DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication

Figure 1 Expression of DNA-dependent activator of interferon-regulatory factors in Huh7 cells can suppress hepatitis B virus replication. A: ELISA analysis of HBV protein synthesis. GFP was transfected to monitor transfection efficiency; B: Real-time PCR analysis of HBV RNA. Huh7 cells were cotransfected with pHBV1.3 and different doses of hemagglutinin (HA)-DAI. Total RNA was extracted 48 h after transfection and HBV RNA was examined by real-time PCR; C: Northern blottings analysis of HBV RNA; Huh7 cells were cotransfected with pHBV1.3 and control DNA or MyD88 and HA-DAI. 1000 IU/mL IFN-α was added 12 h after transfection, and 48 h later, total RNA was extracted for Northern blotting hybridization. The positions of the HBV 3.5-, 2.4- and 2.1-kb RNA were indicated; D: Southern blotting analysis of HBV core particle associated DNA. Huh7 cells were treated as in C. HBV core particle associated DNA was analyzed 48 h later. Southern blotting was performed to detect HBV DNA as described. The positions of relaxed circular (RC), double stranded (DS) and single stranded (SS) DNAs were indicated; E: Effect of DAI on cell growth. Cell number was counted by adding cell counting kit-8 at 1, 2, 3, 4, 5, 6 d after transfection. DAI: DNA-dependent activator of interferon-regulatory factors; HBV: Hepatitis B virus; ELISA: Enzyme-linked immunosorbent assay; PCR: Polymerase chain reaction; IFN: Interferon; GFP: Green fluorescent protein; HBsAg: Hepatitis B virus surface antigen; HBeAg: Hepatitis B virus e antigen; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; MyD88: Myeloid differentiation primary response protein 88.

Figure 2 Interferon regulatory factor 3 signaling pathway is not required for inhibition of hepatitis B virus by DNA-dependent activator of interferon-regulatory factors. A: DAI did not induce IRF-3 phosphorylation. Control DNA, hemagglutinin (HA)-DAI or Flag-interferon-β promoter stimulator 1 (IPS1) was transfected into Huh7 cells. Forty-eight hours later, the phosphorylated form of IRF-3 was analyzed by Western blotting; B: Blockage of IRF-3 signaling did not affect inhibitory effect of DAI on HBV replication. Northern blotting assay was performed as shown in Figure 1C; C: Expression of DAI could not induce IRF-3 nuclear translocation. Cells were harvested 48 h after transfection and IRF-3 was stained as described. DAI: DNA-dependent activator of interferon-regulatory factors; IRF: Interferon regulatory factor; IRF-3ΔN: IRF-3 dominant negative plasmid; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6′-diamidino-2-phenylindole; YFP: Yellow fluorescent protein.

Figure 3 Inhibition of hepatitis B virus by DNA-dependent activator of interferon-regulatory factors depends on activation of nuclear factor-κB. A: NF-κB activity was induced by dependent activator of interferon-regulatory factors (DAI). Forty-eight hours after transfection, NF-κB (p65) was stained as described. NF-κB dependent luciferase reporter plasmid pNF-κB-Luc was co-transfected with control DNA or different doses of hemagglutinin (HA)-DAI into 293T cells. Renilla luciferase-herpes simplex virus thymidine kinase promoter was transfected to monitor the transfection efficiency; B: Blockage of NF-κB activation abolished DAI-mediated suppression of hepatitis B virus (HBV) replication. Forty-eight hours after transfection, the levels of hepatitis B e antigen and hepatitis B surface antigen were examined by enzyme-linked immunosorbent assay. HBV RNA was determined by Northern blotting hybridization. NF-κB: Nuclear factor-κB; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6′-diamidino-2-phenylindole; YFP: Yellow fluorescent protein.

Figure 4 Inhibiting hepatitis B virus replication by DNA-dependent activator of interferon-regulatory factors is an intracellular event. Transwell co-culture experiment was performed: Huh7 cells were seeded in both 6-well plates (below) and transwells (top). In transwell co-culture group, pHBV1.3 was transfected into the cells in 6-well plates while hemagglutinin (HA)-DNA-dependent activator of interferon-regulatory factors (DAI) was transfected into the cells in transwells. Twenty-four hours after the transfection, the cells in 6-well plates and transwells were co-cultured; in the direct cotransfection groups, pHBV1.3 and control DNA or HA-DAI were cotransfected into cells in 6-well plates; in interferon (IFN)-α treatment group, pHBV1.3 was transfected into the cells in 6-well plates, 1000 IU/mL IFN-α was added 12 h later. Seventy-two hours after transfection, all the cells were harvested and hepatitis B virus (HBV) RNA was determined by real-time polymerase chain reaction. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.