猪伪狂犬病毒引起PK-15细胞自噬的研究

[目的]研究猪伪狂犬病毒（PRV）感染猪肾传代细胞系（PK-15）后对细胞自噬的影响，以及细胞自噬在PRV感染复制过程中的作用。[方法]采用Western blot、透射电子显微镜（TEM）、激光共聚焦、siRNA转染等方法研究了PK-15细胞感染PRV后的细胞自噬。利用自噬诱导剂雷帕霉素和自噬抑制剂3-MA分析自噬对病毒复制的影响，再通过沉默自噬相关基因Beclin-1，进一步观察自噬对PRV复制的影响。[结果]PRV感染PK-15细胞后，LC3-Ⅰ显著转化为LC3-Ⅱ，PK-15细胞内自噬小体的数量明显增加。PRV感染增加了LC3阳性斑点的细胞数。同时，变异毒株PRV ZJ01与传统毒株PRV LA诱导的自噬存在差异。自噬的药理学变化试验和siRNA转染结果还显示自噬能够促进PRV的复制。[结论]PRV感染PK-15细胞可以诱导自噬并有利于病毒的复制。
[Objectives]The aim of this research was to investigate the viral inducible-autophagy on PK-15 cell by pseudorabies virus(PRV)and effect of the autophagy on virus replication.[Methods]In this study,Western blot,transmission electron microscopy(TEM),confocal laser scanning microscopy,and transfection were performed to confirm PRV-induced autophagy. The role of autophagy on virus replication was also analyzed by autophagy inducer(rapamycin)and autophagy inhibitor(3-methyladenine,3-MA). By silencing autophagy-related gene Beclin-1, the effect of autophagy on PRV replication was further observed.[Results]Western blot assay showed that LC3-Ⅰ was significantly converted into LC3-Ⅱ in PK-15 cells inoculated with PRV. While the number of auto-phagosome in PK-15 cells infected with PRV were remarkably increased by TEM. In addition,the numbers of LC3-positive puncta per cell in the PRV infected cells were more than in mock cells. These results indicated that PRV infection could induce a complete autophagic flux in PK-15 cells. Furthermore,the aforementioned studies also confirmed that there were differences between autophagy induced by epidemic strains PRV ZJ01 and traditional strains PRV LA. So,the enhancement to PRV replication by autophagy was confirmed in the pharmacological alteration assay and siRNA transfection.[Conclusions]PRV infection can induce autophagy to facilitate virus replication in PK-15 cells