Table 1.
Primers used for qRT-PCR.
Fig 1.
The functions of MITA/STING and MRP in hepatoma cell line Huh7 which supports HBV replication.
(A) pIRF3-luc and pNF-κB-luc, (B) pIFN-β-luc and (C) pISRE-luc reporter plasmids were cotransfected with a serial concentration of pFlag-MITA/STING plasmids into Huh7 cells. (D) pIRF3-luc and pNF-κB-luc, (E) pIFN-β-luc and (F) pISRE-luc reporter plasmids were cotransfected with a serial density of pHA-MRP plasmids into Huh7 cells. The reporter activity was measured at 18–24 hpt with the Dual-Luciferase reporter assay system. (G-H) A serial increasing amount of pFlag-MITA or pHA-MRP plasmids were transfected into Huh7. The intracellular pIRF3, IRF3, pIκB and IκB protein levels were detected by Western blot. The two ways ANOVA followed by Bonferroni’s test was used to determine the differences in multiple comparisons (*, P <0.05; **, P <0.01; ***, P <0.001).
Fig 2.
MITA/STING and MRP inhibited HBV replication in vitro.
(A-C) The overexpression plasmid pFlag-MITA/STING or pHA-MRP was co-transfected with HBV plasmid pSM2 into Huh7 cells. (A) HBV DNA replicative intermediates (upper) and mRNAs (lower) were detected by Southern blot and Northern blot, respectively. HBV mRNA density signals on Northern blot were normalized to 18S/28S and showed as RNA density ratio. (B) Levels of HBeAg and (C) HBsAg secreted into supernatant were detected by ELISA after 5-fold dilution. Results were presented as the optical density at 450 nm (OD450). (D-G) siRNA specific to human MITA/STING or MRP was transfected into Huh7 for twice. pSM2 was cotransfected with siRNA at the second time. (D) Intracellular HBV core-associated DNA was extracted and quantified with real-time PCR. (E) Intracellular HBV core protein was detected by Western blot. (F) HBsAg and (G) HBeAg expressed in supernatant were diluted 5-fold and measured by ELISA. The dashed line represents the cutoff value (CoV), which was assumed to be 2.1-fold mean value of the negative samples. Significant differences were analyzed using a two-tailed unpaired t-test (*, P <0.05; **, P <0.01; ***, P <0.001).
Fig 3.
MITA/STING and MRP suppressed HBV replication by inducing innate immune signaling.
(A-D) The overexpression plasmid pFlag-MITA/STING or pHA-MRP was co-transfected with HBV plasmid pSM2 into Huh7 cells. (A) Intracellular HBV core protein, pIRF3, IRF3, pIκB and IκB protein levels were detected by Western Blot with indicated antibody. The expression of MRP and MITA/STING were detected with anti-HA and anti-Flag antibody, respectively. (B) The levels of Ifnb, (C) pro-inflammatory cytokines Tnfa, Il-6, chemokine Cxcl2 and (D) ISGs Isg56, Oas1, MxA, Cxcl10 mRNAs in MRP or MITA overexpressed with or without pSM2 transfected Huh7 cells were detected by qRT-PCR. (E-I) HepG2 cells were transfected with pHA-MRP and pSM2 together and then treated with PDTC. (E) HBV DNA replicative intermediates and mRNA transcripts were detected by Southern blot and Northern blot, respectively. (F) The HBsAg and (G) HBeAg level in supernatant were measured by ELISA after 5-fold dilution of the supernatant. (H) HBcAg and capsid were detected by Western blot. (I) The supernatant mature virions were immunoprecipitated with anti-HBs antibodies and subjected to HBV core-associated DNA extraction, then quantified by real-time PCR. Significant differences were analyzed using a two-tailed unpaired t-test (*, P <0.05; **, P <0.01; ***, P <0.001).
Fig 4.
MITA/STING, MRP and c-di-GMP inhibited HBV replication in HepG2.2.15 cells.
(A-E) The overexpression plasmid pHA-MRP, pFlag-MITA/STING or vector plasmid was transfected into HepG2.2.15 cells. The pHA-β-actin was cotransfected as an inner control. (A) The levels of HBV capsid, intracellular core antigen, pIRF3, IRF3, pIκBα, IκBα, phospho-p65, p65, MRP, MITA, GAPDH and β-actin were detected by immunoblotting with indicated antibodies. (B) The HBV mRNA replicative intermediates were detected by Northern blot. (C) Intracellular HBV core-associated DNA were extracted and quantified with real-time PCR. (D) The HBV mature particles in supernatant were immunoprecipitated with anti-HBs, and then HBV core-associated DNA were extracted and quantified with real-time PCR. (E-G) HepG2.2.15 cells were transfected with c-di-GMP of different concentrations and incubated for 24 h. (E) The HBV DNA replicative intermediates (upper) and mRNAs (lower) were investigated by Southern blot and Northern blot, respectively. (F) HBcAg (upper) and HBV capsid (lower) were immunoblotted with indicated antibody. (G) The supernatant HBV mature particles were immunoprecipitated and HBV core-associated DNA were extracted and quantified with real-time PCR. Significant differences were analyzed using a two-tailed unpaired t-test (*, P <0.05; **, P <0.01; ***, P <0.001).
Fig 5.
Overexpressed MRP and MITA/STING significantly suppressed HBV replication in vivo.
6~8 weeks old wild type C57BL/6 mice were hydrodynamically co-injected with 10 μg pSM2 plasmid and 10 μg pHA-MRP or pFlag-MITA/STING or control plasmid by tail-vein. (A) Expression of MRP or MITA/STING protein was detected in liver sample collected at 4 days post-HI by IHC staining with antibody against HA- or Flag-tag. (B) The levels of HBsAg or (C) HBeAg in 10-fold diluted sera were detected by ELISA. (D) The serum HBV DNA in mice was quantified by real-time PCR at the indicated time points. The detection limit for HBV DNA in our system was 1000 copies per milliliter. (E) The percentage of mice with detectable serum HBV DNA at indicated time point was calculated as the percentage of total mice in each group. (F) HBV DNA and RNA replicative intermediates in liver samples collected at 4 days post-HI were measured with Southern blot and Northern blot, respectively. Each lane represented a liver sample from a mouse. (G) Immunohistochemical staining showed HBcAg levels in liver at 4 days post-HI. (H) The dynamic levels of antibody specific to HBV S antigen in 10-fold diluted sera at indicated time points were detected by ELISA. The black arrow indicated the HA-MRP, Flag-MITA or HBcAg expression positive hepatocytes. The dashed line represents the cutoff value, which was assumed to be 2.1-fold mean value of the negative samples. Five or six mice per group were analyzed. The two ways ANOVA followed by Bonferroni’s test was used to determine the differences in multiple comparisons (*, P <0.05; **, P <0.01; ***, P <0.001).
Fig 6.
MITA deficiency induced elevation of serum HBV viral loads and impaired HBV humoral immune response in vivo.
Wild type (MITA+/+) and MITA knockout (MITA-/-) mice were hydrodynamically (HI) injected with 10 μg pSM2 plasmid. (A) The serum HBV DNA in mice was quantified by real-time PCR at the indicated time points. (B) The levels of HBsAg and (C) HBeAg in 10-fold diluted sera were detected by ELISA. (D) The dynamic anti-HBs antibodies in 10-fold diluted sera were monitored and detected by ELISA. (E) The levels of HBsAg-specific IgG subtypes were determined by ELISA. (F) The dynamic of HBsAg-specific IgG1 subtype were also detected by ELISA. The dashed line represents the cutoff value, which was assumed to be 2.1-fold mean value of the negative samples. Six or seven mice per group were analyzed. The two ways ANOVA followed by Bonferroni’s test was used to determine the differences in multiple comparisons (*, P <0.05; **, P <0.01).
Fig 7.
Liver-infiltrating CD8+ T lymphocytes and splenocytes in MITA knockout mice displayed deficient cytokines producing phenotype and impaired specific CTL response.
(A) Wild type (MITA+/+) and MITA knockout (MITA-/-) mice were hydrodynamically injected with pSM2 plasmids. Twenty-eight days after injection, intrahepatic lymphocytes and splenocytes from HBsAg-positive mice were isolated and unstimulated (Mock) or stimulated with HBcAg- or HBsAg-derived peptides ex vivo. The frequencies of IFN-γ+ or TNF-α+ or IL-2+ CD8+ T cells were measured by intracellular staining and analyzed by FACS assay. (B) Quantitative analysis showed the percentages of one or two or three kinds of cytokines (IFN-γ/ TNF-α/ IL-2) simultaneously producing cells within the CD8+ T cell population from liver or (C) spleen. Five or six mice per group were analyzed. Significant differences were analyzed using a two-tailed unpaired t-test (*, P <0.05; **, P <0.01; ***, P <0.001).