Jiang Dong, Guo Haitao, Xu Chunxiao, Chang Jinhong, Gu Baohua, Wang Lijuan, Block Timothy M, Guo Ju-Tao
Drexel Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine, 3805 Old Easton Road, Doylestown, PA 18902, USA.
J Virol. 2008 Feb;82(4):1665-78. doi: 10.1128/JVI.02113-07. Epub 2007 Dec 12.
Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. However, the underlying mechanism of IFN-alpha therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-alpha, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-alpha and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3'-5' exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-l-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.
丙型肝炎病毒（HCV）感染是慢性肝炎的常见病因，目前采用基于α干扰素（IFN-α）的疗法进行治疗。然而，IFN-α治疗的潜在机制仍有待阐明。为了鉴定介导IFN-α抗病毒作用的细胞蛋白，我们构建了一个基于人胚肾293细胞（HEK293）的细胞培养系统，用于诱导表达单个干扰素刺激基因（ISG），并确定它们对HCV的抗病毒作用。通过筛选29个在Huh7细胞中被IFN-α诱导或在HCV感染的肝脏中上调的ISG，我们发现蝰蛇毒蛋白（viperin）、ISG20和双链RNA依赖性蛋白激酶（PKR）可非细胞溶解性地抑制HCV复制子的复制。从机制上讲，ISG20和PKR对HCV复制的抑制分别取决于它们的3'-5'核酸外切酶和蛋白激酶活性。此外，我们的研究首次提供了有力证据，表明蝰蛇毒蛋白是一种推定的自由基S-腺苷-L-甲硫氨酸（SAM）酶。除了证明蝰蛇毒蛋白的抗病毒活性取决于其自由基SAM结构域（该结构域包含协调[4Fe-4S]簇和辅因子SAM的保守基序，对其酶活性至关重要）外，诱变研究还表明，蝰蛇毒蛋白在其C末端需要一个芳香族氨基酸残基才能发挥正常的抗病毒功能。此外，虽然蝰蛇毒蛋白的N末端70个氨基酸残基并非绝对必需，但删除该区域会显著损害其对HCV的抗病毒活性。我们的研究结果表明，蝰蛇毒蛋白代表了一种新的抗病毒途径，它与其他抗病毒蛋白（如ISG20和PKR）共同作用，介导针对HCV感染的IFN反应。
