The USP18 cysteine protease promotes HBV production independent of its protease activity

Yujia Li,Min Yao,Limin Chen,Shilin Li,Yongjun Chen,Haiyan Ye,Xiaoqiong Duan,Chunhui Yang

doi:10.1186/s12985-020-01304-2

Abstract

BackgroundHepatitis B virus (HBV) infection remains as one of the major public health problems in the world. Type I interferon (IFN) plays an essential role in antiviral defense by induced expression of a few hundred interferon stimulated genes (ISGs), including ubiquitin-specific protease 18 (USP18). The expression level of USP18 was elevated in the pretreatment liver tissues of chronic hepatitis B(CHB) patients who did not respond to IFN treatment. Thus, this study was designed to investigate the effects of USP18 on HBV replication/production.MethodsThe levels of wild type USP18(WT-USP18) and USP18 catalytically inactive form C64S were up-regulated by plasmids transfection in HepAD38 cells, respectively. Real-time PCR and ELISA were used to quantify HBV replication. Type I IFN signaling pathway was monitored at three levels: p-STAT1 (western Blot), interferon stimulated response element (ISRE) activity (dual luciferase assay) and ISGs expression (real time PCR).ResultsOur data demonstrated that overexpression of either WT-USP18 or USP18-C64S inactive mutant increased the intracellular viral pgRNA, total DNA, cccDNA, as well as HBV DNA levels in the culture supernatant, while silencing USP18 led to opposite effect on HBV production. In addition, upregulated WT-USP18 or USP18-C64S suppressed ISRE activity and the expression levels of p-STAT1 and ISGs.ConclusionUSP18 promoted HBV replication via inhibiting type I IFN signaling pathway, which was independent of its protease activity.

Highlights

Hepatitis B virus (HBV) infection remains as one of the major public health problems in the world
Confirmation of ubiquitin-specific protease 18 (USP18) expression and its catalytic activity In order to explore the effect of USP18 on HBV infection, we first confirmed whether USP18 and USP18-C64Secoding plasmids were successfully constructed
Since it has been reported [13] that full length USP18 has a conserved catalytic-activity-related site cysteine at Cys64 in its Cys-box, we obtained the mutant form of USP18 by conversing the cyserine into serine

Summary

Methods

The levels of wild type USP18(WT-USP18) and USP18 catalytically inactive form C64S were up-regulated by plasmids transfection in HepAD38 cells, respectively. Real-time PCR and ELISA were used to quantify HBV replication. Type I IFN signaling pathway was monitored at three levels: p-STAT1 (western Blot), interferon stimulated response element (ISRE) activity (dual luciferase assay) and ISGs expression (real time PCR)

Results

Background

Materials and methods

Discussion

Conclusions