Human DBR1 defines a new pathway of intrinsic antiviral immunity in the CNS

描述(申请人提供)：儿童单纯疱疹病毒1型(HSV-1)脑炎(HSE)是HSV-1初次感染的威胁生命的并发症，HSV-1是一种常见病毒，会在大多数儿童中造成无害感染。HSE的发病机制尚不清楚，直到我们最近发现，在一些儿童中，这种疾病可能是由于单基因突变损害了中枢神经系统(CNS)中依赖TLR3的、干扰素介导的对HSV-1的免疫。我们描述了10例患者，每个患者都携带TLR3途径基因的纯合子或杂合突变。我们还证实了患者特异性诱导多能干细胞(IPSC)来源的TLR3缺陷神经元和少突胶质细胞对HSV-1感染高度易感，提示TLR3-干扰素介导的CNS固有抗HSV-1免疫功能受损是先天性TLR3免疫缺陷患者HSE发病的基础。因此，其他儿童的HSE可能是中枢神经系统对HSV-1固有免疫的单基因先天错误的集合，可能但不一定与TLR3-干扰素回路有关。令人惊讶的是，在最近通过全外显子组测序(WES)研究的100名HSE儿童中，有4名携带编码人类脱支酶1(DBR1)基因的纯合(两名患者)或杂合(两名患者)错义突变。人DBR1被认为在套索内含子的快速周转中起关键作用，该内含子从Pre-mRNA中去除。与这一观察结果相关的是，另外七名被研究的HSE儿童携带编码5‘-3’外切核糖核酸酶1(XRN1)基因的杂合性无义突变(两名患者)或错义突变(五名患者)，XRN1是一种在宿主mRNA前衰退途径中与DBR1密切相关的分子。DBR1和XRN1都有特定的CNS表达模式。虽然酵母Xrn1被认为是一种重要的抗病毒分子，但DBR1和XRN1是如何在人的中枢神经系统中控制抗HSV-1免疫的尚不清楚。我们假设人类DBR1和XRN1定义了一种新的中枢神经系统特异性的内在抗HSV-1免疫机制。本申请的目标是在分子和细胞水平上检验这一假说。拟议的工作将 重点探讨DBR1及其相关基因(主要是XRN1)在中枢神经系统固有抗病毒免疫中的具体作用。初步结果进一步表明，与测试的健康对照组相比，1名DBR1I120T突变纯合子患者的皮肤成纤维细胞只表达非常低水平的DBR1蛋白，并增强了HSV-1的易感性。我们将测试所有携带DBR1或XRN1突变的患者的成纤维细胞。我们将在体外实验中，通过TLR3或干扰素介导的免疫间接或直接通过DBR1或XRN1抑制病毒RNA重组来研究DBR1和XRN1的抗病毒活性。我们将研究缺乏DBR1或XRN1的患者成纤维细胞和IPSC来源的神经元中的抗HSV-1免疫反应和HSV-1敏感性，无论是否依赖于干扰素，并与TLR3和干扰素介导的免疫受损的患者进行比较。本研究将揭示HSE发病机制的新分子和细胞机制，为HSE的治疗开辟新的途径。

DESCRIPTION (provided by applicant): Childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE) is a life-threatening complication of primary infection by HSV-1, a common virus that causes innocuous infections in most children. The pathogenesis of HSE remained unclear until we recently showed that the disease may result from single-gene mutations impairing TLR3-dependent, IFN-¿/¿-mediated immunity to HSV-1 in the central nervous system (CNS), in some children. We described 10 patients, each carrying homozygous or heterozygous mutations in a TLR3 pathway gene. We also demonstrated that patient-specific induced pluripotent stem cells (iPSC)-derived TLR3-deficient neurons and oligodendrocytes were highly susceptible to HSV-1 infection, suggesting that impaired TLR3-IFN-mediated CNS intrinsic anti-HSV-1 immunity underlies the pathogenesis of HSE in patients with inborn errors of TLR3 immunity. HSE in other children may therefore result from a collection of single-gene inborn errors of CNS- intrinsic immunity against HSV-1, probably but not necessarily related to the TLR3-IFN circuit. Surprisingly, out of the 100 HSE children recently studied by whole exome sequencing (WES), four carry homozygous (two patients) or heterozygous (two patients) missense mutations in the gene encoding human debranching enzyme 1 (DBR1). Human DBR1 has been proposed to play a key role in the rapid turnover of lariat introns excised from pre-mRNA. Related to this observation, seven other HSE children studied carry heterozygous nonsense (two patients) or missense (five patients) mutations in the gene encoding 5'-3' exoribonuclease 1 (XRN1), a molecule closely connected to DBR1 in a host pre-mRNA decay pathway. Both DBR1 and XRN1 have particular CNS expression patterns. Although yeast Xrn1 is known as an essential antiviral molecule, it is completely unknown how DBR1 and XRN1 can control anti-HSV-1 immunity in the human CNS. We hypothesize that human DBR1 and XRN1 define a new, CNS-specific mechanism of intrinsic anti-HSV-1 immunity. The goal of the present application is to test this hypothesis at the molecular and cellular levels. The proposed work will be focused on the exploration of the specific role of DBR1 and its related genes (mainly XRN1) in CNS-intrinsic anti-viral immunity. The preliminary results further showed that dermal fibroblasts from one patient homozygous for a DBR1 I120T mutation express only very low levels of DBR1 protein and enhanced HSV-1 susceptibility, as comparing to the healthy controls tested. We will test fibroblasts from all patients carrying mutations in DBR1 or XRN1. We will investigate the antiviral activity of DBR1 and XRN1, indirectly via TLR3- or IFN-mediated immunity, or directly via the suppression of viral RNA recombination by DBR1 or XRN1, in in vitro assays. We will investigate anti-HSV-1 immune responses and HSV-1 susceptibility, both IFN-dependent or not, in patients' fibroblasts and iPSC-derived neurons deficient for DBR1 or XRN1, in comparison with those with impaired TLR3-, IFN-mediated immunity. This research will shed light on a novel molecule and cellular mechanism of the HSE pathogenesis, which will open new therapeutic avenues.