An efficient rapid system for assaying HBx-mediated transactivation
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.
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.
Collectively, the GFP-based reporter system provides a rapid and accurate approach for analyzing transactivation ability of HBx.
KeywordsCytomegalovirus promoter GFP Hepatitus B virus X protein HBx gene Transactivation Virus X protein
This work was supported by the National Nature Science Foundation of China (No. 81570567, 81571994), the Natural Science Foundation of Guangdong province, China (No. 2014A030313483, 2015A030313447), Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases, and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We would like to thank Dr. Stanley Lin for kindly revising this manuscript.
Supplementary Figure 1—Melting curves of qPCR showed good specificity of EGFP and HBx primers.
Supplementary Figure 2—ELISA standard curve for HBsAg (R square > 99%).
- Aufiero B, Schneider RJ (1990) The hepatitis B virus X-gene product trans-activates both RNA polymerase II and III promoters. EMBO 9:497–504Google Scholar