| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 98 === Dengue virus (DENV) is one of the most common infectious pathogens worldwide. One major clinical and pathogenic feature of DENV infection is the elevation of IL-8 expression; however, little is known about the molecular mechanism of DENV-induced chemokine production. The positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T1, stimulates gene expression by enhancing RNA polymerase II (RNA pol II) processivity. This study examined the possibility that P-TEFb mediates DENV-induced IL-8 expression. The treatment of either a pharmacological inhibitor of P-TEFb, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or cyclin T1 siRNA prior to DENV infection abolished the elevation of IL-8, indicating that P-TEFb is essential for IL-8 induction during DENV infection.
The core protein is the building block of the nucleocapsid of dengue virus. DEN2 core protein was reported to be detectable in the cytoplasm, the nucleus and the nucleolus. To investigate the effect of DEN2 core protein on different transcriptional factors, PathDetectR in vivo Signal Transduction Pathway cis-Reporting Systems consisting of luciferase reporter plasmids with enhancer elements for transcription factors were used. DEN2 core protein was found to increase the luciferase reporter activity of reporter plasmid containing the enhancer elements of NF-κB in a dose-dependent manner. To confirm this activation, electrophoretic mobility shift assay was done with nuclear extract of HeLa cells transfected with DEN2 core protein and DEN2 core protein was found to increase the DNA-binding affinity of NF-κB. It was reported that NF-κB binds P-TEFb to stimulate transcription elongation by RNA Pol II. To find out whether DEN2 core protein had a role in the recruitment of P-TEFb by NF-κB, a co-immunoprecipitation assay and immunostaining were performed and DEN2 core protein was found to bind and colocalized with cyclin T1, the regulatory subunit of P-TEFb. In addition, DEN2 core protein had no effect on NF-kB activity in the presence of DRB (P-TEFb inhibitor) and siCyclinT1. To investigate whether DEN2 core protein was involved in the NF-κB-induced increased IL-8 production, luciferase assay was performed with a reporter plasmid carrying a fragment (-133 to –10) of IL-8 promoter and a mutant plasmid of IL-8 promoter with the NF-κB binding site mutated. The luciferase activity was found to be increased in the presence of DEN2 core protein, whereas DEN2 core protein had no such effect in the presence of DRB or siCyclinT1 or in the mutant. Moreover, DENV core protein participated in the activation of IL-8 promoter in a P-TEFb-dependent manner. Finally, chromatin immunoprecipitation (ChIP) results indicated that P-TEFb and DENV core protein were recruited to the transcriptionally active IL-8 gene promoter. Taken together, this study showed that P-TEFb and DENV core protein work in concert to enhance IL-8 gene expression by DENV infection. This is the first demonstration of P-TEFb being directly involved in virus-induced host gene expression by interacting with a viral structural protein.
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