Infection with hepatitis B virus (HBV) is a major cause of liver disease worldwide and affects more than 1 million people in the United States. Hepatitis caused by hepatitis B virus infection is a complex and intricate process involving interaction of multiple host factors with the virus and/or the viral gene products. The HBV X (HBX) gene plays a crucial role in the life cycle and oncogenic potential of HBV. Since virus-host interactions are central to the pathogenesis of viral infection and host injury, this project aims to elucidate the cellular and molecular mechanisms of HBX-host interactions during HBV infection. We have previously shown that HBX interacts with the proteasome complex in vitro and in vivo. The 26S proteasome complex is the predominant cellular machinery which degrades cellular proteins in both ubiquitin-dependent and -independent pathways. It has been implicated in the regulation of a variety of transcriptional and cell cycle factors, cellular stress response, and antigen presentation. HBX interacts with the proteasome subunits through a mutually competitive structural relationship. The crucial HBX sequences involved in the interaction with the proteasome complex are important for its function as a transcriptional coactivator. We have also shown that HBX functions as a substrate as well as an inhibitor of proteasome. Using the woodchuck model, we demonstrated that the X-defective mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behave like attenuated viruses in the woodchuck model. However woodchucks inoculated with X mutants including those with no serologic evidence of infection were protected from the challenge, suggesting prior infection resulting in protective immunity. To further study the role of the proteasome in the biology of HBX, we analyzed the effects of the proteasome inhibitors on the replication of hepadnaviruses both in vitro and in vivo. For in vitro study, recombinant adenovirus or baculovirus expressing replicating HBV or WHV genome were generated to study viral replication in culture. In HepG2 cells infected with either the adeno-HBV or bv-WHV, the replication level of the X-negative virus was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. For in vivo study, HBV transgenic mice expressing either replicating wild-type or X-negative HBV were injected intravenously with proteasome inhibitor MLN-273 (Millennium Pharmaceuticals) at the age of 6 to 8 weeks. In general, the HBV DNA levels in the sera and the replication levels in the livers of the X-negative mice were much lower than those of the wild-type mice at this age. The sera and livers were collected at 0, 1, and 4 weeks post-injection. The sera were tested for HBV DNA by quantitative PCR and the livers were analyzed for replicative intermediates. In the wild-type HBV mice injected with proteasome inhibitor MLN-273, the HBV DNA level in the sera and the replication level in the livers were not significantly affected. At week 1 post-injection of proteasome inhibitor MLN-273, the level of HBV DNA in the serum of the X-negative mice was enhanced to more than 100-fold of the week 0 level. This increase was also reflected in a significant higher level of replicative intermediates in the liver. At week 4 post-injection, the HBV DNA levels in the sera and livers returned to the baseline level. Together, our data suggest that HBX functions in hepadnaviral replication through a proteasome-dependent pathway in both tissue culture and HBV transgenic mouse model. Because of the importance of HBX in HBV life cycle, we aim to develop potential anti-HBV agents by targeting the functions of HBX using a random combinatorial approach. We developed a modified yeast two-hybrid disruptor system to screen a random peptide aptamer library which uses the bacterial protein TrxA as a platform to display the randomly synthesized peptide aptamers. The peptide aptamers which disrupted HBX-PSMA7 (a proteasome subunit) interaction were cloned into CMV expression vector for transfection studies. The effects of these peptide aptamers on HBX transaction, HBV replication, transcription, and antigen expression were characterized in HepG2 cells. By screening 1.5 x 10E7 yeast colonies with HBX and PSMA7 as interacting pair, and a random peptide aptamer library as disruptors, 367 yeast tranformants were isolated. On secondary screening, 21 colonies were confirmed to show specific disruption of the HBX-PSMA7 interaction. The peptide aptamers from these yeast colonies were isolated, sequenced, and cloned into a CMV-driven construct for transfection in HepG2 cells. Transactivation assays showed that these peptide aptamers could interfere with the effect of HBX transactivation on RSV-Luc reporter by increasing or decreasing the luciferase activities. When co-transfected with a HBV replication competent construct, many of the peptide aptamers which inhibited the HBX transactivation could suppress HBV DNA replication by about 50 to 60%. Our results demonstrate that selection of random peptide apatamers based on disruption of the HBX-proteasome interaction in a modified yeast two-hybrid system may identify potential therapeutic drugs for HBV infection.
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