Effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus.
- Author:
Qiujing WANG
1
;
Shibo LI
;
Hongping HUANG
;
Shenpei LIU
;
Yan YANG
;
Dongliang YANG
Author Information
- Publication Type:Journal Article
- MeSH: Carrier Proteins; metabolism; DNA-Binding Proteins; genetics; metabolism; Down-Regulation; Gene Expression Regulation; Hep G2 Cells; Hepatitis B Surface Antigens; isolation & purification; Hepatitis B e Antigens; isolation & purification; Hepatitis B virus; physiology; Humans; Interleukin-6; metabolism; NF-kappa B; metabolism; Plasmids; RNA, Small Interfering; genetics; Signal Transduction; Transfection; Virus Replication
- From: Chinese Journal of Hepatology 2015;23(12):934-938
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.
METHODSThe hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.
RESULTSDAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.
CONTROL0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.
CONTROL0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.
CONCLUSIONDown-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.
