解旋酶DDX3X在慢性活动性EB病毒感染中的作用及机制研究

Epstein-Barr virus (EBV) transient infection is very common in the healthy population and can be cleared by NK cells. As a rare and severe lymphoproliferative disease, the molecular mechanism of NK cells unable to clear EBV in chronic active EB virus infection (CAEBV) is still unclear and there are no effective treatments for CAEBV. We have found that 30% of CAEBV (n=20) patients carried somatic loss-of-function mutations of the DDX3X gene, which can play a role in antiviral immune response, in NK cells of CAEBV. The RIG-I like receptor (RLR) signaling pathway, which is sensitized by DDX3X, was downregulated in DDX3X mutated NK cells of CAEBV patients. Based on these, we hypothesize that helicase DDX3X is the key factor for EBV persistent infection in CAEBV. In this project, we intend to perform exome sequencing and transcriptome sequencing to study the effect of DDX3X mutation on NK cell activity/cytotoxic functionality. And further investigate the pathogenetic roles of mutated DDX3X which can dysregulate the RLR signaling pathway in NK cells. The functional role of DDX3X in NK cells of CAEBV will be confirmed by a series of essays. It is expected that in this study, the role of helicase DDX3X in the NK cells of CAEBV will be systematically interpreted for the first time and to gain a deeper understanding of the molecular mechanism of CAEBV and other EBV positive diseases and to provide clues for finding new drug targets.

健康人群中普遍存在短期的EB病毒感染，通常可被NK细胞清除。而作为一种罕见的恶性淋巴组织增殖性疾病，慢性活动性EB病毒感染(CAEBV)中NK细胞无法清除EBV的分子机制不明。我们前期20例样本研究发现30%的CAEBV患者的NK细胞在具有抗病毒功能的DDX3X上发生失活体细胞突变，其所调控的RLR免疫通路表达水平下调。据此提出假说：DDX3X是降低NK细胞功能导致EBV无法清除的关键因子。我们将在扩大的样本(n=60)中，通过外显子组测序刻画DDX家族基因突变谱；通过转录组测序明确DDX3X突变后表达水平下调的免疫通路(RLR signaling etc)。并通过体外实验验证DDX3X及下游关键基因对NK活性、细胞因子释放和EBV载量的影响，进而阐明DDX3X介导RLR免疫通路导致EBV持续感染的分子机制。本项目为CAEBV及EBV谱系疾病机制研究提供新思路，并为寻找新药物靶点提供线索。

EB病毒相关淋巴组织增殖性疾病（EBV-T/NK-LPDs）是一组由EB病毒（EBV）持续感染引发的罕见恶性血液病。EBV-T/NK-LPDs主要包括EBV相关噬血细胞综合征（EBV-HLH）和慢性活动性EB病毒感染（CAEBV）。EBV-T/NK-LPDs的临床表现多样，具有从惰性到急性进展不同病程。目前该类疾病的发病分子遗传基础研究不足，制约了对该类疾病的精准分层和采用合适的治疗方案。项目前期，我们在小样本CAEBV (n=20) 研究中发现解螺旋酶DDX3X发生重现性体细胞突变，该基因可参与免疫调节。我们提出科学假说：DDX3X变异是EBV-T/NK-LPDs中EBV持续感染的重要机制之一。在本项研究中，我们收集了119例大样本成人EBV-T/NK-LPDs样本，整合全外显子组测序、靶向测序和转录组测序来探索EBV-T/NK-LPDs的先天突变和获得性突变遗传图谱。并采用一系列功能试验验证我们的发现。我们发现41.1%的病人存在RIG-I-like受体信号通路突变（DDX3X基因的体细胞突变和IFIH1基因的先天突变）。RIG-I-like受体信号通路异常是首次在成人EBV-T/NK-LPDs被发现。存在RIG-I-like受体信号通路突变的病人血浆和感染NK细胞的EB载量高，提示该通路与EBV复制和载量高低具有关联性。此外，存在上述突变患者RIG-I-like受体信号通路活性下降，DHX58, TRAF6, TBKBP1和MAP3K等关键基因表达下调，EBV编码基因LMP1等上调。DDX3X敲低细胞系试验结果与病人中发现一致。另外，存在RIG-I-like受体信号通路相关基因突变的患者，生存率更差。综上，本项研究发现了存在RIG-I-like受体信号通路突变是EBV-T/NK-LPDs新的遗传基础，并发现一组突变基因有望用于该类疾病的预后分层。