IFIT1负反馈上调干扰素β表达促进抗HSV-1病毒保护

Ⅰ型单纯疱疹病毒（HSV-1）是危害人类健康的常见病原体之一，能够通过受损皮肤或黏膜感染宿主细胞并引起多种疾病。HSV-1的侵入激活先天免疫模式识别受体，诱导干扰素β（IFN-β）的产生，通过表达干扰素刺激基因（ISG）发挥抗病毒功能。近年来，干扰素诱导的四肽重复蛋白1（IFIT1）在病毒感染过程中的作用引起了广大研究者的关注。然而，其具体机制尚未完全清楚。本研究利用CRISPR/Cas9技术构建了小鼠成纤维细胞（L929）IFIT1敲除细胞株，免疫印迹方法检测敲除细胞株IFIT1在蛋白质水平的表达。转染HT-DNA和Poly［I:C］刺激L929 WT和IFIT1敲除细胞株，实时定量PCR技术检测发现，HT-DNA刺激敲除细胞时，IFN-β及下游ISGs的表达量显著升高。IFN-β的表达量比 L929-WT组平均高出13.4倍，IFIT1和趋化因子10（CXC chemokine ligand-10，CXCL10）的表达量比L929 WT组分别平均高出 6.7倍和21倍(P<0.001），而Poly［I:C］刺激无明显变化（P>0.05），表明IFIT1是通过DNA信号通路来行使其负反馈调节作用。为研究IFIT1基因的抗病毒作用，利用CRISPR/Cas9技术改造的HSV-1-VP26 mCherry 病毒感染该敲除细胞株，通过测定病毒荧光数及病毒拷贝数，发现IFIT1敲除细胞株与L929 WT细胞相比，存活率提高了60%（P<0.001），病毒增殖能力在48 h后降低28.6倍（P<0.001）。该结果表明，IFIT1基因的缺失有利于抵抗HSV-1的感染。综上所述，IFIT1通过DNA信号通路负反馈上调IFN-β及ISG的表达，IFIT1的缺失对病毒入侵发挥了保护作用。该结果为后续研究开发治疗HSV-1感染相关的治疗药物提供了一个新思路。

IFIT1 Deficiency Promotes Antiviral Protection Against HSV1 Infection by Enhancing Interferon β Production

Herpes simplex virus typeⅠ (HSV-1) is one of the common pathogens, which endanger human health and infect host cells through damaging skin or mucous membranes, therefore causing a variety of diseases. The invasion of HSV-1 activates innate immune pattern recognition receptors，induces the production of interferon-β (IFN-β)，which performs antiviral function by expressing interferon stimulation gene (ISG). In recent years, the role of interferon- induced protein with tetratricopeptide repeats 1 (IFIT1）family in the viral infection process has attracted the attention of researchers，but the details of mechanism remain unclear. This study constructed the mouse fibroblast (L929) IFITI knockout cell line by the CRISPR/Cas9 technology and evaluated the expression of IFIT1 proteins by Western blotting in the knockout cell line．The mRNA levels of interferon-β and downstream ISGs increased significantly in L929 IFIT1 knockout cell line than WT cell line. IFN-β was 13.4 fold higher than the control group on average，IFIT1 and CXCL10 were 6.7 fold higher and 21 fold higher than the control group，respectively, P<0001. When HT-DNA was transfected into cells, Poly［I:C］ showed no significant change (P>0.5)．The results indicated that IFIT1 exercised its negative feedback regulation through the DNA sensing signaling pathway. In order to study the antiviral effect of the IFIT1 gene, the knockout cell line was infected with the CRISPR/ Cas9 technology-modified HSV-1-VP26-mCherry virus. By measuring the viral fluorescence number and the virus copy number，results indicated that the survival rate of IFIT1 knockout cell line was 60% higher than that of L929 WT cell line (P < 0.001)．The results showed that the deletion of IFIT1 gene was beneficial to resist HSV-1 infection．In conclusion，IFIT1 up-regulates the expression of IFN-β and ISG through negative feedback of the DNA signaling pathway，thereby deletion of IFIT1 protects cells from virus invasion. This result provides a new idea for further research and development of therapeutic drugs for the treatment of HSV-1 infection.