HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B

Abstract

At least 250 million people worldwide are chronically infected with HBV, a small hepatotropic DNA virus that replicates through reverse transcription. Chronic infection greatly increases the risk for terminal liver disease. Current therapies rarely achieve a cure due to the refractory nature of an intracellular viral replication intermediate termed covalently closed circular (ccc) DNA. Upon infection, cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (RC) DNA genome in incoming virions. Its fundamental role is that as template for all viral RNAs, and in consequence new virions. Biosynthesis of RC-DNA by reverse transcription of the viral pregenomic RNA is now understood in considerable detail, yet conversion of RC-DNA to cccDNA is still obscure, foremostly due to the lack of feasible, cccDNA-dependent assay systems. Conceptual and recent experimental data link cccDNA formation to cellular DNA repair, which is increasingly appreciated as a critical interface between cells and viruses. Together with new in vitro HBV infection systems, based on the identification of the bile acid transporter sodium taurocholate cotransporting polypeptide as an HBV entry receptor, this offers novel opportunities to decipher, and eventually interfere with, formation of the HBV persistence reservoir. After a brief overview of the role of cccDNA in the HBV infectious cycle, this review aims to summarise current knowledge on cccDNA molecular biology, to highlight the experimental restrictions that have hitherto hampered faster progress and to discuss cccDNA as target for new, potentially curative therapies of chronic hepatitis B.

A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

“…HBV covalently closed circular DNA (cccDNA), the major form of the viral genome in infected hepatocytes, represents an intractable barrier to eradication of the virus and is resistant to the currently available therapies [6]. Several previous studies have explored the potential of the CRISPR/Cas9 system to target HBV cccDNA in mice by hydrodynamic injection of plasmid DNA [7][8][9][10][11]; however, therapeutically relevant in vivo delivery methods are still lacking [12].…”

Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus

“…Cells were transfected using polyethylenimine with the following combinations of plasmids: 150 ng pCMV-GFP, 250 ng HDR donor templates and 2.3 μg of each plasmid expressing the left and right subunits of the TALENs, or 4.6 μg of pUC118. Cells were maintained under mildly hypothermic conditions (30 °C and 5% CO 2 ) [4]. Growth medium was replaced and HBsAg concentrations were measured on days 3, 5 and 7.…”

“…Corroboration of the intended HDR was carried out by sequencing the amplicons (Inqaba Biotechnologies, Pretoria, South Africa). To measure targeted indel formation, HBV DNA was extracted from HepG2.2.15 cells and subjected to analysis using a T7E1 assay as has been described [4].…”

“…Although these therapies diminish replication of HBV, treatment interruption usually results in relapse with proliferation of the virus. The main reason for the modest cure rate of licensed regimens is that efficacy of the drugs is short-lived and the transcriptional template of HBV, comprising covalently closed circular DNA (cccDNA), is unaffected by available drugs [4] and [5]. Licensed vaccination against the virus usually prevents HBV infection, but has little or no therapeutic benefit.…”

Immune therapy for hepatitis B

“…Most antiviral drugs for CHB are capable of reducing or completely blocking replication of replicating HBV. However, they are not so effective to eliminate cccDNA from HBVinfected hepatocytes (19,20 (27)(28)(29). Host immunity is dichotomous during CHB infection, altering between pathogenic and protective/therapeutic states.…”