Ancestral allele of DNA polymerase gamma modifies antiviral tolerance

Mitochondria are arising as critical modulators of antiviral tolerance by releasing mtDNA/mtRNA fragments to cytoplasm upon infection, activating virus sensors and type-I interferon response. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here, we investigated mitochondrial recessive ataxia syndrome (MIRAS), caused by a common European founder mutation in DNA polymerase gamma (POLG1). The patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages-of-onset and symptoms, indicating unknown modifying factors contributing to disease manifestation. We report that the mitochondrial DNA (mtDNA) replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV, SARS-CoV-2) and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release to cytoplasm and slow interferon response in MIRAS allow an early replicative advantage for viruses, leading to augmented pro-inflammatory response, subacute loss of GABAergic neurons, liver inflammation and necrosis. A population databank of ~300,000 Finns demonstrates enrichment of immunodeficient traits in p.W748S carriers. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications to mitochondrial disease spectrum, including epilepsy, ataxia, and parkinsonism. To investigate the in vivo response of MIRAS to viral infection, we generated a MIRAS-knock-in mouse model and subjected the mice to TBEV infection. These animals carry a homozygous knock-in mutation and the accompanying polymorphic variant homologous to the human ancestral MIRAS allele (p.W726S+E1121G in mice and p.W748S+E1143G in humans). We performed RNA sequencing on the mouse brain (cerebral cortex) isolated from the control (wildtype) and MIRAS mice at uninfected (baseline) condition and 4 days post TBEV infection (n=5 mice per condition). Comparative gene expression analysis of RNA-seq data for control vs MIRAS mouse cerebral cortex at baseline versus Day 4 after TBEV infection

线粒体作为抗病毒耐受的关键调节因子逐渐受到关注：感染发生时，线粒体向细胞质释放线粒体DNA（mtDNA）/线粒体RNA（mtRNA）片段，激活病毒感知通路与I型干扰素（type-I interferon）应答。目前，此类机制与线粒体疾病的关联仍有待深入探究。
本研究针对由DNA聚合酶γ（POLG1）上常见欧洲奠基者突变引发的线粒体隐性共济失调综合征（MIRAS）展开研究。携带MIRAS变异位点p.W748S纯合基因型的患者，其发病年龄与临床症状存在显著异质性，提示存在尚未明确的疾病修饰因子参与疾病表型的形成。
我们发现，线粒体DNA复制酶POLG1在对抗双链DNA与正链RNA病毒（包括单纯疱疹病毒1型（HSV-1）、蜱传脑炎病毒（TBEV）、新型冠状病毒（SARS-CoV-2））感染的防御机制中发挥作用，而其p.W748S变异会削弱固有免疫应答。
患者样本与敲入小鼠模型的数据表明，p.W748S变异会破坏线粒体DNA复制体的稳定性，引发线粒体DNA耗竭，且病毒感染会进一步加重该表型。细胞质中线粒体DNA与线粒体RNA的释放减少、干扰素应答延迟，使得MIRAS患者体内病毒获得早期复制优势，进而加剧促炎应答、引发亚急性γ-氨基丁酸能神经元丢失、肝脏炎症与坏死。
一项涵盖约30万芬兰人群的人群数据库研究显示，p.W748S携带者的免疫缺陷相关表型富集。我们的研究结果提示，POLG1功能缺陷会损害抗病毒耐受能力，进而诱发癫痫与肝脏疾病。这一发现对涵盖癫痫、共济失调与帕金森综合征在内的线粒体疾病谱具有重要意义。
为探究MIRAS在体内的抗病毒应答特征，我们构建了MIRAS敲入小鼠模型，并对模型小鼠进行蜱传脑炎病毒感染。该模型小鼠携带纯合敲入突变，且伴随与人类祖先MIRAS等位基因同源的多态性变异（小鼠对应位点为p.W726S+E1121G，人类对应位点为p.W748S+E1143G）。
我们对未感染（基线状态）与蜱传脑炎病毒感染4天后的对照（野生型）小鼠及MIRAS模型小鼠的大脑皮层组织进行了RNA测序（RNA-seq），每组各包含5只小鼠。
本研究针对上述RNA测序数据，开展了对照（野生型）与MIRAS模型小鼠在基线状态及蜱传脑炎病毒感染后第4天的大脑皮层组织的基因表达比较分析。