A CRISPR-based system to investigate HBV cccDNA biology

Abstract

ABSTRACT Chronic hepatitis B virus (HBV) infections affect over 250 million people worldwide, and over 800,000 are expected to die yearly from complications including cirrhosis and primary hepatocellular carcinoma. While clinically available nucleoside analog-based antiviral therapies inhibit virus production, they cannot cure infections due to the persistence of covalently closed circular (cccDNA) in the nuclei of infected hepatocytes. Hence, the goals of future curative antiviral therapies for chronic hepatitis B are to either eliminate or permanently silence transcription from cccDNA. Novel antiviral approaches building on a better understanding of the mechanisms underlying transcriptional regulation and the stability of HBV cccDNA will be necessary to accomplish such goals. To advance our understanding of cccDNA biology, we have employed a CRISPR/Cas9-based system to produce extrachromosomal circular (ec) DNA from plasmid DNA integrated into the host genome originally developed by Moller et al. [Nucleic Acid Research 46:(22) e131, 2018]. We modified the enhanced green fluorescent protein (EGFP) reporter system to include portions of the HBV genome encoding wild-type and inactive hepatitis B virus X (HBx) proteins on ecDNA to mimic cccDNA. We demonstrated that expression of EGFP is inhibited by the SMC5/6 complex, as is cccDNA, and that HBx expression can reverse transcriptional silencing of ecDNA. Moreover, we demonstrated that ecDNA is lost during cell division. The system described in this report permits investigations on ecDNA and enables large-scale screening of compound libraries for small molecules targeting host factors involved in ecDNA and, by inference, cccDNA transcription, maintenance, and loss during cell division. IMPORTANCE Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.