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

中文翻译：

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USP18半胱氨酸蛋白酶不依赖其蛋白酶活性而促进HBV产生。

乙型肝炎病毒（HBV）感染仍然是世界上主要的公共卫生问题之一。I型干扰素（IFN）通过诱导表达数百种干扰素刺激基因（ISG），包括泛素特异性蛋白酶18（USP18），在抗病毒防御中起重要作用。在对IFN治疗无反应的慢性乙型肝炎（CHB）患者的预处理肝组织中，USP18的表达水平升高。因此，本研究旨在研究USP18对HBV复制/产生的影响。通过在HepAD38细胞中转染质粒，分别上调了野生型USP18（WT-USP18）和USP18催化失活形式C64S的水平。实时PCR和ELISA用于定量HBV复制。监测I型IFN信号传导途径的三个水平：p-STAT1（western Blot），干扰素刺激的反应元件（ISRE）活性（双荧光素酶测定）和ISGs表达（实时PCR）。我们的数据表明，WT-USP18或USP18-C64S无活性突变体的过表达增加了培养上清液中细胞内病毒pgRNA，总DNA，cccDNA以及HBV DNA的水平，而沉默USP18则对HBV产生了相反的影响。另外，上调的WT-USP18或USP18-C64S抑制了ISRE活性以及p-STAT1和ISGs的表达水平。USP18通过抑制I型IFN信号传导途径促进了HBV复制，这与其蛋白酶活性无关。我们的数据表明，WT-USP18或USP18-C64S无活性突变体的过表达增加了培养上清液中细胞内病毒pgRNA，总DNA，cccDNA以及HBV DNA的水平，而沉默USP18则对HBV产生了相反的影响。另外，上调的WT-USP18或USP18-C64S抑制了ISRE活性以及p-STAT1和ISGs的表达水平。USP18通过抑制I型IFN信号传导途径促进了HBV复制，这与其蛋白酶活性无关。我们的数据表明，WT-USP18或USP18-C64S无活性突变体的过表达增加了培养上清液中细胞内病毒pgRNA，总DNA，cccDNA以及HBV DNA的水平，而沉默USP18则对HBV产生了相反的影响。另外，上调的WT-USP18或USP18-C64S抑制了ISRE活性以及p-STAT1和ISGs的表达水平。USP18通过抑制I型IFN信号传导途径促进了HBV复制，这与其蛋白酶活性无关。