单纯疱疹病毒编码的miRNA拮抗宿主cGAS-STING固有免疫信号的新机制研究

The cGAS-STING signaling is critical for restricting herpes simplex virus (HSV), whereas HSV-encoded miRNA could probably inhibit this pathway to subvert immune surveilling. However, the relevant mechanism needs to be further explored. Unexpectedly, HSV-1 encoded miR-H5-3p was fished out to potentially modulate STING expression in our preliminary bioinformatics analysis, which was preliminarily validated by pilot functional assays. Combing molecular, cellular and infection model approaches, we will firstly reveal the regulation relationship between miR-H5-3p and STING by using mutation experiments in binding site sequences and detecting the effect of miR-H5-3p on STING expression. The expressions of pathway key or downstream genes, the dynamic variations of protein modification, and the changes of signal molecule interaction will be systematically analyzed then in the presence of miR-H5-3p, whereby elucidating the mechanism by which this miRNA inhibits the STING-mediated innate immune response. Finally, the exploration of new functions of miR-H5-3p in pathogenesis, latency and transmission of HSV will reflect the significance of this “virus miRNA-STING” cross kingdom regulation in viral immune evasion. Hopefully, these insights will be instrumental for understanding the new mechanisms of HSV immune escape, and for designing novel anti-infective drugs.

cGAS-STING信号通路介导宿主抗病毒免疫反应，单纯疱疹病毒（HSV）可能利用自身miRNA抑制该通路实现免疫逃逸，相关作用机制有待探索。申请人通过生物信息学分析，推测HSV-1编码的miR-H5-3p可能特异靶向STING；初步的荧光素酶报告基因实验等暗示了推测的正确性。本项目将从分子、细胞与感染模型层面，利用结合位点序列突变实验与STING表达变化检测，揭示miR-H5-3p对STING的靶向调控功能；系统分析miR-H5-3p对该通路关键分子及下游基因的表达、蛋白修饰的动态变化、信号分子相互作用变化等的影响，阐明这一miRNA抑制STING介导的固有免疫反应的具体机制；探索miR-H5-3p在HSV致病、潜伏及传播过程中的新功能，系统诠释“病毒miRNA-STING”跨界调控在HSV免疫逃逸中的重要性。本研究将有助于深刻理解HSV免疫逃逸的新机制，为设计新型抗感染药物提供新思路。

I型单纯疱疹病毒（HSV-1）作为一种在人群中广泛感染与传播的病原体，能够利用多种方式逃避宿主的抗病毒免疫反应。与HSV-1编码的多种蛋白相比，其编码的miRNA分子在HSV-1逃避宿主免疫监视的过程中所发挥的作用仍然很不清楚。通过生物信息学分析，我们发现HSV-1编码的miR-H5-3p能够靶向宿主固有免疫信号中的关键分子STING。在多种HSV-1感染的宿主细胞中，我们均检测到了miR-H5-3p的存在。在L929以及MEF细胞中转入miR-H5-3p的类似物能够有效降低STING的蛋白表达水平，然而其mRNA的表达水平几乎不变，提示这一病毒编码的miRNA可能是通过翻译抑制的方式对STING的表达进行调控。在使用htDNA或cGAMP等STING激动剂刺激细胞后，miR-H5-3p能够有效抑制宿主细胞cGAS-STING通路的激活，降低细胞中被诱导产生的IFN-β以及相关干扰素刺激基因（ISGs）的表达水平。在STING KO MEF细胞中进行的实验表明，miR-H5-3p对感染过程中宿主细胞免疫应答的抑制需要依赖宿主细胞STING的参与。我们的研究首次发现了HSV-1编码的miRNA对宿主cGAS-STING固有免疫信号的调控作用，有助于深刻理解HSV免疫逃逸的新机制，为设计新型抗感染药物提供新思路。