Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

Cloning and sequencing of full-length HBV and different HBV-ORFs

From an archival serum sample of a treatment-naïve CHB patient infected with HBV of genotype D, HBV-DNA was extracted using QIAamp DNA Mini kit (Qiagen, CA, USA). Full-length HBV genome (~ 3.2 kb) was amplified from the extracted DNA by high-fidelity Taq DNA polymerase (Thermo Fisher Scientific, MA, USA) and primers HBVP1 and HBVP2 (Additional file 1: Table S1), each bearing unique SapI restriction enzyme site [11]. The PCR product was purified by QIAquick gel extraction kit (Qiagen) and cloned into pJET1.2/blunt vector with CloneJET PCR cloning kit (Thermo Fisher Scientifc). Complete nucleotide sequences of three HBV clones were determined using BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) and different internal primers (Additional file 1: Table S1) on an automated DNA sequencer (3130, Genetic analyzer, Applied Biosystems). The sequences were compared with representative sequences of HBV D-genotype retrieved from GenBank to reconfirm their genotypic affiliations and one of the HBV/D clones was used for subsequent experiments. The sequence of that HBV/D clone is available in GenBank under Accession Number MF618339. Additionally, the complete PreS1/PreS2/S-ORF and ORF-P were PCR amplified from HBV/D clone with specific primer pairs (Additional file 1: Table S1) and cloned into pEGFP-N1 and pcDNA3.1/myc-His(B) vectors at HindIII/PstI and KpnI/HindIII restriction enzyme sites respectively (Additional file 1: Table S1) to generate pEGFPN1-HBs and pcDNA3.1/myc-His(B)-HBV-P clones. The clones were verified by sequencing. Plasmids pcDNA3.1/myc-His(B)-HBx and pCMV-HBc comprising HBX and core genes respectively [12] were gifted by Prof. Soma Banerjee.

Cell culture and transfection

Human hepatoma cells Huh7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hi-Media Laboratories Pvt. Ltd.) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). The full-length HBV linear monomer was released from pJET1.2/blunt vector by digestion with SapI enzyme (Thermo Scientific) at 37 °C for 12 h followed by gel purification using QIAquick gel extraction kit (Qiagen). The released HBV monomer or plasmid containing HBV-ORF was transfected individually into Huh7 cells seeded in 12 well plate at a concentration of 2 × 105 cells/well using Lipofectamine 3000 (Invitrogen). Untransfected or empty vector transfected Huh7 cells were maintained as control. Six hours post transfection, the media was replaced with fresh DMEM media containing 10% FBS and after seventy-two hours transfected as well as untransfected Huh7 cells were harvested. All experiments were performed in triplicate and repeated at least three times. The Huh7 cell line was authenticated by STR profile analysis method from National Centre for Cell Science, Pune, India.

HBV production and infection

HepG2.2.15 and HepG2hNTCP cell lines were kindly gifted by Prof. Shyam Kottilil, Institute of Human Virology, University of Maryland. HepG2.2.15 cells (stably transfected with the complete HBV genome) were cultured in DMEM containing 10% FBS for 4 days. The supernatant containing HBV particles was collected and HBV stock titre (genome equivalents per millilitre) was assessed by Real Time PCR. HepG2hNTCP cells expressing human sodium taurocholate cotransporting polypeptide (NTCP) are susceptible to HBV infection and HBV derived from culture supernatant of HepG2.2.15 cells was used as an inoculum for infection of HepG2hNTCP cells. The HepG2hNTCP cells were seeded in DMEM supplemented with 10% FBS and 1% L-Glutamine and after 72 h, the cells were infected with HBV particles (at 100 virus genome equivalent per cell) combined with 8% polyethylene glycol (PEG) 8000 and 2.5% dimethyl sulfoxide (DMSO) [13]. Six hours after infection the cells were washed with PBS, cultured in DMEM with 10% FBS, 1% L-Glutamine and 2.5% DMSO for 72 h and then harvested for further experiments. HBV uninfected HepG2hNTCP cells served as control.

Real-time PCR

Total RNA was extracted from harvested Huh7 and HepG2hNTCP cells using TRIzol (Invitrogen) and cDNA was generated by reverse transcription with RevertAid Reverse Transcriptase (RT) enzyme (Thermo Scientific). The mRNA expression of different complement components (C5-C9) as well as that of other host genes were determined by Real-time PCR using the cDNA, SYBR Green Master mix (Applied Biosystems), and primer-pairs specific to each gene (Additional file 1: Table S2). Each sample was assessed in triplicates and the values were normalized by 18S rRNA expression level.

Study subjects and samples

Treatment-naïve chronically HBV infected patients (HBsAg positivity > 6 months) representing different phases of chronic HBV infection were recruited from Hepatology Clinic of School of Digestive and Liver Diseases (SDLD), Institute of Post Graduate Medical Education and Research (I.P.G.M.E.&R.), Kolkata, India and were categorized into distinct cohorts namely-

Immune tolerant (IT)

Patients having HBeAg-positivity, high HBV-DNA (> 106 IU/ml), normal serum alanine transaminase (ALT) levels (≤ 40 IU/L) and minimal or no necro-inflammation.

HBeAg-positive chronic hepatitis B (CHB)

Patients with high HBV-DNA > 104 copies/ml, HBeAg-positive, elevated ALT > 40 IU/L and evidence of active hepatic necro-inflammation.

Inactive carrier (IC)

Patients negative for HBeAg and having low HBV-DNA (< 104 copies/ml), normal ALT (≤ 40 IU/L) and minimal or no necro-inflammation.

HBeAg-negative chronic hepatitis B

Patients having HBV-DNA > 104 copies/ml, HBeAg-negative, ALT > 40 IU/L and evidence of active hepatic necro-inflammation.

Patients with HIV/HCV co-infection, significant co-morbidity like diabetes mellitus, chronic alcoholism, intravenous drug abuse or any prolonged (> 7 days) drug therapy, evidences of any carcinoma, overt infection or autoimmune disorders were excluded. As comparison/control group sera samples were collected from healthy volunteers (HC) without any viral/bacterial infection and chronic/acute illness in preceding 6 months.

Five millilitre blood was collected from each study subject and sera was isolated, aliquoted and stored at − 80 °C until use. Prior informed written consents were obtained from the participants or from parents or legal guardians of minor participants prior to study inclusion. The access to human samples and all experimental protocols were carried out in accordance with the approved guidelines of the Ethical Review Committee of I.P.G.M.E.&R.

Liver biopsy tissue samples were collected from selected CHB patients during Endoscopic ultrasound-guided routine biopsy as a part of their diagnostic workup. In addition, liver tissues collected from patients undergoing cholecystectomy and those depicting normal liver histology and absence of liver metastasis served as control group. The tissues were collected in RNAlater solution (Invitrogen), kept at 4 °C overnight and then preserved at − 80 °C for future use.

Measurement of C9 level in patient sera

Sera from chronically HBV infected patients and HC were used for estimation of C9 level by commercially available ELISA kits (Elabscience).

Expression of C9 in liver tissue

The liver tissues stored in RNA later were homogenized, RNA was extracted using TRIzol reagent, cDNA was generated and the expression of C9 mRNA was determined by Real-time PCR as described previously.

Western blot analysis

Huh7 cells transfected with pcDNA3.1/myc-His(B)-HBx or empty-vector were lysed in RIPA buffer [150 mM NaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0)] supplemented with 1X protease inhibitor cocktail (Roche, Switzerland). Protein concentrations in the whole cell lysates were measured by Bradford protein assay using Bradford reagent (Sigma Aldrich, MO, USA). Equal amount of total proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred to nitrocellulose membranes which was blocked using 5% BSA at room temperature for 1 h, and then treated with mouse anti-human anti-C9 (1:500) or mouse anti-human anti-USF-1 (1:500) primary antibody for overnight at 4 °C (Santa Cruz Biotechnology). The membranes were washed by PBST [PBS plus 0.1% Tween-20] and incubated in horseradish peroxidase (HRP)-labelled goat anti-mouse IgG (1:1000) at room temperature for 1 h, and then treated with chemiluminescent HRP substrate (Thermo Fisher Scientific). Finally, X-ray films were exposed, developed, dried, scanned and analysed with ImageJ software. Cellular α-tubulin served as loading control for the Western blot.

Treatment of HBx transfected Huh7 cells with 5-Aza-2′-deoxycytidine and Trichostatin A

To determine the role of DNA methylation and histone deacetylation in C9 downregulation, HBx transfected Huh7 cells were treated with 5 μM DNA methylation inhibitor, 5-Aza-2′-deoxycytidine or 0.1 μM histone deacetylation inhibitor, Trichostatin A (TSA) while untreated cells served as control. At first, fresh DMEM media containing 5 μM 5-Aza-2′-deoxycytidine or 0.1 μM TSA were added separately to the Huh7 cells at the time of seeding the cells onto cell culture plates. Huh7 cells were then transfected with pcDNA3.1/myc-His(B)-HBx and 6 h after transfection, the cells were washed with PBS and the culture medium was replaced with media containing 5 μM 5-Aza-2′-deoxycytidine or 0.1 μM TSA. The media was replaced every 24 h with fresh media containing the same concentration of 5-Aza-2′-deoxycytidine or TSA. Seventy-two hours post transfection, the cells were harvested, total RNA was extracted and cDNA was prepared as before. The mRNA expression of C9 and USF-1 from both 5-Aza-2′-deoxycytidine and TSA treated cells were analyzed by Real-time PCR and compared with untreated cells.

Mutagenesis of C9 promoter sequence

The C9 promoter region (− 786 to + 40 bp) [8] was first amplified from Huh7 genomic DNA with primers C9-prom_F and C9-prom_R (Additional file 1: Table S2) and inserted into KpnI/HindIII sites of pGL3-Basic vector (Promega, WI, USA) to generate the pGL3-C9-Prom construct. In the pGL3-C9-Prom construct, USF-1 binding site, E-box (CACGTG) was deleted to create pGL3-C9-Prom-mt plasmid with Agilent Site Directed Mutagenesis Kit (Agilent, CA, USA) and were confirmed by sequencing. The oligonucleotides used for the generation of mutated promoter construct are listed in Additional file 1: Table S2.

Dual luciferase reporter assay

To study C9 promoter activity in presence of HBX, Huh7 cells were co-transfected with pGL3-C9-Prom, pcDNA3.1/myc-His(B)-HBx and pRL-CMV Renilla Luciferase Reporter vector (Promega, WI, USA). Seventy-two hours post transfection, the cells were harvested in Passive Lysis Buffer (Promega) and the firefly and Renilla luciferase activities were measured sequentially using the Dual-Luciferase Reporter Assay kit (DLR) (Promega) in a luminometer (Promega). The activity of Renilla luciferase was used to normalize the firefly luciferase enzyme activity.

Analysis of CpG island distribution and transcription factors (TF) binding at C9 promoter

Putative CpG islands in the promoter region of C9 were analyzed using online tool Methprimer [14].

Transcription factors (TF) binding at the promoter region of C9 were identified bioinformatically with PROMO and TFBIND software using threshold of < 5% dissimilarity and string length > 10. The level of expression of TFs in liver was determined from Human Protein Atlas database. In vitro studies were performed with selected hepatic TFs binding to C9 promoter.

Overexpression and knockdown of USF1

The pCMV-USF1 plasmid encoding USF-1 was a generous gift of Prof. Marie-Dominique Galibert. Huh7 cells were transfected with pcDNA3.1/myc-His(B)-HBx, pGL3-C9-Prom and pRL-CMV Renilla Luciferase Reporter vector along with different concentrations (0, 400, 800 and 1200 ng/ml) of pCMV-USF1 plasmid and luciferase activity of C9 promoter was assessed from the harvested cells. The mRNA expression of C9 was also measured from Huh7 cells co-transfected with pcDNA3.1/myc-His(B)-HBx and pCMV-USF1 plasmid (in different concentration). Additionally, USF-1 antisense oligonucleotide (USF-1_ASO) (Additional file 1: Table S2) directed against the exon1 of USF-1 was designed from USF-1 gene sequence obtained from UCSC genome browser. Huh7 cells were transfected with pGL3-C9-Prom, pRL-CMV Renilla Luciferase Reporter vector and USF-1_ASO (0–1200 ng/ml) and C9-promoter luciferase activity was measured as described earlier. Further, C9 mRNA expression was evaluated in Huh7 cells transfected with USF-1_ASO (1000 ng/ml) or scrambled oligo control (ASO_Ctrl) (1000 ng/ml) (Additional file 1: Table S2), having no known targets in HBV or human transcriptome.

Chromatin immunoprecipitation (ChIP) assay

To clarify the interaction between USF-1 and the promoter region of C9, ChIP assay was performed using EpiQuik Chromatin Immunoprecipitation kit (EpigenTek, NY, USA) according to the manufacturer's instructions. Firstly, the assay strips were prepared by coating with mouse anti-human anti-USF1 antibody (Santa Cruz Biotechnology) and non-immune IgG (provided with the kit) that served as negative control. 2 × 106 Huh7 cells were transfected with pcDNA3.1/myc-His(B)-HBx or empty vector and after 72 h the cells were collected and the chromatin was crosslinked with 1% formaldehyde. 1.25 M glycine was added for 5 min to terminate the crosslinking process. The cells were incubated in lysis buffer supplemented with 1X protease inhibitor cocktail (Roche). The cross-linked DNA–protein complexes were sheared into ~ 200–1000 bp fragment by sonication. 5% input reference was removed from the sonicated DNA–protein complexes. 100 μl of the supernatant from each sample were then added to the antibody coated assay strips for immunoprecipitation. The DNA crosslinks were reversed and the proteins were removed by treatment with Proteinase K at 65 °C for 2 h in all the sample tubes including the input DNA vials and DNA was recovered using DNA purification spin columns. Finally, the DNA was quantified by Real-time PCR using primers specific for the C9 promoter as listed in Additional file 1: Table S2. Data obtained were then normalized to the negative control and expressed as percent recovery relative to the input.

Bisulfite sequencing

Genomic DNA was isolated from pcDNA3.1/myc-His(B)-HBx or empty vector transfected Huh7 cells using DNA extraction kit (FAVORGEN Biotech Corp., Taiwan). 2ug of isolated genomic DNA was bisulfite treated and purified using EZ DNA methylation kit (Zymo Research, CA, USA). The bisulfite-modified genomic DNA was amplified with bisulfite specific primers of USF-1 promoter (Additional file 1: Table S2) for 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 20 s and the amplified products were cloned into pJET1.2 blunt vector. Six different clones from each group pcDNA3.1/myc-His(B)-HBx /empty vector transfected cells were screened by sequencing and DNA methylation status was analyzed.

Immunofluorescence

HBV transfected Huh7 cells were grown as monolayer on poly-l-lysine coated coverslips in 6-well plate for 48 h and then treated with 50 μl sera from HC or CHB patients or with CHB patient sera supplemented with recombinant C9 protein at final concentration of 15 ng/ml of culture media for 60 min at 37 °C. The cells were fixed with 4% paraformaldehyde for 10 min and after washing with ice cold TBS, the masked antigens were retrieved by treatment with 0.05% trypsin-calcium chloride solution [trypsin 0.5%, calcium chloride 1% (pH 7.8)] at 37 °C for 20 min. After washing with TBS containing 0.025% TritonX-100, blocking was done with 1% BSA in TBS for 2 h. The cells were stained overnight at 4 °C with 1:500 dilution of mouse anti human anti-C5b-9 primary antibody (Santa Cruz Biotechnology) followed by incubation with 1:1000 dilution of goat anti-mouse IgG secondary antibody conjugated to Alexa fluor 488 (Thermo Fisher Scientific) for 1 h at 37 °C. Pro-Long Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology) was used for nuclear counterstaining and mounting. Images were captured in Thunder imager (Leica) at 40× magnification.

Complement-mediated cytolysis assay

Huh7 cells were transfected with full-length HBV monomer and distributed in 96 well plate at a density of 1 × 104 cells/well. After 3 days, the cells were treated with sera from HC or CHB patients or with CHB sera supplemented with recombinant C9 protein (final concentration 15 ng/ml) for 1 h at 37 °C. Finally, the cells were treated with 40 μg/well MTT solution (Sigma Aldrich) and incubated for 4 h at 37 °C in dark. Optical density of the solution was measured on an ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm.

C9 deposition on E. coli

1 × 107 cells of E. coli DH5α were incubated with CHB patient or HC sera at 37 °C for 60 min. After a wash with PBS, the bacteria were plated and adhered onto 96-well enzyme immunoassay (EIA) plates at ~ 5 × 106 cells per well. The nonspecific binding sites were blocked with 200 µl of 0.5% bovine serum albumin (BSA)–PBS and incubated for 30 min at 37 °C. After washing the plates were sequentially incubated at room temperature with 100 µl of 1:100 (v/v) mouse anti-human anti-C9 antibody for 60 min, 100 µl of 1:1000 HRP-conjugated goat anti-mouse secondary antibody for 30 min followed by addition of 100 µl of substrate solution for visualizing HRP activity. The reaction was stopped with 50 µl of 10% H2SO4, and the colour reaction was detected by reading the optical density at 490 nm in ELISA reader.

Bactericidal assay

1 × 104 cells of Escherichia coli strain DH5α were incubated with 100 µl of sera (1:500 dilution) of CHB patient or HC or heat inactivated sera of HC (for complement inactivation) at 56 °C for 60 min. After washing, serial tenfold dilutions of bacterial suspensions were prepared in saline solution and 100 µl was spread on LB plates. Colony forming units (CFU) were counted from the plates after 16 h of incubation at 37 °C. The experiment was performed in triplicate and repeated thrice.

Determination of bacterial load and endotoxin titre

Bacterial DNA in sera samples of CHB patients of different phases and healthy volunteers was isolated by incubation of the sera samples with lysozyme at 56 °C for 30 min followed by DNA isolation using QiaAmp Blood DNA mini kit (Qiagen). The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified with primers 338F (5′ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [15] using Real-time PCR. The bacterial load was determined from standard curve prepared using E. coli DNA of known bacterial load as standard. Endotoxin levels in sera of chronically HBV infected patients and healthy individuals were measured by ELISA method using Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific). Briefly, endotoxin standard solutions were prepared by reconstitution with endotoxin free water and then 50 μl of each standard as well as serum sample of HC and chronically HBV infected individuals were added to 96 well plate at 37 °C waterbath. Amoebocyte lysate reagent was added to each well and incubated at 37 °C, followed by addition of chromogenic substrate and finally reaction was stopped by 25% acetic acid. Optical density was measured at 405 nm in optical plate reader.

Longitudinal assessment of serum C9 level and virological/bacteriological parameters in CHB patients receiving tenofovir

From 10 CHB patients treated with tenofovir (300 mg daily), blood samples were collected at day 0 before the initiation of therapy (= baseline) and at the end of 12 months of treatment respectively and serum C9, HBV DNA, ALT, bacterial load and endotoxin titre were assessed as described previously.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Paired Student's t tests and Repeated Measures ANOVA were used to determine statistical significance. Comparison between groups was done by one‐way ANOVA followed by Tukey's Multiple Comparison Test. Linear regression was performed for correlation analysis. Statistical analysis was performed using GraphPad Prism Software, version 5.0. For all tests, p < 0.05 was considered statistically significant.

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