| Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
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Author:
Date:
2017-12-05
[Abstract] Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein interactions (Duggal and Emerman, 2012). The potency of the IFN signaling response determines the outcome of TBFV infection (Best, 2017; Carletti et al., 2017). Interestingly, data from our lab show that TBFV replication is significantly restricted in cells of the reservoir species Peromyscus leucopus thereby suggesting a potent antiviral response (Izuogu et al., 2017). We assessed the relative contribution of IFN ...
[摘要] 蜱传黄热病病毒（TBFV）的细胞感染导致干扰素（IFN）信号传导途径的激活和随后称为IFN刺激基因（ISG）（Schoggins等人，2011）的众多基因的上调。许多ISG通过蛋白质 - 蛋白质相互作用以广泛或特定的方式起作用来防止病毒发病（Duggal和Emerman，2012）。 IFN信号反应的效力决定了TBFV感染的结果（Best，2016; Carletti等人，2017）。有趣的是，我们实验室的数据显示TBFV复制在储库物种Peromyscus leucopus的细胞中显着受到限制，从而表明有效的抗病毒应答（Izuogu等人，2017）。我们评估干扰素信号对抗性的相对贡献。通过敲低IFN反应途径中的主要转录因子来抑制白血病。信号转导和转录激活因子1（STAT1）是专门针对在P。 leucopus细胞通过shRNA技术。我们进一步测试了基因敲低对细胞对IFN反应和限制病毒复制的能力的影响;结果表明当STAT1表达被改变时，leucopus细胞对IFN刺激的反应降低，并且对TBFV复制显着更敏感。
【背景】IFN信号是抵抗侵入宿主细胞的黄病毒的第一道防线（Robertson等人，2009; Lazear和Diamond，2015）。通过模式识别受体（PRR）检测与病毒颗粒相关的分子标记，然后通过转录因子引发下游信号从细胞释放1型IFN（Kawai ...
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| Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA
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Author:
Date:
2014-03-05
[Abstract] Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. ...
[摘要] TLR3-Flag或Myc-MSR1与HCV RNA的免疫沉淀测定用于鉴定病毒RNA与识别病毒RNA以启动干扰素（IFN）信号传导（宿主细胞的关键抗病毒反应）的宿主蛋白的直接相互作用。 Toll样受体3（TLR3）和A类1型清道夫受体（MSR1）蛋白识别病毒双链RNA（dsRNA），其可以释放到细胞外环境或从HCV感染的细胞扩散到未感染的邻近细胞细胞间接触，导致限制病毒繁殖的IFN-β活化。我们已经发现MSR1结合细胞外dsRNA，介导其胞吞作用并向内吞体转运，其中TLR3参与其中，从而在感染和未感染的细胞中引发IFN应答。我们使用这个测定来证明MSR1在介导HCV RNA的TLR3识别中的关键作用。该方案中描述的测定基于具有条件缓冲液的常规蛋白质免疫沉淀方案，其防止裂解物中存在的RNA酶引起的非特异性RNA降解。与Flag-标记的蛋白相关的RNA分子被特异性抗体捕获，随后是蛋白G捕获，提取并通过RT-PCR测定定量检测，随后是用于可视化的琼脂糖凝胶电泳。这种方法也可以应用于其他蛋白质-RNA相互作用的检测。
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