LAUSR

BACKGROUND Current antiviral therapies help keep hepatitis B virus (HBV) under control, but they are not curative. A cure for chronic hepatitis B (CHB) is lacking due to the inability of current therapies to eliminate the intracellular viral replication intermediate termed covalently closed-circular DNA (cccDNA). Therefore, there is an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS Here, we screened 2000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanized liver mouse model. RESULTS Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanized liver mouse model. Mechanistic study found that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited cccDNA transcription activity, which was correlated with establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS This study reports a novel small molecule that targets HBx to combat chronic HBV infection and reveals a function of NQO1 in HBV replication through the regulation HBx protein stability.