Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70

Abstract

Background: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication.

Methods: We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA.

Results: Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells.

Conclusions: Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.

Figure 1

Effect of siRNAs on the expression of HBV surface open reading frame in HEK293 and T98G cells. (A) As shown in Additional file 2: Figure S2. (B) Fluorescence micrographs of cells transfected with reporter plasmids and cotransfected with either the corresponding or non-corresponding siRNA with Lipofectamine ^TM 2000 (Invitrogen). At 24 hrs after transfection, the cells were observed with an Olympus BH-2 microscope, and representative bright-field images (left column) and relative fluorescent-field images (right column) were recorded by tenfold amplification. (C) Flow cytometry analysis of siRNA-mediated gene silencing of EGFP. Level of EGFP expression in cells cotransfected with (a) pEGFP-N1 vector; ( b) pS-EGFP-N1 and pU6; (c) pS-EGFP-N1 and S1; (d) pS-EGFP-N1 and S2; (e) pS-EGFP-N1 and S3 (heterologous siRNA). (D) Cells were analyzed for EGFP expression by fluorescence-activated cell sorting and the level of fluorescence relative to the control was quantitated. The mean fluorescence intensity of control siRNA was taken as 100% and adopted as control. Data represent means±SD from three independent experiments were performed in triplicate.*Significant differences compared to the heterologous siRNA control: Student’s t-test; P < 0.05.

Figure 2

Synergistic inhibition of HBV protein expression by siRNA targeting viral gene in combination with siHsc70 in HepG2.2.15 cells. (A) The expression of Hsc70 in HepG2.2.15 cells transfected with siHsc70 or siCtrl after 72 hrs was identified by Western blotting with anti-Hsc70. GAPDH expression was used as an internal control. (B) The reduction of HBsAg expression. HepG2.2.15 cells transfected with pU6, S1, S2, siHsc70, S2 with siHsc70, and also the heterologous S3. (C) The reduction of HBeAg expression. HepG2.2.15 cells transfected with pU6, S1, S2, siHsc70, S2 with siHsc70, and also the heterologous S3. Data represent means±SD from three independent experiments were performed in triplicate.*Significant differences compared to the heterologous siRNA control: Student’s t-test; P < 0.05.

Figure 3

Specific inhibition of HBV genes by siRNA targeting viral gene in combination with siHsc70 in HepG2.2.15 cells. (A) The inhibition of HBVS mRNA by siRNAs at different time points. HepG2.2.15 cells were transfected with pU6, S1, S2, siHsc70, S2 with siHsc70, and also the heterologous S3. Data represent means±SD from three independent experiments were performed in triplicate. *Significant differences compared to the heterologous siRNA control: Student’s t-test; P < 0.05. (B) Inhibition of HBV DNA by siRNAs in cell culture supernatants. HepG2.2.15 cells were transfected with pU6, S1, S2, siHsc70, S2 with siHsc70, and also the heterologous S3. Data represent means±SD from three independent experiments were performed in triplicate.

Figure 4

(A, B, C, D) Effects of siRNAs on IFN-α, IFN-β, TNF-α in HEK293 and HepG2.2.15 cells. (A) HEK293 and HepG2.2.15 cells were respectively transfected with pU6, S1, S2, S3, siHsc70, EGFP-siRNA and the positive-control poly (I:C). The concentrations of IFN-α, IFN-β and TNF-α in cell culture supernatants from transfected cells were determined by ELISA assay. (B) The effects of siRNAs on the mRNA concentrations of IFN-α, IFN-β, and TNF-α in HEK293 cells. HEK293 and HepG2.2.15 cells were respectively transfected with pU6, S1, S2, S3, siHsc70, EGFP-siRNA and the positive-control poly (I:C). The mRNA concentrations of IFN-α, IFN-β and TNF-α were examined by RT-PCR amplification with the SYBR RT-PCR Kit. Data represent means±SD from three independent experiments were performed in triplicate. (C) T98G cells were transfected with pU6, S1, S2, S3, siHsc70, EGFP-siRNA and the positive-control poly (I:C). The concentrations of IFN-α, IFN-β and TNF-α in cell culture supernatants from transfected cells were determined by ELISA assay. (D) The effects of siRNAs on the mRNA concentrations of IFN-α, IFN-β, and TNF-α in HEK293 cells. HEK293 cells were transfected with pU6, S1, S2, S3, siHsc70, EGFP-siRNA and the positive-control poly (I:C). The mRNA concentrations of IFN-α, IFN-β and TNF-α were examined by RT-PCR amplification with the SYBR RT-PCR Kit. Data represent means±SD from three independent experiments were performed in triplicate.