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

Abstract

Background: Hepatitis 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.

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: Our 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.

Conclusion: USP18 promoted HBV replication via inhibiting type I IFN signaling pathway, which was independent of its protease activity.

Keywords: HBV; Interferon; Persistent infection; Type I IFN signaling pathway; USP18.

Fig. 1

Over-expression of USP18 and its catalytic activity. HepAD38 cells were transfected with WT-USP18 plasmid, USP18-C64S plasmid or empty vector (MOCK) or left untreated. a: Twenty-four hours after transfection, USP18 mRNA was determined by real-time PCR (normalized by GAPDH). b: Forty-eight hours after transfection, USP18 protein expressions were analyzed by western blot (left). The relative expression levels of USP18 (normalized by GAPDH) were calculated by densitometry analysis (right). c: Cleavage of ISG15-GST fusion in vitro. USP18, ISG15/GST and WT-USP18 (or USP18-C64S) were co-transfected into Hela cells. Total intracellular protein was collected to perform Western blot. WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). ** p ≤ 0.01; *** p ≤ 0.001

Fig. 2

Over-expression of USP18 in HepAD38 cells promoted HBV production independent of its protease activity. HepAD38 cells were transfected with USP18 plasmids as indicated. Real-time PCR was performed to quantify HBV production. Total supernatant HBV DNA (a), total intracellular HBV DNA (b), HBV cccDNA (c) and HBV pgRNA (d) 48 h post transfection with 4 μg WT-USP18 plasmid. Supernatant HBV total DNA 48 h post transfection with 4 μg WT-USP18 or 4 μg USP18-C64S plasmid, respectively (e). WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). * p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001

Fig. 3

Over-expression of USP18 in HepAD38 cells did not affect expression of HBV proteins. HepAD38 cells were transfected with the 4 μg WT-USP18, 4 μg USP18-C64S or 4 μg MOCK, respectively. Forty-eight hours later, culture medium was collected to quantify HBsAg (a) and HBeAg (b) expression level by ELISA assay. Intracellular HBcAgwas detected by western blot (c, left) and (c, right) analyzed by densitometry analysis. WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3)

Fig. 4

Silencing USP18 in HepAD38 suppressed HBV production. HepAD38 cells were seeded at 3 × 10⁵/ml, 2 ml per well in 6-well plates. 24 h later, the cells were left untreated or transfected with 20 nM siUSP18 or 20 nM NC. 48 h post transfection, knockdown efficiency was evaluated by detecting USP18 mRNA expression (a, left), which was further confirmed by western blot (a, right). Intracellular total RNA and DNA, as well as supernatant DNA, were collected respectively. Real-time PCR was performed to detect supernatant HBV total DNA (b), intracellular HBV total DNA (c), HBV cccDNA (d) and pgRNA (e). siUSP18, USP18 small inhibitory RNA; NC, the negative control siRNA. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Fig. 5

USP18 upregulation inhibited IFN-induced Jak/STAT signaling pathway. HepAD38 cells and HepG2 cells were seeded in 6-well plate without any treatment, respectively. Forty-eight hours later, supernatant IFNα (a, left) and IFNβ (a, right) and intracellular mRNA expression of ISGs (b) were analyzed by ELISA assay and real-time PCR, respectively. To investigate the effects of USP18 on STAT phosphorylation, HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for 30 min before harvested. Western blot was used to analyze the expression of STAT1 and p-STAT1 (c, left). The interferon stimulated response element (ISRE) activity was quantified by dual luciferase reporter gene assay. Briefly, HepAD38 cells were co-transfected with WT-USP18, USP18-C64S or MOCK and ISRE-luc reporter plasmid /pRL-TK reporter plasmid for 24 h, and then left untreated or treated with 100 IU/ml or 1000 IU/ml IFNα for 24 h before the cells were lysed (c, middle). HepAD38 cells were transfected with WT-USP18, USP18-C64S or MOCK for 48 h and treated with 500 IU/ml IFNα for additional 24 h. Expression of ISGs mRNA including MxA and OAS2 were detected by real-time PCR (c, right). WT-USP18, wide type USP18; MOCK, empty plasmid. Results are presented as means ± SD (n ≥ 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001