Division of Gastroenterology, Department of Medicine, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL, USA.
J Viral Hepat. 2011 Oct;18(10):e523-9. doi: 10.1111/j.1365-2893.2011.01452.x. Epub 2011 Mar 16.
To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)-mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES-mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES-mediated translation.
为了研究信号转导子和转录激活子 1（STAT1）诱导的抗丙型肝炎病毒（HCV）效应的功能，建立了一个稳定的 Huh7.5 细胞系（Huh7.5-STAT1ER），该细胞系持续表达 STAT1 和小鼠雌激素受体（ER）的融合蛋白（STAT1ER），在 4-羟他莫昔芬（4-HT）处理后形成 STAT1ER 同源二聚体。这种诱导型和细胞因子/受体非依赖性 STAT1 激活系统使我们能够在诱导 IFN 刺激基因（ISG）表达后研究 STAT1ER 激活的抗 HCV 效应。通过 HCV（JFH-1）感染后实时 PCR 以时间依赖性方式确定二聚化 STAT1ER 融合蛋白的抗 HCV 效应。在 4-HT 处理后 72 小时，HCV（JFH-1）RNA 减少了 48%。为了区分 STAT1ER 激活对 HCV RNA 复制或 HCV 内部核糖体进入位点（IRES）介导的翻译的抑制作用，在研究中使用了双顺反子 pRL-HL 构建体。在 STAT1ER 细胞系中，在 STAT1ER 激活后 72 小时，细胞（Cap 依赖性）和 HCV IRES 介导的（Cap 非依赖性）翻译分别降低了 63%和 57%。在我们之前的研究中，发现干扰素诱导的跨膜蛋白 3[（IFITM3）（1-8U）]抑制 HCV RNA 复制。随后，通过 Western blot 在 Huh7.5-STAT1ER 细胞系中证实了 1-8U 基因的上调表达。为了进一步研究 1-8U 在体内和体外研究中的功能，发现 1-8U 基因抑制细胞和 HCV IRES 介导的翻译。
