Journal: PLoS Pathogens

Article Title: HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

doi: 10.1371/journal.ppat.1006658

Figure Lengend Snippet: C3A hNTCP 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 pan e l , 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.

Article Snippet: Alpha-interferon (IFN-α) was purchased from PBL Assay Science.

Techniques: Infection, Real-time Polymerase Chain Reaction, Hybridization, Southern Blot, Labeling, Molecular Weight, Marker, Northern Blot

Journal: PLoS Pathogens

Article Title: HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

doi: 10.1371/journal.ppat.1006658

Figure Lengend Snippet: (A) C3A hNTCP 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) C3A hNTCP 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).

Article Snippet: Alpha-interferon (IFN-α) was purchased from PBL Assay Science.

Techniques: Infection, Real-time Polymerase Chain Reaction