Dr. Alan McLachlan
My experience and qualifications make me particularly well-suited for my role as Principal Investigator in this project for a number of reasons. 1. I have worked on hepatitis B virus (HBV) gene regulation and its relationship to HBV replication since 1984. 2. In this time, my group has generated a large number of mutant viral genomes that permit the role of specific viral gene products and cellular transcription factors in viral biosynthesis to be determined under various conditions. 3. My group developed the first new HBV replication system in two decades which permitted the role of specific liver-enriched transcriptional factors in HBV biosynthesis to be determined. This complementation system utilizing essentially any transfectable nonhepatoma cell line established that nuclear receptors are the only transcription factors on their own that are capable of supporting viral transcription and replication in this system accounting for a major determinant of viral tropism. In addition, this system was used to demonstrate that the liver-enriched FoxA/HNF3 transcription factors (when transfected without coactivators) inhibit nuclear receptor-mediated HBV transcription and replication which led us to initiate our investigation into their role in vivo (which will be continued as part of the current application). Recently, we used this system to demonstrate FoxA/HNF3 in the presence of the appropriate coactivators was actually capable of supporting viral transcription and replication in nonhepatoma cells demonstrating that coactivators are limiting factors for HBV biosynthesis under certain circumstances. Furthermore, the utility, flexibility and innovation of this system has revealed that there are multiple independent mechanisms of transcriptional regulation of HBV biosynthesis that are possible (i.e. nuclear receptor and FoxA/HNF3 plus coactivators) explaining the failure to detect a single simple pathway of transcriptional regulation of HBV biosynthesis in hepatoma cell lines and HBV transgenic mice. Without the development of the HBV replication system in nonhepatoma cell lines, the identification of multiple independent pathways for the transcriptional regulation of HBV biosynthesis would not have been achieved. This is the great power of this system which permits individual aspects of the transcriptional regulation of HBV biosynthesis to be isolated from each other and characterized in detail prior to their evaluation in human hepatoma cell lines or in vivo in the transgenic mouse model of chronic HBV infection. 4. In addition, the nonhepatoma cell line HBV replication system has been used to support the conclusion that the human hepatoma cell lines, HepG2 and Huh7, display distinct transcriptional regulation of HBV biosynthesis indicating that these cell lines reflect two distinctly different physiological states that may or may not be relevant to the situation in vivo. These observations clearly demonstrate that the utilization of in vivo models of HBV biosynthesis are essential if a clearer understanding of the regulation of HBV biosynthesis during infection is to be achieved. This is essential if new antiviral therapeutics are to be developed. 5. Based on our various cell culture observations, it is clear that findings in cell culture must be supported by in vivo studies. The only available small animal model of chronic HBV infection is the HBV transgenic mouse model which I have used extensively to support our cell culture studies. Indeed, we demonstrated the essential requirement for HNF4 in the nonhepatoma cell culture system and in vivo using HBV transgenic mice. These studies clearly support the utility of both systems and the manner in which together they greatly enhance our understanding of the transcriptional regulation of HBV biosynthesis in a way that is not possible using only one of these systems. This has been the great strength of my program and it has permitted us to make significant progressive over the past few years. In addition, the analysis of FoxA/HNF3 in vivo in a natural extension of our previous studies. Indeed, FoxA/HNF3 appears an extremely interesting potential target for antiviral therapy as FoxA-deficiency results in the complete loss of HBV biosynthesis while largely maintaining normal liver physiological functions. 6. Of particular importance to the current proposal, we have demonstrated that FoxA-deficienct HBV transgenic mice have a hypermethylated genome which probably accounts for the lack of viral biosynthesis in the liver of these animals (i.e. they can be considered “cured” despite the presence of the HBV transgene). To my knowledge, this is first definitive demonstration of the connection between a specific “pioneer” transcription factor family, FoxA, and gene silencing by DNA methylation. 7. Furthermore, my group has generated a unique collection of transcription factor-null and coactivator/corepressor-null HBV transgenic mice that have been used to establish the in vivo role of these factors in the only available small animal model of chronic HBV infection. (Although hydrodynamic injection of HBV DNA into mice represents an alternative model system, it is simply not reproducible enough to be used for the types of analysis we perform). Importantly, we have previously generated and characterized HNF1-null HBV transgenic mice which synthesize nuclear HBV CCC DNA. These mice will be employed to examine the effects of FoxA-deficiency on the methylation status of HBV CCC DNA, the natural template for viral transcription during natural infection. 8. Most importantly, over the past several years we have generated and partially characterized several FoxA/HNF3-deficient HBV transgenic mice which are central to this proposal. The most interesting to date is the conditional liver-specific single-allele FoxA3-expressing HBV transgenic mice which lacks HBV biosynthesis. Determining the liver developmental stages where viral genome DNA methylation and chromatin structure alterations lead to the loss of viral biosynthesis in FoxA/HNF3-deficient HBV transgenic mice is a central goal of this proposal and may help to define convenient targets which are amenable to the development of antiviral therapeutics. Based on the above comments, it is clear that my laboratory has unique reagents and approaches that we can exploit to understand the role of the liver-enriched FoxA/HNF3 transcription factors in governing HBV biosynthesis. Understanding the molecular details of the transcription factors controlling HBV transcription and replication in vivo will identify potential targets for the development of antiviral therapeutics.