树鼩抗病毒先天免疫RLR通路基因的功能鉴定
文献类型：学位论文
| 作者 | 许凌 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-10 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 姚永刚 |
| 关键词 | 树鼩 先天性免疫 病毒感染 动物模型 |
| 其他题名 | Functional characterization of RLR signaling genes in the Chinese tree shrew |
| 学位专业 | 遗传学 |
| 中文摘要 | 树鼩作为具有潜在应用价值的新型实验动物，是灵长类动物的近亲，日渐受到重视。通过前期高质量的树鼩基因组解析，全面获取了这一新型实验动物的遗传特性，解释了其用于人类疾病动物模型，尤其是病毒感染模型创建的遗传基础。通过对树鼩基因组分析，我们发现树鼩天然免疫系统基因中RIG-I基因缺失。RIG-I是细胞内识别病毒RNA的关键受体，它与线粒体接头蛋白MAVS（也称为Cardif、IPS-1或VISA）相互作用，诱导I型干扰素产生，在抗病毒先天然免疫中发挥了重要的作用。树鼩基因组上该基因缺失，可能对抗病毒先天性免疫信号通路产生了影响，导致多种病毒可以感染树鼩，也可能因为有新的RIG-I功能替代物出现，（部分）补偿了其功能，导致目前某些病毒感染树鼩受到限制。 本论文基于RIG-I缺失，从RLR（RIG-I-like receptors）信号通路出发，进行较为系统的研究发现： 1. 树鼩RIG-I基因的缺失是在中缅树鼩（Tupaia belangeri）中普遍存在的现象，并且该缺失是在树鼩进化成为独立物种过程中发生的。树鼩RIG-I的缺失，同一家族成员树鼩MDA5（tMDA5）受到了正选择，过表达tMDA5能有效的激活IRF3、NF-κB 和IFN-β，并在仙台病毒（sendaivirus，SeV）刺激下协同SeV诱导I型干扰素的表达，且明显抑制丙型肝炎病毒（Hepatitis C virus， HCV）的复制。tMDA5受到正选择的位点是Lys188和Ala402，突变体功能实验证实tMDA5的抗病毒能力要强于未受到正选择的突变体tMDA5 K188Q和tMDA5 A402K，提示着树鼩中可能是tMDA5部分替代了树鼩RIG-I的功能，在细胞中发挥主要识别病毒的功能。 2. 我们检测了树鼩RIG-I缺失后，RLR的唯一下游接头蛋白树鼩MAVS（tMAVS）在HCV感染后的功能变化。克隆和功能结果显示了tMAVS在结构和功能上的保守性。从HCV逃避先天性免疫的角度，我们比较了人MAVS（hMAVS）和tMAVS的异同点，从分子水平探讨树鼩作为丙型肝炎动物模型的可能性。在树鼩肝源性细胞中，HCV NS3/4A蛋白酶可与tMAVS共定位，并通过识别保守的第508位半胱氨酸切割tMAVS。tMAVS的第506位谷氨酸，虽然相对hMAVS不是保守的缬氨酸，但仍然支持NS3/4A对tMAVS的切割。出乎意料的是，HCV NS3/4A只能削弱IRF3介导诱导的IFN-β，并不影响NF-κB信号的传递，这也许可以解释树鼩HCV长期感染率低的现象。 3. 我们深入研究了这条信号通路中另一个抗病毒通路必不可少的蛋白MITA（也称为STING/TMEM173/MPYS/ERIS）在树鼩抗病毒先天性免疫中的功能。我们克隆了树鼩MITA（tMITA）基因，意外发现tMITA存在两种不同的转录本，除全长的tMITA（tMITA-FL）外，还存在一个缺乏C末端的截断剪切突变体（tMITA-mini），tMITA-mini在静息状态下表现出对tMITA-FL的抑制功能，发挥负调控子的功能。有趣的是，在抗病毒感染过程中，tMITA-FL主要表现出对HSV-1的抗病毒能力，而tMITA-mini则在不同RNA病毒刺激下表现出强烈的抑制VSV和HCV复制的能力。这些初步的实验结果提示着tMITA-FL和tMITA-mini分别在不同的信号通路中分别发挥抗病毒功能。 总之，我们深入探讨了树鼩先天性免疫RLR信号通路关键基因和分子调控机制，取得了一系列原创性结果，从先天性免疫的角度，证明了树鼩可以感染HCV，及树鼩RIG-I缺失后关键抗病毒蛋白功能的变化。研究结果将为树鼩病毒感染动物模型建立提供新的思路。 |
| 英文摘要 | The Chinese tree shrew (Tupaia belangeri chinensis), the closest relative of primates, possesses great potential as an experimental animal in biomedical research and has attracted increasing attention in modeling human diseases and therapeutic responses. A publicly available annotated genome sequence for the Chinese tree shrew was recently presented by the team Kunming institute of Zoology and BGI. Key factors and signaling pathways in immune systems were investigated Tree shrews possessed both shared common and unique features. The RIG-I gene was lost in the Chinese tree shrew genome, which functions to trigger the transduction cascade involving the signaling pathway mediated by the MAVS (also known as Cardif, IPS-1 and VISA). The functional loss of RIG-I in tree shrews suggests that the interruption of immune response might serve as one potential reason causing the capability of HCV infection in this animal. Based on the absence of RIG-I gene in the Chinese tree shrew, we firstly identified the absence of RIG-I was a common phenomenon in the tree shrew and this event occurred after they had evolved independently. Evolutionary analysis of tMDA5 showed that positive selection signal was identified by both the branch and branch-site models. The analysis of the location of positively selected sites in the tMDA5 gene relative to domain organization was Lys 188 and Ala 402. tMDA5 overexpression led to the significant activation of the NF-κB, ISRE and IFN-β reporters, and increased the induction of type I interferons in response to various RNA virus infection, which potent than its mutants tMDA5 K188Q and tMDA5 A402K. Hepatitis C virus (HCV) copies much lower when tMDA5 was overexpressed. These data highlighted the relevance of tMDA5 could replace RIG-I in sensing RNA virus. We also identified and characterized the functions of the tree shrew MAVS (tMAVS), as the RLR unique adaptor protein in the context of HCV infection. Conservatively, RNA and DNA viruses induce the expression of tMAVS. Ectopic-expression of tMAVS markedly potentiates the virus-triggered activation of IRF3, NF-κB and IFN-β, whereas knockdown of tMAVS displays the opposite effects. Notably, HCV co-localizes with tMAVS in tree shrew liver-derived cells, and cleaves the tMAVS at its Cys-508 via the corresponding NS3/4A protease. Interestingly, site Glu-506 in tMAVS, although not conserved relative to Val-506 in hMAVS, can support the cleavage role of NS3/4A. Unexpectedly, HCV NS3/4A could only impair the IRF3-mediated induction of IFN-β, but not the activation of the NF-κB signaling in tree shrew, which might account for the low infection rate and HCV titers in tree shrew model. Collectively, this study reveals the conservation and diversification of the MAVS antiviral signaling pathways, demonstrating the practicality of the tree shrew for studying HCV infection. Mediator of IRF3 activation (MITA) (also known as STING, TMEM173, MPYS, or ERIS) has been found to be a key adaptor protein for most DNA-sensing pathways. We also cloned this key gene in the Chinese tree shrew. During cloning of the full-length cDNA of tMITA (tMITA-FL) by RT-qPCR, a cDNA clone with a short sequence was identified. Sequence analysis of the clone revealed that it contained a splice variant of tMITA coding sequence and lack amino acid at the carboxyl terminal. In unifected cells, tMITA-FL activated ISRE, NF-κB and IFN-β-promoter but tMITA-mini inhibits tMITA-FL-induced IFN-β-promoter activation. Intriguingly, tMITA-FL mediates IFN-β induction in the HSV-1 infection, but tMITA-mini plays a major role in effectively inhibit VSV and HCV replication. These results suggest that tMITA employ differential transcripts to mediate of different virus-triggered induction of type I IFNs and cellular antiviral responses. Our results uncovered the mechanisms of tree shrew’s susceptibility to virus infection and offers an opportunity to decipher the genetic basis of the tree shrews’ suitability as an animal |
| 源URL | [http://159.226.149.26:8080/handle/152453/10190]  |
| 专题 | 昆明动物研究所_重大疾病机理的遗传学 |
| 推荐引用方式 GB/T 7714 | 许凌. 树鼩抗病毒先天免疫RLR通路基因的功能鉴定[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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