Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators

Abstract

Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.

Epigenetic regulation of hepatitis B virus covalently closed circular DNA: Implications for epigenetic therapy against chronic hepatitis B

“…Inhibition of deacetylation by HDACis has been shown to enhance HBV transcription and replication in vitro. (37,62) In addition, HDACis may also potentially reactivate latent DNA virus infection in patients receiving them as anticancer drugs. (63) IFN-a is the only approved immunomodulator for CHB treatment.…”

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

“…Using improved methods (e.g., digital droplet PCR), this may be even further improved for high-throughput screening approaches. Thus, the direct cccDNA assay complements a recently developed cell culture system with HepDE19 and HepBHAe82 cells in which HBeAg serves as a surrogate marker for cccDNA transcriptional activity (45)(46)(47).…”

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

“…To overcome this problem, others [80] and we (Dörnbrack, K.; Costa, C.; Verrier, E.; Nassal, M; unpublished data) have engineered coding sequences for the small hemagglutinine (HA) tag into the precore regions of HBV and/or DHBV such that only HBeAg becomes HA-tagged and the negative impact on replication via the precore-overlapping ε sequence remains limited. Figure 5C shows the accumulation of nuclear cccDNA (and RC-DNA) in a TetOFF HepG2 line producing HA-tagged DHBeAg.…”