Fig 1.
HBV capsid assembly modulators efficiently inhibit cccDNA formation during de novo HBV infection of C3AhNTCP cells.
C3AhNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with 100nM of MyrB, 1 μM of ETV, 2.5 μM of Bay 41–4109, 1 μM of GLS-4, 5 μM of ENAN-34017 or 1,000 IU/ml of IFN-α, starting from 24 h before infection until harvesting at 3 or 6 days post infection (dpi). HBV cccDNA (A), pgRNA (B) and cytoplasmic core DNA (C) were quantified by real-time PCR assays. Differences in viral cccDNA, core DNA or pgRNA between mock-treated control and treated groups were statistically analyzed (t-test, * p <0.05, ** p <0.01, *** p <0.001). (D) Hybridization analyses of HBV replication intermediates in cells harvested at 6 dpi. Upper panel, Hirt DNA extracted from the cells harvested at 6 dpi were denatured at 88°C for 5 min to denature DP-rcDNA to single-stranded DNA and followed by restriction with EcoRI to convert cccDNA into unit-length double stranded linear DNA and detected by Southern blot hybridization (labeled as CCC/EcoRI). Unit-length HBV linear DNA served as a molecular weight marker. Lower panel, HBV RNAs, pre-genomic RNA (pgRNA), 2.4 and 2.1kb mRNA specifying envelope proteins were determined by Northern blot hybridization. 28S and 18S ribosomal RNA (rRNA) served as loading controls.
Fig 2.
Inhibition of cccDNA formation by HBV capsid assembly modulators is time- and concentration-dependent.
(A) C3AhNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with 100 nM of MyrB, 1 μM of ETV, 2.5 μM of Bay 41–4109, 1 μM of GLS-4, 5 μM of ENAN-34017 or 1,000 IU/ml of IFN-α., starting from 24 h before infection, at infection or 24 h post infection until harvesting at 3 days post infection. HBV cccDNA was quantified by a real-time PCR assay. (B) C3AhNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with the indicated concentrations of Bay 41–4109, GLS4 or ENAN-34017, starting from 24 h before infection until harvesting at 3 days post infection. HBV cccDNA were quantified by a real-time PCR assay. Differences in viral cccDNA between mock-treated control and treated group cultures under each treatment schedule were statistically analyzed (t-test, * p <0.05, ** p <0.01, *** p <0.001).
Fig 3.
HBV capsid assembly modulators accelerate cccDNA formation through the intracellular amplification pathway.
As depicted in panel (A), HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 after removal of tet from medium. On day 6, PFA-containing medium were removed and the cells were mock-treated or treated with 1 μM of ETV, 2.5 μM of Bay 41–4109 or 5 μM of ENAN-34017 and harvested at 0, 3, 6, 9 and 12 h of the treatment. The cytoplasmic core DNA (B), cccDNA and DP-rcDNA (C) were analyzed by Southern blot hybridization. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated. For cccDNA and DP-rcDNA analysis, before loading, Hirt DNA were denatured at 88°C for 5 min, which denatures DP-rcDNA into single-stranded DNA (labeled as denatured DP-rc).
Fig 4.
Quantitative analyses of the effects of CpAMs on cccDNA synthesis.
HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 after removal of tet from medium. On day 6, PFA-containing medium were removed and the cells were mock-treated or treated with 1 μM of ETV, 1 μM of GLS4, 2.5 μM of Bay 41–4109 or 5 μM of ENAN-34017 and harvested at 0, 6, 12 and 24 h of treatment. (A) Hirt DNA were extracted and resolved in 1.5% agarose gel electrophoresis. HBV DNA were detected by Southern blot hybridization. (B) The amounts of cccDNA in each sample were quantified with Tyhoon FLA 7000 and plotted as arbitrary units. (C) Hirt DNA extracted from cells treated with the indicated compounds for 12 h after PFA removal were denatured at 88°C for 5 min and followed by EcoRI digestion. The DNA samples were then resolved in agarose gel and HBV DNA were detected by Southern blot hybridization (left panel). The amounts of cccDNA in each samples were quantified and normalized to the levels of mock-treated controls. The relative amounts of cccDNA were plotted (right panel). The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and cccDNA (CCC) species were indicated. * p < 0.05, ** p < 0.01 (by t-test).
Fig 5.
HBV capsid assembly inhibitors do not inhibit completion of positive-strand DNA synthesis, but render the mature forms of HBV DNA sensitive to DNase I digestion.
HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 post removal of tet from medium. Intracellular capsids were purified by sucrose gradient centrifugation. (A) Endogenous DNA polymerase reactions with the purified HBV capsids were performed in the absence or presence of dNTP, 2.5 μM of Bay 41–4109, 5 μM of ENAN-34017 or 25 μM of AT-61 at 37°C for 16 h. Three-fourths of each reaction were subjected for extraction of viral DNA without or with prior DNase I digestion at 37°C for 30 min. The viral DNA were detected by Southern blot hybridization with full-length riboprobe recognizing minus strand DNA. The remaining one-fourth of each reaction was analyzed by a particle gel assay to detect HBV capsids. (B) Endogenous DNA polymerase reactions were performed in the presence of dNTP and indicated concentrations of Bay 41–4109, ENAN-34017 or AT-61 at 37°C for 16 h. Viral DNA and capsids were analyzed as described above. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated.
Fig 6.
HBV capsid assembly modulator treatment confers HBV virion DNA sensitivity to DNase digestion.
(A) Each of the endogenous DNA polymerase reaction contains approximately 108 HBV virions prepared from chronic HBV carriers and incubated in the presence or absence of dNTP and indicated concentrations of Bay 41–4109, ENAN-34017 or GLS4 at 37°C for 16 h. Viral DNA were extracted without or with prior DNase I digestion at 37°C for 30 min and detected by Southern blot hybridization with full-length riboprobe recognizing minus strand DNA. (B) Approximately 108 HBV virions in each endogenous DNA polymerase reaction were incubated in the absence of dNTP (except for lanes 2, 3, 19 and 20) and absence or presence of the indicated concentrations of Bay 41–4109, ENAN-34017 or GLS4 at 37°C for 16 h. Viral DNA were extracted and analyzed as described above.
Fig 7.
CpAM treatment confers double-stranded DNA in cytoplasmic progeny nucleocapsids sensitive to DNase I digestion in a concentration- and time-dependent manner.
HBV capsids purified from HepAD38 cells were mock-treated or treated with the indicated concentrations of GLS4, Bay 41–4109 or ENAN-34017 in reactions containing 150 mM NaCl, 50 mM Tris-HCl, pH8.0, 10 mM MgCl2, 1 mM DTT and 0.1% NP-40 at 37°C for 16 h (A) or harvested at the indicated time of incubation (B). Viral DNA were extracted without or with prior DNase I digestion at 37°C for 30 min and detected by Southern blot hybridization with a full-length riboprobe that hybridizes to negative strand of HBV DNA. Partial DS: partially double-starnded DNA. Partial DS: partially double stranded DNA.
Fig 8.
Differential effects of different chemotypes of CpAMs on integrity of mature double-stranded DNA-containing nucleocapsids.
HBV capsids prepared from HepAD38 cells were mock treated (A) or incubated with 5 μM ENAN-34017 (B), 3 μM Bay 41–4109 (C) or 3 μM GLS4 (D) in a reaction containing 150 mM NaCl, 50 mM Tris-HCl, pH8.0, 10 mM MgCl2, 1 mM DTT and 0.1% NP-40 for at 37°C for 16 h. The reactions were then fractioned by sucrose gradient centrifugation. HBV capsids in each of viral DNA-containing fractions, as detected by a qPCR assay, were determined by a 1.5% native agarose gel electrophoresis-based particle gel assay. HBV DNA in the fractions were detected by Southern blot hybridization with a riboprobe that specifically hybridizes to negative strand of HBV DNA. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated. Sucrose concentration (w/v %) of each of the fractions is provided. The mature forms of HBV DNA species (RC and DSL) are highlighted in red-dash lined box.
Fig 9.
A schematic representation of the multiple mode of action of CpAMs on HBV capsid assembly and disassembly as well as their effects on cccDNA biosynthesis.
The red arrowed lines indicate the normal processes in viral replication. The black arrowed lines indicate CpAMs-induced processes. See text for detailed explanation.