The coding regions for HBsAg (nt 129–842) and HBcAg (nt 1901–2348) were amplified from a HBV genome of subtype ayw (GenBank accession no. U95551) and cloned into pCR2.1 vector (Invitrogen, Karlsruhe, DE). The primers S6C, S7D, HcNCO, and Hc-149s used for PCR amplification are listed in Table 1 . Clones with the inserts in both orientations with regard to the T7 promoter were selected and sequenced to verify that the construction was correct. EsiRNAs targeting the coding region of HBsAg and HBcAg (SesiRNA and CesiRNA) were prepared by using Silencer siRNA Cocktail Kit (RNase III) (Ambion, Darmstadt, DE) according to the manufacturer's instructions. Briefly, single strand RNAs were transcribed from the plasmids using T7 polymerase and then annealed to form double strand RNA. EsiRNAs were generated finally by digestion of the purified dsRNA with RNase III at 37 °C for 1 h, and verified by gel electrophoresis on a 4% agarose gel. HCV-C5, a plasmid containing HCV core region (nt 631–900) (Lu et al., 1995) was used to prepare HCVesiRNA as a scramble esiRNA control.Table 1

Primers used for cloning of the coding region of HBsAg and HBcAg and RT-PCR

The sequences of 3 siRNAs targeting the coding region of S antigen of HBV adw strain were designed by using an online software from Ambion (www.ambion.com) and combined with the algorithm described by Reynolds et al. (2004). The sequences of the siRNAs are: siHBs1, 5′-GAA TCC TCA CAA TAC CGC A-3′ (nt 225–243); siHBs2, 5′-GGT ATG TTG CCC GTT TGT C-3′ (nt 458–496); siHBs3, 5′-GGA ACC TCT ATG TAT CCC T-3′ (nt 542–560). A siRNA against green fluorescent protein, siGFP (5′-CGG CAA GCT GAC CCT GAA GTT-3′), was used as control. All siRNAs were purchased from Qiagen.

BHK cells, HepG2 cells, and HepG2.2.15 cells (provided by Prof. G. Arc; HBV serotype ayw, genotype D; GenBank accession no. U95551) (Sells et al., 1987) were maintained in Eagle's minimal essential medium or RPMI 1640 medium, respectively, supplemented with 10% of fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded in an 8-well chamber slide or 24-well plate at about 60% confluence. After 24 h, cells were transfected with lipofectamine 2000 (Invitrogen, Karlsruhe, DE) following the manufacturer's instructions. 0.2 μg or 0.5 μg of plasmid DNA and 0.5 μl or 2 μl of lipofectamine 2000 were placed in each well of an 8-well chamber slide or in a 24-well plate, respectively.

A replication-competent HBV construct pHY106 + wta was generated by inserting a full length wild type HBV genome from pSM2 (kindly provided by Prof. Hans Will, Genotype D, subtype ayw, GenBank accession no. V01460) into pHY106 vector (H. Yang et al., 2004). The pHY106 vector contains a cytomegalovirus (CMV) promoter upstream of a short, recombinant HBV sequence that allows the in-frame insertion of a full length HBV genome following SapI digestion. The CMV promoter upstream of the precore initiation site allows an efficient transcription of the 3.5-kb pregenomic RNA following transfection of liver cell lines. Two vectors pHBeex and pHBcex were constructed for the expression of HBeAg and HBcAg, respectively. The regions of the HBV genome (nt 1814–2451) and (nt 1901–2451) were amplified with primers HpreC-EV1, HBc-EV2 and HBc-EV1, HBc-EV2, respectively (Table 1), digested with EcoRV and XhoI, and inserted into the pcDNA3 vector via the EcoRV and XhoI sites. The expression plasmids encoding HBsAg 1056Sp (Genotype D, subtype ayw), HK188 (Genotype C, subtype adr), and 91-4696 (Genotype A, subtype adw) were described by Ireland et al. (2000).

Levels of HBsAg and HBeAg in cell supernatants were determined by using the Architect system and HBsAg and HBeAg CMIA kits (Abbott Laboratories, Wiesbaden-Delkenheim, DE) according to the manufacturer's instructions.

Hepatitis B core antigen was visualized by IF staining with specific antibodies. Transfected cells were cultured for 24 h, then fixed with 50% of methanol in phosphate-buffered saline (PBS), and stained with polyclonal rabbit anti-HBc antibody (DAKO, Hamburg, DE). Goat anti-rabbit immunoglobulin G-fluorescein isothiocyanate (Sigma, Munich, DE) was used as a secondary antibody for the experiments. Staining was visualized under a fluorescence microscope (Nikon, Tokyo, JP) with an excitation wavelength of 490 nm.

Total RNA was extracted from transfected cells with TRIzol reagent (Invitrogen, Karlsruhe, DE) according to the manufacturer's instruction. Northern blot analysis was carried out by agarose–formaldehyde method. Briefly, 5 μg of total RNA per sample were separated on 1% agarose–formaldehyde gel and blotted to a Hybond-N⁺ nylon membrane (Amersham, Buckinghamshire, GB). HBV transcripts were detected by using a ³²P-labeled full length HBV probe. Hybridization signals were visualized and analyzed by a Phospho-Imager (Cyclon, Parkard Instrument).

HBV replicative intermediates were purified from intracellular core particles according to the method described by Sterneck et al. (1998) with minor modification. Briefly, cells were washed in ice-cold PBS and lysed in 0.4 ml of lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% NP-40 at 4 °C for 15 min. Nuclei were pelleted by centrifugation. The supernatant was adjusted to 10 mM MgCl₂ and treated with 100 μg/ml DNase I (Roche, Mannheim, DE) at 37 °C for 30 min. The reaction was stopped by the addition of EDTA to a final concentration of 25 mM. Proteins were then digested with 0.5 mg/ml proteinase K (Qiagen, Düsseldorf, DE) and 1% sodium dodecyl sulfate at 55 °C for 2 h. HBV nucleocapsid-associated DNA was purified by phenol/chloroform (1:1) extraction followed by isopropanol precipitation by adding 15 μg of tRNA and 1/10 volume of 3 M sodium acetate, pH 5.2. The isolated HBV DNA was subjected to agarose gel electrophoresis, followed by denaturation and Southern blotting. HBV DNA was detected by hybridization with a ³²P-labeled full length HBV probe. Hybridization signals were visualized and analyzed by a Phospho-Imager (Cyclon, Parkard Instrument).

Two micrograms of total RNA per sample was reverse transcribed by using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Promega, Mannheim, DE) and Oligo (dT) (Invitrogen, Karlsruhe, DE) as primer. The cDNA fragments of STAT1 and ISG15 were amplified by using the primers listed in Table 1. Cycle parameters were (i) 1 cycle: 94 °C, 4 min; (ii) 30 cycles: 94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s; and (iii) 1 cycle: 72 °C, 10 min. PCR products were subjected to agarose gel electrophoresis and visualized by ethidium bromide staining. Quantitative PCR was carried out using Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, Karlsruhe, DE) in a Roche Lightcycler V.3. The PCR was performed with the following cycling parameters over 45 cycles: 95 °C for 5 s, 58 °C for 10 s, and 72 °C for 10 s. The specificity of the PCR products was verified by melting curve analysis and agarose gel electrophoresis.

To examine the inhibitory effect of esiRNAs on the HBV gene expression, the expression plasmids encoding HBsAg and HBeAg were cotransfected with esiRNA into HepG2 cells. The expression levels of HBsAg and HBeAg in cell culture supernatants were determined 72 h later by CMIA. Cotransfection with SesiRNA and CesiRNA at the concentration of 100 nM reduced the expression levels of HBsAg and HBeAg to 20% and 10% of the control without transfection of esiRNA, respectively (Fig. 1a and b). The inhibition of the HBcAg expression by CesiRNA was verified by IF staining of cells transfected with the HBcAg expression plasmid with and without CesiRNA (Fig. 1c). The HBcAg expression in transfected cells was strongly reduced by CesiRNA.Fig. 1

The effects of esiRNAs on HBV S and core antigen expression. HepG2 cells were cotransfected with 0.5 μg of the HBsAg or HBeAg expression plasmid and esiRNAs at concentrations of 50 nM or 100 nM. The HBsAg (a) and HBeAg (b) levels in cell culture media were measured by CMIA 72 h post-transfection. (c) BHK cells in 8-well chamber slide were cotransfected with 0.2 μg of the expression plasmid encoding HBcAg and CesiRNA at concentration of 50 nM or 100 nM, or 100 nM of SesiRNA as a control, and fixed 24 h post-transfection. HBcAg was stained with a polyclonal rabbit anti-HBc serum. Sesi, SesiRNA; Cesi, CesiRNA.

To determine whether esiRNAs are able to inhibit the HBV replication, HepG2 cells were transfected with pHY106 + wta in the presence of SesiRNA or CesiRNA. The expression of HBsAg and HBeAg in cell culture supernatants was reduced significantly by esiRNAs (Fig. 2a). A decrease of 76.0% and 68.1% of the HBsAg levels in supernatants was measured by cotransfection with SesiRNA and CesiRNA, respectively. Likewise, SesiRNA and CesiRNA suppressed the expression levels of HBeAg to 20% and 22% of the control, respectively (Fig. 2a). The amount of HBV replicative intermediates was reduced in HepG2 cells by both esiRNAs in a dose-dependent manner (Fig. 2b). Only 10% of HBV replication intermediates was detected in HepG2 cells treated with 100 nM of SesiRNA and CesiRNA. Northern blot analysis showed that HBV transcripts were reduced to a level of about 40% of the control (Fig. 2c).Fig. 2

The effects of esiRNAs on HBV gene expression and replication. HepG2 cells were cotransfected with 0.5 μg of pHY106 + wta and esiRNAs at concentrations of 50 nM or 100 nM, or 100 nM of HCVesiRNA as a control. The HBsAg and HBeAg levels in cell culture supernatants (a), HBV replicative intermediates (b), and HBV transcripts (c) were analyzed 72 h post-transfection, by CMIA, Southern blot, and Northern blot analysis, respectively. The relative strength of signals detected in Southern blot and Northern blot was calculated by setting the untransfected control as 100% and given in (b) and (c). Sesi, SesiRNA; Cesi, CesiRNA; HCVesi, HCVesiRNA.

A number of specific siRNAs have been shown to knock down the corresponding HBV transcripts efficiently in vitro and in vivo. Therefore, the ability of esiRNA and synthetic siRNAs to inhibit HBV gene expression and viral replication was compared. Three synthetic siRNAs siHBs1–3 targeting the coding region of HBsAg inhibited the HBsAg expression with different effectiveness (Fig. 4a). However, the silencing of the HBsAg expression by synthetic siRNAs was impaired strongly by nucleotide variations (Fig. 4b). A G to A substitution at the position 17 in the target sequence reduced the ability of siHBs1 to inhibit the expression of HBsAg. Similarly, the two nucleotide substitutions in the target sequence of siHBs2 abolished the silencing effect completely. In contrast, these mutations did not affect the silencing effect of SesiRNA (data not shown, see below).Fig. 4

Comparison of esiRNA and siRNAs. HepG2 cells were cotransfected with 10 nM of siRNA targeting HBV S gene (siHBs1–3) and an expression plasmid encoding wild type HBsAg (HBs-wt) (a) or mutated HBsAg (HBsm1 and HBsm2) (b). To compare the gene silencing by synthetic siRNAs and esiRNAs, HepG2 cells were cotransfected with pHy106 + wta and SesiRNA or siHBs1 (c–e). The HBsAg and HBeAg levels in cell culture supernatants (c), HBV replicative intermediates (d), and HBV transcripts (e) were analyzed 72 h post-transfection by CMIA, Southern blot, and Northern blot analysis, respectively. The sequences of siRNA and their targets were shown. siRNA targeting green fluorescence protein (siGFP) was used as a scramble siRNA control. The relative strength of signals detected in Southern blot and Northern blot was calculated by setting the untransfected control as 100% and given in (d) and (e).

Large dsRNA with a length over 35 bp might induce the interferon response in cells. Thus, it is necessary to examine whether the use of esiRNAs induces ISGs in HepG2.2.15 cells to exclude the off-target effect. RT-PCR analysis was carried out to monitor the induction of STAT1 and ISG15 in cells. The expression of STAT1 and ISG15 in HepG2.2.15 cells was increased after a 24-h treatment with 100 units of IFN-α. The transfection of HepG2.2.15 cells with SesiRNA and CesiRNA did not increase the expression of STAT1 and ISG15 (Fig. 6 ). Thus, esiRNA was not able to activate the interferon response in hepatoma cells.Fig. 6

RT-PCR analysis of STAT1, ISG15, and β-actin mRNA in cells transfected with esiRNAs. Total RNAs were purified from HepG2.2.15 cells transfected with or without esiRNA. cDNAs were prepared by reverse transcription with Oligo (dT) primer. The cDNA fragments of STAT1, ISG15, and β-actin were amplified and examined by agarose gel electrophoresis. In addition, the relative amounts of STAT1 mRNAs were determined by real-time RT-PCR as shown in copies per microgram RNA. M, 123 bp DNA Ladder; lane 1, no RNAi; lane 2, SesiRNA 50 nM; lane 3, SesiRNA 100 nM; lane 4, CesiRNA 50 nM; lane 5, CesiRNA 100 nM; lane 6, HCVesiRNA 100 nM; lane 7, negative control; lane 8, positive control: total RNA purified from HepG2.2.15 cells stimulated with interferon-alpha at a concentration of 100 units/ml.