马传染性贫血病毒强毒和疫苗毒激活NLRP3炎症小体差异的分子机制

The successful application of the Chinese attenuated vaccines against equine infectious anemia virus (EIAV) has practically overcome the difficulties in inducing protective immunities to lentiviruses. The development of EIAV vaccines can be a valuable reference for studies on pathogenicity and immunogenicity of other lentiviruses, such as the human immunodeficiency virus (HIV). Accumulating evidences indicate that the characters and strengths of inflammation stimulated by infected viruses greatly involve in the outcome of host responses. We observed that significantly higher proinflammatory cytokines were existed in sera and various tissues, combining with the appearance of pathological changes, such as apoptosis, necrosis and inflammatory lesions, in multiple organs from horses inoculated with EIAV virulent, but not vaccine strains. In addition, the virulent strains induced significantly higher expression level of the inflammasome NLRP3 than that induced by the vaccine strains. All these data implicate that there are different mechanisms between EIAV virulent and vaccine strains in activating inflammasome to stimulate inflammation. In the proposal, we will investigate molecular mechanisms on the cytokine storm stimulated by EIAV virulent strains and the immunity beneficial inflammation induced by EIAV vaccine strains. The following three objectives will be performed by comparative analysis: 1) the activation of NLRP3 and its downstream signaling; 2) the viral proteins interacted with NLRP3 and the details of the interaction; 3) the influence on NLRP3 reactivation and viral replication by replacing key domains between the virulent and vaccine strains. The successful performance of this proposal is expected to be able to obtain essential mechanisms on inflammation reaction during the infection of different EIAV strains, which may provide useful information for novel antiviral strategies design.

我国研发的马传染性贫血病毒(EIAV)疫苗的成功应用，从实践上克服了慢病毒不能诱导免疫保护的难点，可为艾滋病毒等其他慢病毒致病及免疫机制研究提供借鉴。病毒诱导炎症反应的特性和程度很大程度左右了宿主应答的转归。我们的前期研究发现，EIAV强毒感染马的血清和组织中特异性高表达多种促炎因子，且重要脏器出现明显的炎性坏死，疫苗株却未见此现象。此外，强、弱毒株感染对炎症小体NLRP3表达的上调显著不同，推测强、弱毒株激活炎性小体和炎症反应的差异是不同致病性的重要原因。本项目拟从EIAV对NLRP3的激活及相关信号通路；与该炎性小体互作的相关病毒蛋白及作用机制；病毒蛋白关键结构域的置换对NLRP3激活及病毒复制影响等三个方面，通过对比分析，探讨强毒株刺激炎症因子风暴及疫苗株诱导适度炎症反应的分子机制。该研究的开展可获得抗EIAV感染的炎症反应相关分子信息，促进炎症在慢病毒感染与机体应答中作用的理解。

马传染性贫血病毒（EIAV）弱毒疫苗是目前为止唯一成功应用的慢病毒疫苗。该疫苗克服了强毒株引发的炎性风暴（IL-1β为主），获得了对炎症应答调控的平衡。因此，本项目从EIAV对炎症小体的激活作用和调控角度，通过强毒株和疫苗株系统性对比分析，探讨EIAV强毒株诱发“炎症风暴”的诱因及疫苗株精确调控炎症反应的分子机制。通过项目的开展获得了以下重要发现：1）EIAV感染可以激活NLRP3炎症小体，且强弱毒株诱导产生IL-1β的差异是对NLRP3炎症小体激活差异决定的；2）发现EIAV是通过其编码的囊膜蛋白（Env），协同另一个炎症相关调控分子NEK7与NLRP3结合，共同激活NLRP3炎症小体；3）NEK7对于EIAV-Env激活NLRP3炎症小体是必不可少的，且受K+的调控；4） EIAV 强毒株致弱过程中，决定毒力的几个关键氨基酸位点的差异（235S/R，236D/-，237D/K，246D/K），是导致EIAV 强弱毒激活NLRP3炎症小体差异的关键因素。上述研究结果，不仅全面的解析了EIAV激活NLRP3炎症小体的分子机制，发现了导致EIAV 强毒株强烈激活NLRP3炎症小体的关键位点。而且也为靶向NLRP3炎症小体的调控提供有效策略。