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The pHBV1.3 (serotype adw, genotype B) plasmid was generated from the HBV genome (GenBank accession number JN406371) inserted into the pUC18 plasmid. The Core-HA plasmid contained the HBV core protein gene, was digested with Kpn Ⅰ/Hind Ⅲ and inserted into pXJ40-HA. Filamin B-GFP (designated FLNB-GFP) was provided as a generous gift from Professor Arnoud Sonnenberg (Netherlands Cancer Institute). We used the wild-type Core-HA plasmid (designated WT-HA) as a template to generate mutated versions of the Core-HA plasmid (designated M1-HA, M2-HA, M3-HA, M4-HA; each involving one of the four blocks of arginine residues), and the primers for the mutants are listed in Supplementary Table S1. Small interfering RNAs (siRNAs; siFLNB-1, siFLNB-2, siFLNB-3) and the negative control (siNC) were purchased from RiboBio (Guangzhou, China).
Table S1. Primer sequences used in the construction of mutants
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HEK 293T cells, Huh7 cells and HepG2 cells (from the American Type Culture Collection [ATCC]) were maintained in Dulbecco's modified Eagle's medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco) at 37 ℃ and 5% CO2. Cells were seeded in 6-well plates (4 × 105 cells/well) or 12-well plates (2 × 105 cells/well) and grown to ~ 70% confluence at the time of transfection. The siRNA and plasmids were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China).
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Cells grown in 10-cm2 culture dishes, and FLNB-GFP or GFP (10 μg) were co-transfected with the Core-HA plasmid (10 μg) using Lipofectamine 2000 reagent according to the manufacturer's protocol. Transfected cells were harvested at 48 h post transfection, then washed with PBS and lysed with 1 mL Nonide P-40 supplemented the 10 μL 1009 Cocktail (Biyuntian, Shanghai, China) and 10 μL 1009 PMSF (100 mmol/L) (Biyuntian). Cell lysates were centrifuged (13, 000 ×g) at 4 ℃ for 10 min to remove the nuclei, and then incubated with 40 μL proteinA/G (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 8 μg antiGFP antibody at 4 ℃ overnight. After centrifugation, the sediment was washed three times with washing buffer (50 mmol/L Tris [pH 7.4] supplemented with 150 mmol/L NaCl).
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Cells were lysed in radioimmunology precipitation assay (RIPA) buffer with SDS. The protein samples were then separated by SDS polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane (GE Healthcare, Waukesha, WI, USA). The membrane was then blocked with skim milk and probed with antibodies. The antibodies were anti-HA (Santa Cruz Biotechnology), anti-GFP (Santa Cruz Biotechnology), anti-filamin B (GeneTex, CA, USA), anti-GAPDH (Sigma, St. Louis, MO, USA) and anti-tubulin (Sigma). The immunoblot signals were detected using an enhanced chemiluminescence (ECL) substrate (Bio-Rad, Shanghai, China).
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Cells were cultured on glass coverslips and transfected with FLNB-GFP and Core-HA plasmids respectively, or co-transfected pair-wise, using Lipofectamine 2000 for 24 h. Cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. Then cells were then incubated with polyclonal rabbit anti-HA antibody (Santa Cruz Biotechnology), followed by 5 μg/mL Alexa 568 anti-rabbit antibody (Invitrogen) for 1 h at room temperature. At last, cells were permeabilized and stained with DAPI. Coverslips were mounted using Gel Mount (Biomedia), and the fluorescence was viewed with a Leica confocal microscope (LEICA Spectral Confocal TCS-SL, Serveis Cientı ´fico Te'cnics de la Universitat de Barcelona). Colocalization between filamin B and core protein was quantified by using Leica confocal microscope software. R values are shown as Pearson correlation coefficient ± SD averaged from 5 to 8 cells.
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Quantitative real-time PCR analysis was performed to determine the level of HBV total RNAs and HBV pgRNA. Cells were grown in 6-well plates and transfected with the following plasmids: pHBV1.3, GFP, and pXJ40-HA; pHBV1.3, Core-HA, and GFP; pHBV1.3, FLNB-GFP, and pXJ40-HA; pHBV1.3, FLNB-GFP, and Core-HA. Total RNAs were isolated from cells using TRIzol Reagent (Invitrogen). The RNA (1 μg) was reverse transcribed with RT primer mix (oligo dT primer and random 6 mers) (TaKaRa). Quantitative PCRs were performed with SYBR Select Master Mix (Life Technologies) using the StepOne Real-Time PCR System (Applied Biosystems). The mRNAs levels were normalized to the GAPDH expression level.
PCR was performed at 95 ℃ for 3 min followed by 40 cycles at 95 ℃ for 15 s, 58 ℃ for 30 s, and 72 ℃ for 30 s. Each set of PCRs was performed in triplicate, and the CT values were obtained for each PCR. The ΔΔCT method was used to calculate the ratios of gene expression relative to the control set (pHBV1.3, GFP, and pXJ40-HA group) in the experiments. The primers used are list in Supplementary Table S2.
Table S2. Primer sequences used in quantitative real-time PCR
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Cells were grown in 6-well plates and transfected with the following plasmids: pHBV1.3, GFP, and pXJ40-HA; pHBV1.3, Core-HA, and GFP; pHBV1.3, FLNB-GFP, and pXJ40-HA; pHBV1.3, FLNB-GFP, and Core-HA. The culture medium was collected at 48 h post transfection. The level of HBsAg and HBeAg in the culture medium was determined using ELISA kits (Beijing Wantai Biotech, Beijing, China) according to the manufacturer's instructions.
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Cells were grown in 12-well plates and transfected with the following plasmids: pHBV1.3, GFP, and pXJ40-HA; pHBV1.3, Core-HA, and GFP; pHBV1.3, FLNB-GFP, and pXJ40-HA; pHBV1.3, FLNB-GFP, and Core-HA. For the Southern blot analysis, HBV DNA from intracellular core particles was extracted at 96 h post-transfection. Cells were lysed in 400 μL lysis buffer (50 mmol/L Tris–Cl [pH 7.4], 1% Nonidet P-40, 1 mmol/L EDTA) at 4 ℃ for 30 min and subsequently centrifuged (13, 000 ×g) at room temperature. The supernatant was then transferred to fresh tubes and incubated with 10 μL DNase Ⅰ (1 U/μL; Sigma) and 5 μL RNase A (10 mg/mL; Sigma) at 37 ℃ for 2 h. Subsequently, 20 μL EDTA (0.5 mol/L) was added to inactivate DNase Ⅰ, and the mixture was then incubated with 10 μL protease K (20 mg/mL Sigma) and 20 μL 20% SDS at 55 ℃ overnight. The digestion mixture was extracted with phenol/chloroform, and DNA was precipitated with ethanol and dissolved in 15 μL TE buffer (10 mmol/L Tris–HCl [pH 8.0], 1 mmol/L EDTA). All the extracted DNA was separated on 0.8% agarose gels. After standard denaturation and neutralization procedures, the DNA was transferred onto a positively charged nylon membrane (GE Healthcare, Waukesha, WI, USA) and probed with a digoxigenin (DIG)-labeled HBV RNA probe. The probe preparation and subsequent DIG detection were conducted using the DIG Northern Starter Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. pHBV1.3 plasmid was used as HBV DNA marker. HBV DNA marker bands are shown for relaxed circular DNA (rcDNA), double stranded DNA (dsDNA), and single stranded DNA (ssDNA). Ratios were quantified by gray analysis using GeneTools from Syngene software (GeneGnome5).