<italic>In vitro</italic> experimental models for the study of hepatitis B virus cccDNA

Abstract

Chronic infection of hepatitis B virus (HBV) remains a global public health problem and affects at least 240 million people worldwide. HBV is a small enveloped DNA virus belonging to the Hepadnaviridae family. The virus has a narrow host range, limited to humans and chimpanzees. In the nucleus of infected hepatocytes, the partially double-stranded DNA (rcDNA) HBV genome is converted to covalent closed circular DNA (cccDNA), which serves as the sole template for HBV transcription. HBV cccDNA exists as an episomal minichromosome in the nucleus, with a turnover rate that correlates with the mitosis or death of virus-infected hepatocytes. It is thus regarded as a primary molecular mechanism for viral persistence. In the past decade, significant progress has been made in HBV research and the related antiviral development. Sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a receptor for HBV entry into hepatocytes. It was recently shown that HBV x (HBx), an enigmatic viral protein, achieves its regulatory function by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the Smc5/6 complex for degradation. Although still controversial, type I interferon has been implicated to either affect the epigenetic control of cccDNA minichromosome, or induce cytidine deaminases-mediated specific degradation of the episomal cccDNA. Of particular note, core protein allosteric modulator (CpAM) has been most widely investigated. These studies represent a new frontier in the development of antivirals against HBV infection. Despite continuing progress in HBV research, a complete cure of HBV infection is still not achievable. Current antiviral therapies with nucleos(t)ide analogs (NUC) and pegylated IFN-α can efficiently inhibit HBV replication, but the treatments fail to eliminate the preexisting cccDNA pool that is responsible for a viral rebound after therapy cessation. Therefore, it is widely held that cccDNA elimination is a prerequisite for complete cure of chronic HBV infection. To this end, it is essential to understand the mechanisms involved in cccDNA metabolism and transcriptional regulation. Studies of HBV cccDNA have often been hampered by the low copy number of cccDNA per cell and the lack of convenient techniques with high sensitivity and specificity for cccDNA detection. Development of appropriate experimental models is thus fundamental for HBV cccDNA studies. Herein, we reviewed recent advances in cell culture models for the study of HBV cccDNA, including models of HBV de novo infection and cccDNA biogenesis, cccDNA formation (rcDNA repair-related molecular events), HBV replicon-based transfection models to study established cccDNA, and the surrogate model of cccDNA produced from site-specific DNA recombination. Animal models to study HBV cccDNA will be further discussed by other authors in this issue. We hope this review may help clinicians and researchers who are interested in the study of cccDNA and antiviral development against HBV.