宿主RBM14蛋白促进流感病毒vRNP复合体出核转运的机制研究

The viral RNA polymerase (vRNP) complex is an important determinant of virus pathogenicity and host adaptation. Previously, we demonstrated that host protein MOV10 inhibited the nuclear import of the vRNP, thereby restricted the replication of influenza A virus (doi:10.1128/JVI.03137-15). However, the mechanism by which the nuclear export of vRNP is regulated is poorly characterized. In our preliminary study, we found that RBM14 was co-purified with NP, one of the vRNP components, using TAP-MS assay. Further investigation indicated that RBM14 participates in the replication of influenza A viruses. We also found that the depletion of RBM14 resulted in a significant inhibitory effect on nuclear export of vRNP. Thus, we proposed that RBM14 participates in the nuclear export of vRNP complex and subsequently facilitates the replication of influneza A viruses. Our project will focus on the interaction between RBM14 and CRM1 transport system, the binding domain of RBM14 to NP, and the relationship between paraspeckle bodies and the replication of influenza virus, aiming at finding the particular mechanism that RBM14 participates in the nuclear export of vRNP complex, which may provide a new strategy for anti-influenza viral drug design.

流感病毒的vRNP复合体与宿主蛋白的相互作用可以决定流感病毒的传染力和毒力。前期我们已证明宿主MOV10蛋白通过抑制病毒vRNP复合体的入核转运，进而抑制流感病毒的复制过程（已发表）。然而，vRNP复合体的出核转运的机制尚不完全清楚。在本项目中，我们初步发现①宿主RBM14蛋白能够与vRNP复合体的NP亚基结合；②RBM14蛋白能够辅助流感病毒的复制；③RBM14表达降低能够抑制vRNP复合体的出核转运。因此，我们首次提出了RBM14蛋白促进了流感病毒vRNP复合体的出核转运过程，进而辅助流感病毒复制的科学假说。本项目拟明确：①RBM14蛋白与宿主CRM1核转运系统的互作关系；②RBM14蛋白与NP蛋白互作的结构域；③RBM14蛋白所在的核旁斑小体与流感病毒复制的关系。本项目旨在深层解析RBM14蛋白促进vRNP复合体的出核转运的分子机制，这将为抗流感药物的研发提供新的设计策略和科学依据。

流感病毒的vRNP复合体与宿主蛋白的相互作用可以决定流感病毒的传染力和毒力。前期我们已证明宿主MOV10蛋白通过抑制病毒vRNP复合体的入核转运，进而抑制流感病毒的复制过程（已发表）。然而，vRNP复合体的出核转运的机制尚不完全清楚。在本项目中，我们初步发现宿主RBM14蛋白能够与vRNP复合体的NP亚基结合,并辅助vRNP复合体的出核转运,进而促进流感病毒的复制。因此，我们提出了流感NP蛋白为药物筛选靶标,我们确定了一种新型的抗流感化合物，叫做ZBMD-1，来自于细胞的甲型流感感染测定法的20,000个化合物的文库。我们发现ZBMD-1可以抑制流感病毒的复制复制，包括H1N1和H3N2甲型流感病毒株的体外复制过程，其IC50浓度为0.41-1.14 uM。此外，ZBMD-1可以抑制了聚合酶活性，并特别抑制NP的出核过程。进一步的研究表明ZBMD-1与出核信号3（NES3）域和NP口袋的二聚体界面相结合。ZBMD-1还可以保护小鼠受到致命剂量A/PR/8/1934（H1N1）病毒的感染，可有效缓解肺组织病理学变化，并强烈抑制促炎性细胞因子/趋化因子的产生，而不会在小鼠中引起毒性作用。这些结果表明ZBMD-1是一种很有前途的抗流感化合物，可以作为将来针对甲型流感病毒的有用策略进行进一步研究。