Launching HBV with RNA to assess antiviral resistance and explore fundamental aspects of virus-host biology

Rockefeller University, New York NY

Investigators

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Abstract

Project Summary This proposal addresses the monumental health burden of chronic hepatitis B virus (HBV) infection, for which there is no effective cure. Long-term objectives are to develop and apply new methods to study HBV biology and make discoveries that inspire curative therapies. In this proposal, we build on the HBV RNA launch method and our experience with deep mutational scanning to address key points of interest among HBV advocates. These include understanding HBV genetic diversity and how it influences drug resistance. How mutations that commonly arise under immune pressure in the chronic phase of infection may alter disease pathogenesis. And how HBV organizes its genome into a multilayered masterpiece of economy. One specific goal of this proposal is to study mechanisms of resistance to core protein (Cp) allosteric modulators (CpAMs), a class of compounds that interfere with HBV nucleocapsid assembly or stability. We will develop a new cell culture method to accomplish this and complement and confirm results with in vivo studies in human liver chimeric mice (huFNRG). Our approach uses self-amplifying RNAs to deliver complex libraries of HBV variants to cultured cells, which overcomes certain obstacles with existing methods. These studies will provide a comprehensive, low-biased view of Cp sequence requirements. While the primary goal is to study mechanisms of CpAM resistance, the method is not limited to studying Cp, and comprehensive fitness maps generated in the process also have the potential to uncover basic truths of HBV biology. A second goal is to apply the HBV RNA launch method to study the effects of mutations in the HBV precore mRNA, which arise under immune pressure and reduce the production of the HBV e antigen. We hypothesize that besides reducing e antigen levels, certain clinically relevant mutations may also increase the production of double-strand linear viral DNA and, consequently, the frequency of viral DNA integration into host chromosomes. The dominant view in the literature is that HBV integrants are incapable of producing infectious viruses; however, they provide an alternative source for viral protein expression with important implications for ongoing clinical trials and the definition of a cure. Lastly, a third goal of this proposal is to continue applying the already fruitful RNA-based deep mutational scanning approach we established to further “deconstruct” the information-dense HBV genome and define protein and nucleic acid sequence requirements for its replication. Specifically, we focus on identifying requirements for each of the following steps: packaging the viral pregenomic RNA into nucleocapsids, reverse transcription to make DNA, and conversion into the stable, episomal form that persists in infected hepatocytes. The proposed work is important because chronic HBV infection is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide, and existing therapies are rarely curative. New tools and fundamental insights are needed to support a pipeline for therapeutic development leading to a cure, which we know is possible but is inefficient.