在抗病毒模式识别受体RIG-I和PKR通过有限蛋白酶消化和Native PAGE监测激活
Summary
先天防御病毒感染是由模式识别受体（模式识别受体）触发。这两个细胞质模式识别受体RIG-I和PKR结合到病毒签名的RNA，构象变化，低聚，并激活抗病毒信号。方法被描述，其允许方便地监控构象交换和这些细胞质的PRR的低聚。
Abstract
宿主防御病毒感染依赖于快速检测由先天免疫系统的模式识别受体（的PRR）。在细胞质中，PRRS的RIG-I和PKR绑定到特定的病毒RNA配体。该第一介导的构象转换和低聚，然后使活化的抗病毒干扰素应答。而方法来测量抗病毒宿主的基因表达被确立，方法直接监测RIG-I和PKR的激活状态是仅部分和少确立。
在这里，我们描述了两种方法来监测RIG-I和PKR感染刺激后，有一个既定的干扰素诱生剂，裂谷热病毒突变克隆13（以Cl 13）。有限的胰蛋白酶消化使分析改建蛋白酶的敏感性，说明猪蓝耳病的构象变化。裂解液从模拟感染的细胞产生的RIG-I和PKR，wherea的快速降解胰酶消化s氯13感染导致的蛋白酶抗性RIG-I片段的出现。也PKR显示了胰蛋白酶消化，这与其标志的磷酸化苏氨酸在446一致的病毒引起的局部阻力。RIG-I和PKR低聚物是由非变性聚丙烯酰胺凝胶电泳（PAGE）验证的形成。一旦感染，有RIG-I和PKR寡聚物的深厚积累，而这些蛋白仍然是模拟感染样本中单体。
有限蛋白酶消化和天然PAGE，既耦合到免疫印迹分析，允许RIG-I和PKR激活两个不同步骤的敏感和直接测量。这些技术是相对容易和快速执行，并且不需要昂贵的设备。
Introduction
在抗病毒的宿主防御的一个关键事件是由所谓的模式识别受体（模式识别受体）1,2的快速检测病原体。细胞内检测的RNA病毒感染是依赖于两个的细胞质RNA解旋酶，RIG-I（视黄酸诱导基因I）和MDA5（黑色素瘤分化相关蛋白5）3-5。 RIG-I是由两个N-末端caspase募集结构域（卡），一个中央DECH箱型RNA解旋酶结构域和C-末端结构域（CTD）的4,6。而CTD与解旋酶结构域都需要认识到非自（病毒）的RNA，卡介导的下游信号传导，导致建立抗病毒主机的状态。
如果RIG-I是在无声状态下， 即在没有特定的RNA配体，第二张牌交互与中央解旋酶结构域，并保持RIG-I在自动抑制构象7-11。 RIG-I结合，以短双链（DS）的RNA带有5'-三磷酸（5'PPP），长的dsRNA，和理/ UC-丰富的RNA，经典签名结构，其存在于许多RNA病毒12-16的基因组。 RIG-I激活的两个主要特征是一个开关为闭合构象6,17和均聚的低聚6,18,19。构象开关提高了RNA结合，公开卡下游信号，并重新组成一个活跃的ATP酶活性位点8,9,11,20。低聚RIG-I复合物的形成导致下游信号接头分子的增强招募以形成用于抗病毒的信号转导11的一个平台。该RIG-I调节信号链，最终激活转录因子IRF-3的上调对于一个完整的抗病毒反应21,22干扰素（IFN-alpha/beta）基因和干扰素刺激基因，因此该基因的表达（的ISG）的。一个最佳表征ISG的是RNA活化proteiÑ 激酶（PKR）23。 PKR属于真核翻译起始因子2 – α（eIF2α蛋白）激酶家族，它由N-末端的双链RNA结合域和C-末端激酶结构域。激酶结构域构成PKR的激活的二聚化界面的关键，并进行了蛋白质的催化功能。 PKR结合dsRNA病毒导致其构象变化，允许二聚化和自身磷酸化苏氨酸在446等残基中。 PKR介导再eIF2α蛋白磷酸化，从而阻断病毒的mRNA 23-27的翻译。
这两个RIG-I和PKR进行重大的结构重组，形成寡聚复合物，并翻译后磷酸化/去磷酸化和泛素化10,11,19,23,24,26-29修改。为了更好地理解其中的病毒RNA结构被激活RIG-I和PKR（以及在什么阶段病毒拮抗剂可以bë干扰），它精确地确定的激活状态是很重要的。对于这两种的PRR它先前描述的活化导致胰蛋白酶抗性蛋白片段6,17,30和更高阶低聚物6,18,19的出现。然而，由于在抗病毒宿主反应1,2,24这些关键因素的丰富文献，应用直接方法似乎比较罕见的。在刺激更广泛使用的希望，我们提供方便，灵敏协议，以稳健分析RIG-I和PKR的激活状态。干扰素主管人A549细胞感染了RIG-I和PKR，减毒裂谷热病毒突变克隆13（以Cl 13）31,32的既定激活。一个简单的裂解过程后，被感染的细胞的提取物通过有限的胰蛋白酶消化/免疫印迹分析测试，以评估构象转换，并通过蓝色非变性聚丙烯酰胺凝胶电泳（PAGE）/蛋白质印迹analys是测量形成的低聚物。
Protocol
1，播种A549细胞的感染培养的A549细胞的T75烧瓶，在37℃和5％CO 2在细胞培养基（DMEM补充有10％FCS，526.6毫克/升L-谷氨酰胺，50.000 U /升青霉素和50毫克/升的链霉素）。 开始收获细胞前，热身细胞培养基，PBS和0.05％胰蛋白酶-EDTA中加热至37℃的水浴除去培养基并用10ml PBS洗细胞。再次取出PBS。 加3毫升胰蛋白酶-EDTA，并在烧瓶中分发平分。传输烧瓶在培养箱37℃和5％CO…
Representative Results
病毒受体激动剂通过RIG-I或PKR的识别触发构象开关6,17,30和齐聚6,18,27。 ，我们检测这两种活化标志物由有限蛋白酶消化和非变性聚丙烯酰胺凝胶电泳（PAGE）分别。 人类A549细胞被感染裂谷热病毒克隆13（以Cl 13），其特征在于，干扰素拮抗剂奈米35,36的突变。由于缺乏功能性奈米球，克隆13强诱导RIG-I和PKR，导致细胞12,31,32,37建立一个强大的抗病?…
Discussion
感病毒的存在和活化的抗病毒I型IFN系统是成功的先天免疫反应22是至关重要的。病毒检测是由病原体识别受体（模式识别受体），如RIG-I和PKR介导的，从而，实现了快速反应和激活抗病毒防御机制。在这里，我们描述了两种方法来直接评估RIG-I和PKR的激活状态。
有限公司蛋白酶消化作为一种工具来监控RIG-I和PKR首次由M.大风小和T.藤田6,17，和JL科尔30，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢亚历杭德罗·布伦来自中钢协，老龄研究所提供的抗裂谷热病毒血清。在我们的实验室工作是由Forschungsförderung宝石的支持。 §2绝对值。 3 KooperationsvertragUniversitätsklinikum吉森UND马尔堡，莱布尼茨研究生院新生病毒性疾病（EIDIS），东风集团Sonderforschungsbereich（SFB）1021和东风集团Schwerpunktprogramm（SPP）1596。
Materials
| Name | Company | Catalog Number | Comments |
| cell culture | |||
| Dulbecco’s modified Eagle’s medium (DMEM) | Gibco | 21969-035 | |
| OptiMEM | Gibco | 31985-47 | |
| L-glutamine | PAA | 25030-024 | |
| penicillin-streptomycin | PAA | 15070-063 | |
| fetal calf serum (FCS) | PAA | 10270 | |
| 0.05% trypsin-EDTA | Gibco | 25300-054 | |
| chemicals | |||
| L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated (TPCK) trypsin | Sigma Aldrich | T1426 | |
| antibodies | |||
| mouse monoclonal anti-RIG-I antibody (ALME-1) | Enzo Life Sciences | ALX-804-849-C100 | WB: 1:500 in 1% skim milk in TBS |
| mouse monoclonal anti-PKR (B10) | Santa Cruz | sc-6282 | WB: 1:500 in 1% skim milk in TBS |
| rabbit monoclonal anti-P-PKR (Thr446) | Epitomics | 1120-1 | WB: 1:1.000 in 5% BSA in TBS |
| rabbit polyclonal anti-IRF-3 | Santa Cruz | sc-9082 | WB: 1:500 in 1% skim milk in TBS |
| rabbit monoclonal anti-P-IRF-3 (Ser386) | IBL | og-413 | WB: 1:100 in 1% skim milk in TBS |
| rabbit anti-RVFV hyperimmune serum "C2" (MP-12) | Kindly provided by Alejandro Brun (CISA-INIA) | ||
| WB: 1:2.000 in 1% skim milk in TBS | |||
| mouse monoclonal anti-beta-actin (8H10D10) | Cell Signalling | 3700 | WB: 1:1.000 in 1% skim milk in TBS |
| polyclonal peroxidase-conjugated goat anti-rabbit | Thermo Fisher | 0031460 1892914 | WB: 1:20.000 in 1% skim milk in TBS |
| polyclonal peroxidase-conjugated goat anti-mouse | Thermo Fisher | 0031430 1892913 | WB: 1:20.000 in 1% skim milk in TBS |
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Cite This Article
Weber, M., Weber, F. Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE. J. Vis. Exp. (89), e51415, doi:10.3791/51415 (2014).