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 induced by hepatitis B virus infection is a complex and intricate process involving interactions of multiple host factors with the virus and/or the viral gene products. The HBV X (HBV) 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. Using the yeast two-hybrid system we identified that HBX interacts with two subunits of the 26S proteasome (PSMA7 and PSMC1). The 26S proteasome complex is the predominant cellular factor 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. We further demonstrated an association in vivo of HBX with the 26S proteasome complex by co-immunoprecipitation and co- localization upon sucrose gradient centrifugation. Analysis of the interacting domains among HBX and the proteasome subunits by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid disruptor system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 were important for its function as a transcriptional coactivator. Expression of HBX in HepG2 cells caused a modest decrease in the proteasomes chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBX functions as an inhibitor of proteasome. In these cells, HBX is degraded with a half life of 30 min. Proteasome inhibitors retarded this rapid degradation and caused a marked increase in the level of HBX and an accumulation of HBX in polyubiquitinated form. Thus, the low intracellular level of HBX is due to rapid proteolysis by the ubiquitin-proteasome pathway. Surprisingly, the proteasome inhibitors blocked the transactivation by HBX and this effect was not a result of a squelching phenomenon due to HBX accumulation. Therefore, proteasome function is possibly required for the transactivation function of HBX. To further study the effects of HBX in vivo, we established a HBX transgenic mouse model using the mouse major urinary protein (MUP) promoter to express HBX in a liver-specific and developmentally regulated manner, i.e post-partum. The hepatic proteasome activities were significantly inhibited up to 40% in HBX transgenic mice. Similar to our tissue culture studies, both 20S and 26S activities were affected. Interestingly, the inhibition of proteasome activities by HBX gradually disappeared in older mice, suggesting an adaptive response of the mice to the effect of HBX on proteasome in vivo. We plan to use the cDNA microarray technology to evaluate the changes in gene expression patterns of these transgnenic mice. The inhibition of proteasome function by HBX may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation. Our laboratory is also conducting experiments to study the biological significance of this interaction in the woodchuck model. Finally, experiments are under way to target the HBX-proteasome interaction as a novel antiviral approach. - Virus-Host Interaction/Viral Persistence/Proteolysis/Proteasome
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