Functional analysis of DEN2 core protein

• Main Authors: Li-Li Li, 李麗麗
• Other Authors: Shiau-Ting Hu
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/94244058808853563772

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 93 === 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. It was also reported to up-regulate the transcription factor C/EBPβ (NF-IL6). To investigate the effect of DEN2 core protein on different transcriptional factors, PathDetect(R) 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 DEN2 core protein transfected HeLa cells 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 was performed and DEN2 core protein was found to bind the cyclin T1, the regulatory subunit of P-TEFb. In addition, it was observed that DEN2 core protein had no effect on NF-kB activity in the presence of DRB (P-TEFb inhibitor). NF-κB activation in dengue virus infected cells was reported to correlate with the increase in chemokines production, like IL-8, which was also reported to be elevated in the plasma of patients with Dengue hemorrhagic fever or Dengue shock syndrome. To investigate whether DEN2 core protein was involved in the increased IL-8 production by NF-κB, luciferase assay was performed with a reporter plasmid carrying a fragment (-133 to –10) of IL-8 promoter. 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. With these results we propose that DEN2 core protein might interact with P-TEFb and NF-κB in regulating the production of IL-8. On the other hand, DEN2 core protein was found to inhibit the luciferase activity of plasmid containing the enhancer elements of p53 in a dose-dependent. Electrophoretic Mobility Shift Assay revealed that the DNA-binding affinity of p53 was decreased in the presence of DEN2 core protein when compared to vector only. Further investigations are needed to provide a plausible explanation of such findings.