Targeting immunophilin- and MAPK-associated pathways to inhibit MERS-CoV replication and prevent lung injury

2002年SARS（严重急性呼吸系统综合征）和2012年MERS（中东呼吸系统综合征）的出现，揭示了冠状病毒可能诱发严重肺炎和肺损伤，死亡率很高。由于新型高致病性冠状病毒被认为是未来对人类的新威胁，因此范围广泛、易于获得的治疗方法至关重要。利用建立的MERS-冠状病毒（MERS - CoV）小鼠感染模型和原代人肺细胞，我们旨在通过诱导干扰素λ (IFNλ)和靶向免疫亲蛋白和下游MAPK途径，在体外和体内研究已知阻断MERS- CoV-宿主相互作用的化合物的抗病毒机制。最终目标是为目前和未来由高致病性人类冠状病毒引起的新发感染制定易于获得的治疗策略。在第一个资助期，我们研究了环孢素A （Cyclosporin A, CsA）对MERS-CoV感染的抗病毒和肺损伤衰减特性。我们发现，csa诱导的强抗病毒作用主要是通过诱导显着的III型干扰素（IFNλ）反应介导的。升高的IFNλ水平刺激干扰素刺激基因（ISG）的IRF1（干扰素调节转录因子1）依赖性上调，并在体外、人原代肺泡上皮细胞中强烈减弱病毒生长，重要的是，在我们最近建立的MERS-CoV小鼠体内模型中（Sauerhering等人，未发表的数据）。此外，我们的数据表明，亲免疫蛋白和mapk依赖途径都传递了csa诱导的MERS-CoV复制阻断。有趣的是，在分析的各种MAPK中，只有JNK抑制导致MERS-CoV颗粒释放的显着损伤。在即将到来的资助期内，我们的目标是研究i) csa刺激的分子信号级联如何导致IFNλ诱导，并研究MERS cov开发的逃逸策略是否会抵消细胞防御机制。我们将进一步研究CsA在MERS-CoV感染的呼吸道上皮细胞中诱导的下游效应，这些效应先前已通过RNA-Seq分析确定。鉴定的差异表达基因将被评估其作为抗病毒策略靶点的适用性。此外，我们将阐明iii) JNK如何通过表征其与宿主细胞因子（包括亲环蛋白A）的相互作用来损害MERS-CoV颗粒的出口，从而在我们的体内和体外感染模型中识别和评估新的分子靶点及其治疗价值。最后，iv)我们将探讨针对csa下游分子的治疗策略在MERS-CoV体内感染中的治疗潜力。获得的数据有望揭示用于治疗MERS-CoV的亲免疫蛋白靶向抗病毒化合物，并可能进一步在体内治疗呼吸道病毒感染。

The emergence of SARS (Severe Acute Respiratory Syndrome) in 2002 and MERS (Middle East Respiratory Syndrome) in 2012 revealed the potential of coronaviruses to induce severe pneumonia and lung injury with high fatality rates. Since novel highly pathogenic CoV are presumed to represent an emerging threat to humans in future, broad-range, readily-available therapeutics are of utmost importance. Using an established MERS-Coronavirus (MERS CoV) mouse infection model and primary human lung cells, we aim to characterize the antiviral mechanisms of compounds known to block MERS CoV-host interactions by inducing interferon lambda (IFNλ) and targeting immunophilin and downstream MAPK pathways in vitro and in vivo. The final goal is to establish readily-available treatment strategies for current and future emerging infections caused by highly pathogenic human coronaviruses. In the first funding period, we investigated the antiviral and lung injury-attenuating properties of Cyclosporin A (CsA) in MERS-CoV infection. We revealed that a CsA-induced, strong antiviral effect was largely mediated by induction of a pronounced type III interferon (IFNλ) response. Elevated IFNλ levels stimulated an IRF1 (interferon regulatory transcription factor 1)-dependent upregulation of interferon stimulated genes (ISG) and strongly attenuated viral growth in vitro, in human primary alveolar epithelial cells ex vivo, and, importantly, in our recently established MERS-CoV mouse model in vivo (Sauerhering et al., unpublished data). In addition, our data indicate that both immunophilin- and MAPK-dependent pathways convey the CsA-induced block of MERS-CoV replication. Interestingly, among various MAPK analyzed, only JNK inhibition led to a significant impairment of MERS-CoV particle release. In the upcoming funding period we aim to investigate i) how CsA-stimulated molecular signaling cascades lead to IFNλ induction and investigate whether MERS CoV-developed escape strategies counteract cellular defense mechanisms. We will further ii) investigate putative downstream effectors induced by CsA in MERS-CoV infected respiratory epithelial cells which were previously defined by RNA-Seq analyses. The identified differentially expressed genes will be evaluated for their suitability as targets for antiviral strategies. Moreover, we will elucidate iii) how JNK impairs MERS-CoV particle egress by characterizing its interplay with host cell factors including cyclophilin A to identify and evaluate novel molecular targets for their therapeutic value in our in vivo and ex vivo infection models. Finally, iv) we will investigate the therapeutic potential of treatment strategies targeting CsA-downstream molecules in MERS-CoV in vivo infection.The obtained data are expected to reveal immunophilin-targeting antiviral compounds for treatment of MERS-CoV, and potentially further respiratory virus infections in vivo.