宿主miR-138通过靶向病毒ICP0和宿主基因促进HSV潜伏的作用及机制

Herpes simplex virus-1 and -2 (HSV-1 and HSV-2) can undergo latent infection in sensory neurons, from which the viruses can reactivate and cause disease. Latency is the key reason why HSV infections have yet to be cured. Recent studies have suggested that some host miRNAs can promote latency for some viruses, yet little is known about how host miRNAs affect HSV latency. Our previous research suggests that the neuron-specific host miRNA, miR-138, suppresses HSV-1 replication and gene expression, both by directly targeting the mRNA for the viral protein ICP0, and most likely by also targeting host mRNAs, leading to our hypothesis that these are mechanisms conserved between HSV-1 and HSV-2 that promote viral latency. The first aim of this project is to investigate the effects of miR-138 on HSV-1 and HSV-2 replication and gene expression in neuronal cultures. In the second aim, we plan to construct sensory neuron specific miR-138 knock-out mice as well as a recombinant HSV-1 that expresses either miR-138 or its antisense sequences. By comparing these mice and viruses with the corresponding control groups in a mouse model of latent infection, we will investigate the effects of miR-138 on levels of HSV-1 or HSV-2 replication and gene expression in mouse trigeminal ganglia, as well as on mouse immune responses and symptoms. In the third aim, we plan to identify the unknown host targets of miR-138 using PAR-CLIP and RNAseq techniques, followed by investigation of the molecular mechanisms by which miR-138 promotes HSV-1 and HSV-2 latency independently of ICP0. The results of this project should elucidate the role miR-138 plays in HSV latency, and might provide ideas on how to treat or even cure HSV latent infections.

单纯疱疹病毒（HSV）I和II型可潜伏于感觉神经元并从中激活引发疾病。潜伏是HSV无法治愈的关键。近年报道显示宿主miRNA可促使一些病毒潜伏，但人们对其如何影响HSV潜伏所知甚少。我们前期研究提示神经特异性宿主miR-138通过靶向病毒ICP0基因和未知宿主基因两种方式下调HSV复制或基因表达，故推测与其潜伏机制有关。本项目首先用培养细胞确定神经元中miR-138对病毒复制和基因表达的影响。然后构建miR-138敲除小鼠和表达及抑制miR-138的重组病毒，创建敲除、过表达和抑制miR-138三种情况下的小鼠潜伏感染模型，检测病毒复制和基因表达水平，观测小鼠免疫指标和症状，并与对照组比较。最后用PAR-CLIP和RNAseq技术识别miR-138的未知宿主靶基因，进而揭示其独立于ICP0促进潜伏的分子机制。结果将明确miR-138促进HSV潜伏的作用及机制，为治疗HSV潜伏感染提供思路。

本项目旨在明确宿主神经特异性miR-138促进单纯疱疹病毒（HSV）潜伏的作用与机制，从而探索HSV潜伏于神经元的原因。研究内容包括在已知miR-138靶向HSV-1 ICP0的基础上，找出miR-138的其他靶基因，并解析miR-138调控HSV潜伏的分子机制。我们将RNA测序（RNA-seq）、光活性核苷增强型交联与免疫沉淀（PAR-CLIP）和小干扰RNA（siRNA）三种高通量技术相结合，识别出宿主Oct-1和Foxc1 mRNA为miR-138的靶标，并发现其基因产物对HSV-1在神经细胞中复制非常重要。Oct-1是已知的可以促进HSV转录的宿主因子。我们发现在神经细胞系、原代神经元和小鼠中，Foxc1均可以显著促进HSV-1复制和基因表达，而且其作用与ICP0的作用有一定互补效应。这些结果提示miR-138通过调控病毒ICP0基因和宿主Oct-1和Foxc1基因等多种通路联合帮助HSV-1在神经元中潜伏。基于这些结果的论文刚发表于Nature Microbiology。在研究HSV-1的同时，我们研究了上述作用在HSV-2感染中的保守性和差异性，发现miR-138对ICP0的作用在HSV-2感染中是保守的，但ICP0非依赖性机制有所不同，如Foxc1对HSV-2的作用相对较弱，但miR-138又可通过靶向HSV-2的UL20抑制病毒复制。这部分研究也基本完成，预计2021年上半年投稿（拟投mBio）。在完成上述主要目标的过程中，我们也用该项目中建立的技术探讨了与病毒潜伏机制相关的一些问题，相关的一篇论文于2019年发表在mBio，另2篇目前在投于Virology和Journl of Virology杂志中，还有三篇中文综述发表在核心中文期刊。综上所述，我们实现了预期目标，研究结果对理解HSV潜伏感染机制有重要意义。

内容获取失败，请点击重试