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

描述（由申请人提供）：儿童单纯疱疹病毒1型（HSV-1）脑炎（HSE）是HSV-1原发性感染的一种危及生命的并发症，HSV-1是一种常见病毒，可导致大多数儿童发生无害感染。HSE的发病机制仍然不清楚，直到我们最近发现，这种疾病可能是由于单基因突变损害TLR 3依赖性，IFN-γ/γ介导的免疫HSV-1在中枢神经系统（CNS），在一些儿童。我们描述了10例患者，每个携带纯合或杂合突变的TLR 3通路基因。我们还证明了患者特异性诱导多能干细胞（iPSC）衍生的TLR 3缺陷神经元和少突胶质细胞对HSV-1感染高度敏感，表明TLR 3-IFN介导的CNS内在抗HSV-1免疫受损是先天性TLR 3免疫缺陷患者HSE发病机制的基础。因此，其他儿童的HSE可能是由CNS-抗HSV-1的内在免疫的单基因先天性缺陷的集合引起的，可能但不一定与TLR 3-IFN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名在编码人类脱支酶1（DBR 1）的基因中携带纯合（2名患者）或杂合（2名患者）错义突变。已经提出人DBR 1在从前体mRNA切除的lactinase内含子的快速周转中起关键作用。与这一观察结果相关的是，研究的其他7名HSE儿童在编码5 '-3'核糖核酸外切酶1（XRN 1）的基因中携带杂合无义（2名患者）或错义（5名患者）突变，XRN 1是一种与宿主前mRNA衰变途径中的DBR 1密切相关的分子。DBR 1和XRN 1都具有特定的CNS表达模式。虽然酵母Xrn 1被认为是一种重要的抗病毒分子，但DBR 1和XRN 1如何控制人类中枢神经系统中的抗HSV-1免疫是完全未知的。我们假设，人DBR 1和XRN 1定义了一种新的，CNS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测试该假设。拟议的工作将 因此，本研究的重点是探索DBR 1及其相关基因（主要是XRN 1）在CNS内源性抗病毒免疫中的特异性作用。初步结果进一步显示，与所测试的健康对照相比，来自一名DBR 1 I120 T突变纯合患者的真皮成纤维细胞仅表达非常低水平的DBR 1蛋白并增强HSV-1易感性。我们将测试所有携带DBR 1或XRN 1突变的患者的成纤维细胞。我们将研究DBR 1和XRN 1的抗病毒活性，间接通过TLR 3或IFN介导的免疫，或直接通过DBR 1或XRN 1抑制病毒RNA重组，在体外试验中。我们将研究抗HSV-1免疫应答和HSV-1易感性，无论是否依赖于IFN，在患者的成纤维细胞和iPSC衍生的神经元中缺乏DBR 1或XRN 1，与TLR 3，IFN介导的免疫受损的人相比。这项研究将揭示HSE发病机制的新分子和细胞机制，这将开辟新的治疗途径。

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.