Expression data from Huh7.5.1 cells transfected with siNDRG1 or siNT

Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc Downstream-Reguated Gene 1 (NDRG1) limits productive HCV infection by inhibiting viral assembly. Interestingly, HCV infection down-regulates NDRG1 protein and mRNA expression. Loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by up-regulating MYC, which directly inhibits the transcription of NDRG1. Up-regulation of MYC also leads to reduced expression of NDRG1-specific kinase SGK1, resulting in markedly diminished phosphorylation of NDRG1. Knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both transcriptional and post-translational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by down-regulating NDRG1 via a MYC-dependent mechanism. We knocked down NDRG1 by transfecting cells in triplicate with siNDRG1 or siNT as control, we then analyzed the results to determine differential gene expression and differentially regulated pathways.

宿主细胞具备多种固有机制，作为抗病毒防御的第一道防线以限制病毒感染。病毒已演化出多种针对这些抗病毒机制的应对策略。本研究发现，N-Myc下游调控基因1（N-Myc Downstream-Regulated Gene 1，NDRG1）可通过抑制病毒组装过程，限制丙型肝炎病毒（Hepatitis C Virus，HCV）的增殖性感染。有趣的是，HCV感染会下调NDRG1的蛋白质与mRNA表达水平。NDRG1的缺失会增加脂滴的大小与数量，而脂滴正是HCV组装的场所。HCV通过上调MYC来抑制NDRG1的表达，MYC可直接抑制NDRG1的转录。MYC的上调还会导致NDRG1特异性激酶血清糖皮质激素调节激酶1（SGK1）的表达降低，进而使NDRG1的磷酸化水平显著下降。在HCV感染期间敲低MYC可恢复NDRG1的表达与磷酸化水平，这表明MYC可在转录和翻译后两个水平上调控NDRG1。综上，本研究结果表明，NDRG1可通过限制脂滴形成来抑制HCV的组装过程。HCV则通过依赖MYC的机制下调NDRG1，从而拮抗这一天然抗病毒防御机制。本研究通过将细胞以三重重复的方式转染siNDRG1或作为对照的siNT以敲低NDRG1，随后对实验结果进行分析，以鉴定差异表达基因与差异调控通路。