Adenosine deaminase acting on RNA-1 (ADAR1) inhibits hepatitis B virus (HBV) replication by enhancing microRNA-122 processing

Guangyan Liu,Xiancai Ma,Zhe Wang,Kousho Wakae,Yaochang Yuan,Zhangping He,Hironori Yoshiyama,Hisashi Iizasa,Hui Zhang,Mami Matsuda,Ryuichi Sugiyama,Zhiyu Yuan,Masamichi Muramatsu,Linghua Li

doi:10.1074/jbc.ra119.007970

Abstract

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes.

Highlights

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing
This study demonstrated, for the first time, that ADAR1 plays an antiviral role against hepatitis B virus (HBV) infection by increasing the level of miRNA-122 in hepatocytes
Because ADAR1 is an interferon-responsive gene (9 –11, 14), we examined the expression of endogenous ADAR1 in which HBV was replicating

Summary

Results

Because ADAR1 is an interferon-responsive gene (9 –11, 14), we examined the expression of endogenous ADAR1 in which HBV was replicating. Two ADAR1 isoforms, p110 and p150, both increased miR-122 levels and suppressed HBV transcript levels when individually overexpressed (Fig. 3D) These results suggest that ADAR1 plays a role in the positive regulation of miR-122 in hepatocytes. Transfection of a miR-122 inhibitor increased HBV transcript levels in a dose-dependent manner (Fig. 5B, lower panel) These results suggest that the constitutively expressed miR-122 contributes to HBV RNA reduction. The silencing of p53 expression by siRNA (sip and -3, but not -1) increased HBV RNA levels in Huh cells that both overexpressed and did not express ADAR1 (Fig. 6H), indicating that p53 expression regulated the level of HBV transcripts more tightly than ADAR1. NAGE analysis revealed an inverse correlation between p53 protein levels and nucleocapsid formation (Fig. 6B)

Discussion

Cell culture and transfection

NAGE assay

Expression vectors and reagents

Immunoprecipitation and Western blotting

Statistical analysis