LGP2正向调控疱疹病毒蛋白vhs降解宿主mRNA的机制研究

Herpes simplex virus type 1 (HSV-1) infection can cause clinical symptoms such as oral herpes and encephalitis. In the lytic infection phase, the viral protein vhs cleaves the host mRNA by exerting its endonuclease function and mediates it degradation, thereby affecting the infection reaction. However, the molecular mechanism of regulating vhs function is still unclear. Our previous data indicated that HSV-1 infection caused a decrease in the abundance of innate immune gene mRNA, and this reduction was significantly inhibited in LGP2 knockout cells. At the same time, LGP2 knocked-out can inhibit the expression of TTP which is a key component of the vhs complex. Therefore, we hypothesize that LGP2 can positively regulate the cleavage function of vhs on the mRNA of innate immune genes by regulating the expression of TTP. In this study, we utilize the gene microarray, qPCR and Northern blot analysis to determine the mRNA abundance changes after virus infection through cell and mice model, combining siRNA interference, Exogenous gene expression and immunofluorescence assay to uncover the positively role of LGP2 in regulating the clevage function of vhs on host mRNA in vivo and in vitro. This study will provide new insights into the antiviral therapy and reveal the mechanism of virus latent reactivation.

I 型单纯疱疹病毒（HSV-1）感染能引起皮肤性疱疹，甚至脑炎等临床疾病。在急性感染期，病毒蛋白vhs发挥核酸内切酶功能对宿主mRNA进行剪切并介导降解，从而调节感染过程。然而调控vhs功能的宿主分子机制尚不明确。我们预实验结果发现HSV-1感染能造成相关天然免疫基因mRNA的丰度减少，而这种减少在LGP2敲除细胞里受到明显抑制；同时LGP2敲除能抑制vhs通路关键蛋白TTP的表达。因此我们提出假设：LGP2可通过调控TTP表达来正向调节vhs对天然免疫基因mRNA的剪切作用。本研究拟采用细胞和小鼠感染模型，通过基因芯片, qPCR和Northern杂交手段确定mRNA丰度变化情况，结合siRNA敲减，外源基因表达，细胞免疫荧光等技术从“体内-体外”水平探究LGP2正向调控vhs剪切宿主mRNA的作用并阐明其分子机制，为寻找抗病毒治疗新靶点及揭示HSV-1潜伏激活机理提供新思路。

在I型单纯疱疹病毒（HSV-1）急性感染期，病毒蛋白vhs能通过发挥核酸内切酶作用对宿主应激mRNA进行剪切并介导降解，从而拮抗宿主抗病毒反应。另外，LGP2基因的缺失能显著抑制HSV-1病毒的复制和对天然免疫相关应激mRNA的剪切降解。基于以上研究，本项目通过HSV-1溶细胞感染细胞模型，运用CRISPR/Cas9基因敲除、外源基因过表达、RNA-seq、WB、qPCR等技术对LGP2如何调控vhs对天然免疫相关基因mRNA的剪切降解作用并影响病毒复制的分子机制进行了进一步研究。本研究主要完成了：从分子水平验证了在病毒感染过程中，LGP2通过正向调控TGF-β通路，继而促进TTP蛋白表达来增强vhs对天然免疫相关应激基因mRNA的剪切作用。同时筛出了MDA5、IFI16和HDAC4等对HSV-1复制起关键调控作用的基因。在本项目基金支持下，共发表3篇SCI论文，其中还有部分工作正准备投稿中。总体而言，该研究为寻找抗病毒治疗或基于HSV-1病毒的肿瘤溶瘤治疗提供了新的靶点和策略。