probes was examined by EMSA, indicated by the triangle (G). The asterisk indicates free probe DNA. The binding curves were calculated (H).

Article Snippet: KSHV thymidine kinase TATAA-less promoter is co-operatively regulated by K-RTA and SP1 ( 50 ), and K-RTA–mediated transcription of KSHV viral interferon regulatory factor (vIRF) gene involves SP1-binding sites in the vIRF promoter ( 51 ).

Techniques: Infection, Transfection, Plasmid Preparation, Control, Luciferase, Expressing, Quantitative RT-PCR, Activation Assay, Activity Assay, Gene Expression, Binding Assay, Labeling, Incubation