Inhibition of hepatitis B virus gene expression and replication by endoribonuclease-prepared siRNA

Abstract

Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.

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.

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.

Fig. 3

The effects of esiRNAs on HBV gene expression and replication in a stable HBV-producing cell line. HepG2.2.15 cells were transfected with SesiRNA, CesiRNA, or 100 nM of HCVesiRNA as a control. The HBsAg and HBeAg levels in cell culture medium (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 control as 100% and given in (b) and (c). Sesi, SesiRNA; Cesi, CesiRNA; HCVesi, HCVesiRNA.

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).

Fig. 5

Effects of esiRNA on naturally occurred HBs mutants. HepG2 cells were cotransfected with SesiRNA and three expression plasmids encoding different types of HBsAg, 1056Sp (genotype D, ayw), HK188 (genotype C, adr), and 91-4696 (genotype A, adw). The HBsAg level in cell culture supernatants was determined by CMIA 72 h post-transfection.

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.