DataSheet_2_CRISPR-Cas– induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.pdf

BackgroundInterferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.
MethodsBy employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.
ResultsWe found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.
ConclusionAside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

背景：干扰素（IFN）应答在病毒感染的清除过程中发挥关键作用，且已被众多病毒作为主动靶向靶点。其同时在疫苗接种后诱导保护性免疫应答中扮演重要角色，并且作为疫苗佐剂已得到成功应用。干扰素应答在进化上高度保守，在硬骨鱼与哺乳动物中的功能机制极为相似。与哺乳动物类似，鱼类细胞中的干扰素应答同样由不同模式识别受体对胞内病毒感染的识别所启动。一旦识别到病毒组分，干扰素应答会被快速诱导以对抗感染。然而，诸多病毒仍可完成复制，并通过多种途径抑制或规避干扰素应答。
方法：本研究借助CRISPR-Cas9技术，对鲑科细胞系CHSE-214中干扰素信号通路的核心蛋白进行了敲除。通过优化核转染递送CRISPR-Cas核糖核蛋白复合物的实验方案，我们成功获得了线粒体抗病毒信号蛋白（MAVS）、转录因子IRF3与IRF7-1的单基因敲除克隆，以及IRF7-1/3双基因敲除克隆。
结果：经基因表达与启动子激活实验检测发现，MAVS与IRF3基因敲除可抑制胞内聚肌胞苷酸（poly I:C）刺激后干扰素及干扰素刺激基因的诱导表达，而IRF7-1基因敲除则未产生明显影响。上述结果表明，MAVS与IRF3是CHSE-214细胞中胞内RNA诱导的干扰素应答启动所必需的核心因子。为阐明病毒对干扰素诱导通路的干扰机制，我们使用鲑鱼甲病毒3型（SAV3）与传染性胰脏坏死病毒（IPNV）对基因敲除细胞系进行感染实验。在对照组与IRF7-1敲除细胞中，SAV3感染后的病毒滴度相似且未出现明显细胞病变效应；而IRF3与MAVS敲除细胞则出现严重细胞病变效应，且在感染SAV3 6天后病毒滴度显著升高。与之相反，IPNV在IRF3与MAVS敲除细胞中的增殖滴度有所降低，这提示在病毒复制过程中，病毒蛋白与模式识别受体信号通路组分间存在相互依赖的相互作用。
结论：除了为鲑科鱼类的干扰素信号通路研究提供更多新见解之外，本研究结果还提示了一种可在鲑科细胞中提升病毒滴度的潜在方法。