Chronic infection with hepatitis B virus (HBV) is associated with a high incidence of liver diseases, including hepatocellular carcinoma (HCC). Hepatitis B virus x antigen (HBxAg), one of the four proteins encoded by the virus, stimulates virus gene expression and replication, and is crucial for the establishment and maintenance of the chronic carrier state. Intrahepatic HBxAg expression also correlates with the intensity and progression of liver disease, suggesting it plays a central role in the pathogenesis of chronic infection. HBxAg transforms liver cells in vitro and in vivo, and results in the development of HCC in X transgenic mice, suggesting it plays a central role in hepatocarcinogenesis as well. Changes in hepatocellular phenotype may result, in part, from the ability of HBxAg to alter host gene expression. Together, this data (accumulated over the past 20 years) demonstrate that HBxAg is an important target for the development of specific inhibitors that would be effective against virus replication, chronic liver disease, and tumor development. The problems in developing such inhibitors involves the inability to stably express intracellular HBxAg at high levels (which is usually toxic) and the inability to isolate enough soluble HBxAg in native (functional) form in vitro to set up screening assays for drug discovery and to conduct crystallographic analysis for rationale drug design. Hence, this application proposes to express and isolate HBxAg from E. coli, to refold it in vitro, and then to test for HBxAg trans-activation function in cell based assays (aim 1). In addition, we propose to establish conditions for scale up production of HBxAg for crystallographic work and for establishing assays that would be useful for high throughput screening (aim 2). The long-term objective of this study is to obtain high-resolution structure of bioactive of HBxAg for small molecular drugs development to treat both viral infection and HCC.
