Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Abstract

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC 50 : 1.4 nM) and interferon-α (IC 50 : 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC 50 : 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus

“…Since HBV envelope proteins enable secretion of HBV subviral particles and support envelopment of the HDV RNPs, we stably transduced the HuH7-HDV with a lentiviral vector harboring a 2.7-kb HBV fragment encoding the HBV envelope proteins under control of their native promoter/enhancer 20 . Such a construct has also been successfully implemented in the fully replication-competent cell line HepNB2.7 16 . Following selection with blasticidin, the cell pool (referred to as HuH7-HDV-Env) secreted HBsAg as well as infectious HDV virions, as shown by quantitative HBsAg ELISA and infection of HepG2-NTCP cells using cell culture supernatants (Supplemental Fig.…”

“…pWPI-NTCP-GFP was constructed by replacing the puromycin resistance gene GFP. Plasmid pLX304-HB2.7 is a lentiviral vector expressing HBV envelope proteins 16 , which was constructed by insertion of the HBV sequence from pT7HB2.7 into the lentiviral vector pLX304 36 . The sequence of all constructs was verified by Sanger sequencing (GATC Biotech).…”

“…To study HDV secretion in vitro , current systems rely on co-transfection of hepatic cells with HDV- and HBV envelope-expressing plasmids 15 or the HepNB2.7 cell line that supports the release of progeny HDV upon infection 16 . While the HBV field has developed several virus-replicating cell lines (such as HepG2.2.15 17 and HepAD38 18 , which have been used for virus production, investigation of the viral replication cycle and identification of antiviral drug candidates), there is currently no equivalent stable cell line that supports continuous HDV replication and secretion of infectious HDV particles.…”

New insights into HDV persistence: The role of interferon response and implications for upcoming novel therapies

“…The three J o u r n a l P r e -p r o o f membrane-embedded HBV envelope proteins small (S-), middle (M-) and large (L-), collectively termed HBsAg, are encoded by the covalently closed circular DNA (cccDNA) in HBV replicating hepatocytes and the integrated HBV DNA in chromosomes. [21][22][23][24][25] The HDV replication cycle is depicted in Fig. 1.…”

“…Application of newly developed cell culture models, e.g. cell lines expressing both, the HDV receptor and the HBV envelope thereby supporting the full HDV life cycle, 23,24 will be helpful to better understand to what extend these HDV-specific countermeasures contribute to the surveillance of immune recognition. 103 Of note, the sensitivity of HDV RNA quantification assay at that time was low 104 and therefore rates of serum RNA negativation were probably overestimated.…”