Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
J Hepatol. 2010 Nov;53(5):797-804. doi: 10.1016/j.jhep.2010.05.022. Epub 2010 Jul 24.
BACKGROUND & AIMS: We previously reported that the NS2 protein of hepatitis C virus (HCV) inhibits the expression of reporter genes driven by a variety of cellular and viral promoters. The aim of the study was to determine whether the broad transcriptional repression is caused by endoplasmic reticulum (ER) stress.
Phosphorylation of the translation initiation factor eIF2α and HCV replication was detected by Western and Northern blot, respectively. De novo protein synthesis was measured by metabolic labeling. Activation of ER stress responsive genes was determined by promoter reporter assay, as well as mRNA and protein measurement by real time PCR and Western blot.
Transient or inducible NS2 protein expression increased eIF2α phosphorylation and reduced de novo protein synthesis. It up-regulated promoter activities and transcript levels of ER stress inducible genes including GRP78, ATF6, and GADD153, as well as GRP78 protein level. The same effect was observed when NS2 was synthesized as part of the core-E1-E2-p7-NS2 polypeptide. NS2 protein also inhibited reporter gene expression from the HCV internal ribosome entry site and consequently reduced HCV replication. The full-length HCV replicon activated GRP78, ATF6, and GADD153 promoters more efficiently than the subgenomic replicon lacking the coding sequence for both the structural proteins and NS2. Abrogation of HCV infection/replication, by an inhibitor of the NS3 protease, relieved ER stress.
HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism.
我们之前报道称，丙型肝炎病毒（HCV）的 NS2 蛋白抑制了多种细胞和病毒启动子驱动的报告基因的表达。本研究的目的是确定广泛的转录抑制是否由内质网（ER）应激引起。
通过 Western 和 Northern 印迹分别检测翻译起始因子 eIF2α的磷酸化和 HCV 复制。通过代谢标记测量从头蛋白质合成。通过启动子报告测定、实时 PCR 和 Western blot 测量 mRNA 和蛋白质来确定 ER 应激反应基因的激活。
瞬时或诱导性 NS2 蛋白表达增加了 eIF2α的磷酸化并减少了新蛋白质的合成。它上调了包括 GRP78、ATF6 和 GADD153 在内的 ER 应激诱导基因的启动子活性和转录水平，以及 GRP78 蛋白水平。当 NS2 作为核心-E1-E2-p7-NS2 多肽的一部分合成时，也观察到了相同的效果。NS2 蛋白还抑制了 HCV 内部核糖体进入位点的报告基因表达，从而降低了 HCV 复制。全长 HCV 复制子比缺乏结构蛋白和 NS2 编码序列的亚基因组复制子更有效地激活 GRP78、ATF6 和 GADD153 启动子。通过 NS3 蛋白酶抑制剂阻断 HCV 感染/复制可以缓解 ER 应激。
HCV 感染可诱导 ER 应激，NS2 蛋白是主要介导物。这种应激可以通过反馈机制得到缓解。
