早幼粒细胞白血病(PML)蛋白是PML核体(PML-NBs)中不可或缺的元素,在多种细胞功能的调节中发挥关键作用,同时协调针对病毒入侵的先天免疫应答。同时,许多病毒通过靶向PML-NB而逃避免疫检测。日本脑炎病毒(JEV)是引起日本脑炎的黄病毒,一种严重的神经系统疾病,影响人类和动物。然而,JEV通过PML-NB逃避免疫的机制几乎没有研究。在本研究中,PK15细胞感染JEV,计数细胞内PML-NBs的数量。免疫荧光结果表明,与病毒抗原阴性细胞相比,JEV抗原阳性细胞中PML-NBs的数量显着减少。随后,将10种JEV蛋白克隆并转染到PK15细胞中。结果表明,JEV非结构蛋白,NS2B,NS3、NS4A、NS4B,和NS5,显著减少了PML-NBs的数量。用五种JEV蛋白和各种猪PML同种型进行共转染。结果表明,NS2B与PML4和PML5共定位,NS4A与PML1和PML4共定位,NS4B与PML1,PML3,PML4和PML5共定位,而NS3和NS5与所有五种PML亚型相互作用。此外,PML亚型的异位表达证实了PML1、PML3、PML4和PML5抑制JEV复制。这些发现表明,JEV通过与PML亚型的相互作用破坏PML-NBs的结构,可能导致宿主抗病毒免疫反应的减弱。
Promyelocytic leukemia (PML) protein constitutes an indispensable element within PML-nuclear bodies (PML-NBs), playing a pivotal role in the regulation of multiple cellular functions while coordinating the innate immune response against viral invasions. Simultaneously, numerous viruses elude immune detection by targeting PML-NBs. Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis, a severe neurological disease that affects humans and animals. However, the mechanism through which JEV evades immunity via PML-NBs has been scarcely investigated. In the present study, PK15 cells were infected with JEV, and the quantity of intracellular PML-NBs was enumerated. The immunofluorescence results indicated that the number of PML-NBs was significantly reduced in JEV antigen-positive cells compared to viral antigen-negative cells. Subsequently, ten JEV proteins were cloned and transfected into PK15 cells. The results revealed that JEV non-structural proteins, NS2B, NS3, NS4A, NS4B, and NS5, significantly diminished the quantity of PML-NBs. Co-transfection was performed with the five JEV proteins and various porcine PML isoforms. The results demonstrated that NS2B colocalized with PML4 and PML5, NS4A colocalized with PML1 and PML4, NS4B colocalized with PML1, PML3, PML4, and PML5, while NS3 and NS5 interacted with all five PML isoforms. Furthermore, ectopic expression of PML isoforms confirmed that PML1, PML3, PML4, and PML5 inhibited JEV replication. These findings suggest that JEV disrupts the structure of PML-NBs through interaction with PML isoforms, potentially leading to the attenuation of the host\'s antiviral immune response.