口蹄疫病毒3A蛋白与ANXA1互作及其对I型干扰素通路的调控

The non-structure protein 3A of foot-mouth disease virus (FMDV) is responsible for natural adaption to pigs of FMDV, and inhibits the signaling pathway of I type interferons (I-IFN) [17], but it is unclear that the regulations and interactions between host proteins and 3A, which are responsible for the functions. Previously, we confirmed that the 3A can interact with host protein ANXA1, meanwhile, we found that ANXA1 regulates I-IFN production and inhibits FMDV replication. To elucidate the molecular mechanism underlying the regulation of innate immune antivral function by interaction between 3A and ANXA1, we will probe the interacting domains or key sites between 3A and ANXA1 using co-immunoprecipitation (co-IP). Furthermore, we will detect the changes of transcription, phosphorylation and ubiquitin of node proteins in the signaling pathway of I-IFN and it's downstream effective molecules which induced by 3A and ANXA1. Then according to the binding sites of 3A and ANXA1, rescue the recombinant virus containing the mutant the sites to estimate the inhibition of the I-IFN production by interacting between 3A and ANXA1. The study will provide strong evidence for regulation of the innate immune by 3A and ANXA1 interaction. This project entry from the interactions of 3A and host proteins, uncover I-IFN signal transduction regulated via 3A of FMDV, it will lay the foundation to reveal the molecular mechanisms for the regulations and interactions between host proteins and 3A.

口蹄疫病毒非结构蛋白3A是适应猪的嗜性变异的决定因素，本团队也揭示3A具有抑制I型干扰素信号转导的功能[17]，但这些功能背后3A与宿主蛋白作用与调控的机制仍然不清楚，为此，本项目前期鉴定出了与3A蛋白互作的宿主蛋白ANXA1，并证实了ANXA1可调节I型干扰素的产生抑制口蹄疫病毒复制。本项目将利用免疫共沉淀等技术鉴定3A与ANXA1互作的区域或位点，检测3A与ANXA1互作对I型干扰素通路节点蛋白转录、表达、磷酸化和泛素化的影响，检测被影响的节点蛋白下游效应分子的变化；拯救3A功能位点突变重组毒株，证实该位点突变对病毒生物学特征的影响，并评价其对I型IFN信号转导的影响。本研究从3A蛋白与宿主蛋白ANXA1互作入手，阐明口蹄疫病毒3A蛋白介导ANXA1调节天然免疫信号转导，为揭示口蹄疫病毒与宿主互作和调控机制奠定基础。

RLRs和cGAS-STING信号通路在病毒天然应答中起着重要作用。本研究发现ANXA1靶向RLRs途径的TBK1和cGAS-STING途径的TBK1/ikkγ，从而抑制RNA和DNA病毒介导的IRF3或NF-κB信号转导。重要的是，本项目证实口蹄疫病毒3A蛋白作为一种新的靶向ANXA1蛋白的抑制因子，抑制干扰素β的产生。我们进一步确定完整的3A与ANXA1相互作用。此外，3A可能通过ANXA1-TBK1复合物的相互作用来裂解TBK1。与ANXA1野生型细胞相比，病毒刺激后ANXA1-/-（敲除细胞）IFNβ的产生降低。同时，我们证实RNA病毒刺激通过TBK1-Irf3轴引起ANXA1对IFNβ产生的调节。对于DNA病毒，通过TBK1-IRF3和NF-κB轴。总之，这些结果表明，ANXA1在RNA和DNA病毒刺激后主动调节IFNβ的上升，FMDV 3A拮抗ANXA1对IFNβ产生的调节，从而调节先天性免疫应答。