Xiaoling Zhou1,2,3, Haijun Shi1,2,3, Shaozhe Yang1,2,3, Pingnan Sun4,5,6. 1. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou, 515041, People's Republic of China. 2. The Center for Reproductive Medicine, Shantou University Medical College, Shantou, 515041, People's Republic of China. 3. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People's Republic of China. 4. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com. 5. The Center for Reproductive Medicine, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com. 6. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People's Republic of China. Pingnan_sun@yahoo.com.
Abstract
OBJECTIVES: To develop a rapid and accurate assay system for screening inhibitors or enhancing agents targeting the transactivation capability of hepatitis B virus X protein (HBx) that activates cellular promoters in host cells to facilitate viral replication. RESULTS: We constructed a new GFP-based reporter system which was different from a luciferase-based reporter system. Firstly, a FLAG-tagged HBx gene was inserted into an expression plasmid, resulting in plasmid pHBx. Next, HBx-FLAG was linked to EGFP by the internal ribosome entry site resulting in plasmid pHBxE. The transactivation effect of HBx-flag on cytomegalovirus (CMV) promoter was verified by EGFP expression using fluorescence quantitation and qPCR. Furthermore, the transactivation ability of the HBx gene was quantified by flow cytometry. Finally, this assay system was tested by known regulators of HBx including DDB1, ID1, and P53. As expected, the GFP reporter level in 293T cells changed with the increasing of HBx regulators. Furthermore, the system modeling the function of transactivation repressor in Hep3B, a HBV-integrated cell line. CONCLUSION: Collectively, the GFP-based reporter system provides a rapid and accurate approach for analyzing transactivation ability of HBx.
OBJECTIVES: To develop a rapid and accurate assay system for screening inhibitors or enhancing agents targeting the transactivation capability of hepatitis B virus X protein (HBx) that activates cellular promoters in host cells to facilitate viral replication. RESULTS: We constructed a new GFP-based reporter system which was different from a luciferase-based reporter system. Firstly, a FLAG-tagged HBx gene was inserted into an expression plasmid, resulting in plasmid pHBx. Next, HBx-FLAG was linked to EGFP by the internal ribosome entry site resulting in plasmid pHBxE. The transactivation effect of HBx-flag on cytomegalovirus (CMV) promoter was verified by EGFP expression using fluorescence quantitation and qPCR. Furthermore, the transactivation ability of the HBx gene was quantified by flow cytometry. Finally, this assay system was tested by known regulators of HBx including DDB1, ID1, and P53. As expected, the GFP reporter level in 293T cells changed with the increasing of HBx regulators. Furthermore, the system modeling the function of transactivation repressor in Hep3B, a HBV-integrated cell line. CONCLUSION: Collectively, the GFP-based reporter system provides a rapid and accurate approach for analyzing transactivation ability of HBx.
Entities:
Keywords:
Cytomegalovirus promoter; GFP; HBx gene; Hepatitus B virus X protein; Transactivation; Virus X protein