猪流行性腹泻病毒劫持DNA损伤通路操纵细胞周期促进自身复制

PEDV hijacks DNA damage pathways and manipulates the cell cycle to promote self-replication

摘要:

【目的】 探究DNA损伤应答(DNA damage response, DDR)对猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)复制的作用。【方法】 应用特异性抑制剂检测DDR通路是否参与PEDV的复制；通过实时彗星试验观察PEDV感染Vero细胞造成DNA损伤的情况；采用蛋白质免疫印迹和流式细胞术分别检测PEDV感染Vero细胞DDR通路中蛋白表达和细胞周期的变化。【结果】 ATM抑制剂KU55933可以极显著抑制PEDV的复制，病毒滴度从(5.50±0.25) log₁₀ TCID₅₀/mL下降到(3.15±0.15) log₁₀ TCID₅₀/mL；PEDV感染Vero细胞12−60 h可极显著引起DNA损伤；PEDV感染Vero细胞可激活ATM、ATR、Chk1、Chk2和p53，尤其是p-Chk2和p-p53在病毒复制过程中高表达；此外，PEDV感染使Vero细胞停滞在S期；细胞周期蛋白Cyclin B1在病毒复制过程中，蛋白表达先减少后极显著增加。【结论】 PEDV可能通过劫持DNA损伤通路中ATM-Chk2途径操控细胞周期促进病毒自身复制，为进一步阐明PEDV感染复制机制及开发新的潜在抗病毒靶点提供了重要依据。

Abstract:

[Objective] To study the effect of DNA damage response (DDR) on the replication of porcine epidemic diarrhea virus (PEDV). [Methods] Specific inhibitors were used to detect whether DDR pathway was involved in PEDV replication. the comet assay was employed to observe the DNA damage caused by PEDV infection in Vero cells. the changes in the expression levels of proteins in the DDR pathway and cell cycle of PEDV-infected Vero cells were determined by Western blotting and flow cytometry, respectively. [Results] The ATM inhibitor KU55933 significantly inhibited the replication of PEDV, with the virus titer decreasing from (5.50±0.25) log₁₀ TCID₅₀/mL to (3.15±0.15) log₁₀ TCID₅₀/mL. PEDV infection caused DNA damage in Vero cells during 12–60 h. ATM, ATR, Chk1, Chk2, and p53 were activated by PEDV infection of Vero cells. Especially, p-Chk2 and p-p53 showcased high expression during virus replication. In addition, PEDV infection led to the stagnation of Vero cells in the S phase. During virus replication, the expression of Cyclin B1 was first downregulated and then upregulated significantly. [Conclusion] PEDV perhaps utilized DNA damage pathway hijacks the ATM-Chk2 to manipulate the cell cycle and promote self-replication. The results provided a basis for elucidating the replication and infection mechanisms of PEDV and developing new potential antiviral targets.