Fig 2. Knockdown of HGS destabilized ESCRT-0 complex and inhibited core protein expression and HBV replication.

(A) Suppression of viral DNA replication was detected by Southern blot analysis in HepG2 and HuH-7 cells transfected with an HBV genomic dimer and siRNAs specific for HGS or STAM2. Signal intensity from RC and SS bandings was quantified by Image J software. (B) Significant reduction of virion-associated viral DNA was detected by qPCR in HepG2 cells upon depletion of ESCRT-0 components. Relative levels of HBV DNA in samples treated with si-HGS or si-STAM2 were normalized to that of the control sample treated with non-targeting siRNA (si-NT). Data here are representative of three independent experiments. (C) A co-depletion phenomenon was observed in HGS-depleted ESCRT-0 complex by SDS-PAGE analysis. Knockdown of HGS resulted in the concurrent reductions of STAM1, STAM2 and HBc protein expressions when the ESCRT-0 complex was destabilized. (D) The siRNA rescue experiment was performed by co-transfecting a constant amount of HBV replicon DNA with different doses of HGS expression vector (up to 0.2 μg per well in 6-well plate) in si-HGS (50 nM) treated HepG2 cells. Complementation of HGS efficiently restored the HGS protein level in a dose-dependent manner (lanes 2–4), as well as HBV replication. (E) Knockdown of ESCRT-0 factors inhibited HBV enhancer II and core promoter activity. A mixture of the reporter plasmid pGL3-EnhII-Cp, a control plasmid pRL-TK, and 50 nM siRNA, were co-transfected into HepG2 cells. Three days post-transfection, cell lysates were analysed by Dual-Glo Luciferase Assay System. The firefly luciferase activity driven by the HBV EnhII-Cp was always normalized first to an internal control of the renilla luciferase activity. The value of the Y-axis represents the relative luciferase activity in each siRNA-treated sample over the control siRNA-treated sample. Data are representative of three independent experiments.