Fig 1.
NTCP expression in HepG2-NTCP12 cells.
The stable expression of C9-tagged NTCP in HepG2-NTCP12 cells were detected by Western blot (A) and immunofluorescence microscopy (B) using antibody against C9 epitope. The parental HepG2 cells served as negative control.
Fig 2.
Quantification of the genome equivalent of HBV virion DNA in concentrated HBV particles.
(A) HBV virions and naked capsids in the indicated volume of virus stock were separated by native agarose gel electrophoresis, and viral DNA was detected by hybridization. The virion DNA to capsid DNA ratio was calculated by ImageQuant IQTL software using the hybridization signal intensity. (B) HBV DNA were extracted from 10 μl of virus stock and subjected to Southern blot analysis, 100 pg of 3.2 kb HBV linear DNA (approximately 3.1×107 HBV DNA copies quantified by qPCR) served as loading marker. HBV DNA replicative intermediates, including relaxed circular (RC) DNA and single stranded (SS) DNA were labeled. The copy number of total HBV DNA was quantified by using the HBV DNA loading marker as standard. (C) Formula for calculation of HBV virion DNA genome equivalent (v.g.e).
Fig 3.
DMSO enhances HBV infection in HepG2-NTCP12 cells.
Approximately 5×105 HepG2-NTCP12 cells were mock infected or infected with HBV (100 g.e/cell) in the absence or presence of 2% DMSO. DMSO was added to the PMM medium 24 h prior to the infection and remained present in the entire culture period until the cells were harvested. (A) Cells were immunostained with antibodies against HBV capsid protein (HBcAg) and visualized under fluorescence microscopy. Nuclei were counterstained with DAPI. The image shown represents five different microscopic fields, the percentage of HBcAg-positive cells was indicated (Mean ± SD) (similarly hereinafter). (B) HBV RNA were extracted from the infected cells and analyzed by Northern blot hybridization by using an [α-32P] UTP-labeled plus-strand-specific full-length HBV riboprobe. HBV 3.5 kb precore/pregenomic RNA and the 2.4/ 2.1 kb subgenomic RNA are labeled. Cellular 28S and 18S ribosomal RNA served as loading control. C9-tagged NTCP expression was detected by Western blot with β-actin serving as loading control.
Fig 4.
HBV infection of HepG2-NTCP12 cells with different viral inoculum size.
Cells were mock infected or infected with HBV at indicated inoculum size (vge/cell) in the presence of 2% DMSO. Seven days later, the cells were subjected to HBcAg immunofluorescence microscopy (A). The intracellular HBV RNA and core DNA were analyzed by Northern and Southern blot, respectively (B).
Fig 5.
Centrifugal inoculation of HBV.
HepG2-NTCP12 cells in 24-well-plate were inoculated by HBV (500 vge/cell) without centrifugation or at different centrifugal force (g) for 30 min. After spinoculation, the cells were transferred to regular culture condition. 7 days later, the infected cells were analyzed by HBcAg immunofluorescence (A), the levels of intracellular HBV RNA transcription and DNA replication were determined by Northern and Southern blot, respectively (B).
Fig 6.
Effect of centrifugation time on HBV spinoculation.
HepG2-NTCP12 cells were inoculated with HBV (500 vge/cell) by centrifugation at 1,000×g for different time as indicated. 8 days post inoculation, HBV infection was analyzed by HBcAg immunofluorescence (A), viral RNA and core DNA hybridization (B), and HBV cccDNA produced by 60-min-spinoculation was detected by Southern blot and validated by heat denature and EcoR I linearization (C).
Fig 7.
Spinoculation-mediated HBV infection is NTCP-dependent.
HepG2 and HepG2-NTCP12 cells were spinoculated with HBV (500 vge/cell, 1,000×g, 60 min) in the absence or presence of indicated compounds. 8 days post inoculation, the infected cells were analyzed by HBcAg immunofluorescence.