Wang Chia-Hung, Naik Nenavath Gopal, Liao Lin-Li, Wei Sung-Chan, Chao Yu-Chan
Department of Life Science and Institute of Genome Science, National Yang Ming University, No. 115, Section 2, Linong St., Beitou District, Taipei 112, Taiwan.
Institute of Molecular Biology, Academia Sinica, No. 128, Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan.
Mol Ther Methods Clin Dev. 2017 Jul 18;6:194-206. doi: 10.1016/j.omtm.2017.07.002. eCollection 2017 Sep 15.
Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.
尽管杆状病毒已被用作哺乳动物细胞中安全便捷的基因递送载体，但杆状病毒介导的转基因表达在各种哺乳动物细胞系中效果较差。鉴定宿主细胞中的负调控因子对于改进基于杆状病毒的表达系统很有必要。在此，我们针对176个抗病毒先天免疫基因进行了高通量shRNA文库筛选，并在人A549肺癌细胞系中鉴定出43个宿主限制因子基因。其中，抑制受体相互作用蛋白激酶1（RIP1，也称为RIPK1）可显著提高杆状病毒转基因表达，且不会导致明显的细胞死亡。沉默RIP1不影响病毒进入或细胞活力，但会抑制IRF3和NF-κB转录因子的核转位。此外，下游信号介质（如TBK1和IRF7）的激活也受到影响，随后干扰素和细胞因子基因表达水平被消除。此外，RIP1激酶活性抑制剂Necrostatin-1（Nec-1）可显著提高RIP1沉默细胞中杆状病毒转基因的表达。本研究以杆状病毒为模型系统，初步研究了大量人类细胞针对“非适应性病毒”的抗病毒先天免疫反应因子。此外，我们的研究使杆状病毒成为一些最常用哺乳动物细胞系统中更有效的基因转移载体。
