Yamane Daisuke, Dansako Hiromichi, Lemon Stanley M
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, USA.
Tumor Virology Department, Okayama University Graduate School of Medicine, Okayama, Japan.
Bio Protoc. 2014 Feb 20;4(4). doi: 10.21769/BioProtoc.1061.
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. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.
采用TLR3-Flag或Myc-MSR1与丙型肝炎病毒（HCV）RNA的共免疫沉淀试验，来鉴定病毒RNA与宿主蛋白的直接相互作用，这些宿主蛋白识别病毒RNA以启动干扰素（IFN）信号传导，这是宿主细胞至关重要的抗病毒反应。Toll样受体3（TLR3）和A类清道夫受体1型（MSR1）蛋白均识别病毒双链RNA（dsRNA），这种双链RNA可能释放到细胞外环境中，或者通过细胞间接触从HCV感染的细胞传播到未感染的邻近细胞，从而导致IFN-β激活，进而限制病毒传播。我们发现，MSR1结合细胞外dsRNA，介导其胞吞作用并将其转运至内体，在那里它与TLR3结合，从而在感染细胞和未感染细胞中触发IFN反应。我们使用该试验来证明MSR1在介导TLR3识别HCV RNA中的关键作用。本方案中描述的试验基于传统的蛋白质免疫沉淀方案，使用条件缓冲液防止裂解物中存在的核糖核酸酶（RNase）对RNA进行非特异性降解。与Flag标签蛋白相关的RNA分子被特异性抗体捕获，随后通过蛋白G捕获，提取后通过逆转录聚合酶链反应（RT-PCR）试验进行定量检测，然后通过琼脂糖凝胶电泳进行可视化。该方法也可应用于检测其他蛋白质-RNA相互作用。
