摘要
目的探究蛋白激酶D(PKD)在人类冠状病毒229E(HCoV-229E)复制中的作用及潜在分子机制。方法以MRC-5细胞作为模型,采用qPCR和Western blot检测PKD家族基因和蛋白的表达水平;通过siRNA敲除Prkd3及PKD抑制剂(CRT0066101)抑制PKD活性,检测HCoV-229E感染细胞后的复制水平;通过CCK8检测细胞活性,TCID_(50)测定病毒滴度,免疫荧光染色检测HCoV-229E的表达和反式高尔基体网络(TGN)的结构;通过检测磷脂酰肌醇4,5-二磷酸(PI(4,5)P2)的水平反映磷脂酰肌醇4-激酶IIIβ(PI4KIIIβ)活性;采用PI4KIIIβ抑制剂(BQR695)抑制PI4KIIIβ活性,检测HCoV-229E感染细胞后的TGN的结构变化和HCoV-229E复制水平;采用CRT0066101抑制Vero-E6细胞的PKD活性,检测HCoV-229E感染后的的复制水平。结果PKD家族基因和蛋白表达水平在HCoV-229E感染过程中明显增加,包括Prkd1、Prkd2和Prkd3;与对照组相比,敲低PKD3显著抑制HCoV-229E的mRNA水平和滴度(t=8.999,P<0.001;t=6.920,P<0.001),过表达PKD3则增加了HCoV-229E的mRNA水平和滴度(t=6.630,P<0.001;t=5.794,P<0.001);CRT0066101也显著抑制HCoV-229E的mRNA水平和滴度(t=6.931,P<0.001;t=4.055,P<0.01),免疫荧光染色结果表明,CRT0066101抑制HCoV-229E感染导致的TGN裂变;抑制PI4KIIIβ活性显著降低PI(4,5)P2水平,且BQR695挽救了PKD过表达导致的PI(4,5)P2水平升高(t=6.671,P<0.01),BQR695降低了HCoV-229E感染细胞的TGN裂变,HCoV-229E mRNA水平和滴度(t=5.151,P<0.001;t=7.744,P<0.001);CRT0066101抑制HCoV-229E在Vero-E6细胞中的mRNA水平和滴度(t=7.480,P<0.001;t=7.228,P<0.01)。结论本研究揭示了PKD通过调控PI4KIIIβ影响TGN的裂变参与HCoV-229E复制,并证明PKD抑制剂和PI4KIIIβ抑制剂对降低HCoV-229E的复制具有显著作用,这些发现为今后研究冠状病毒感染的治疗提供重要的参考依据。
This study investigated the role of protein kinase D(PKD)in the replication of human coronavirus 229E(HCoV-229E)and its potential molecular mechanisms.Using MRC-5 cells as a model,we assessed the expression levels of PKD family genes and proteins through qPCR and western blotting.We knocked down Prkd3 with siRNA and inhibited PKD activity with CRT0066101 to examine the replication levels of HCoV-229E-infected cells.Cell viability was assessed with CCK8,viral titration was determined with the TCID_(50),and immunofluorescence staining was used to assess HCoV-229E expression and the structure of the trans-Golgi network(TGN).The level of phosphatidylinositol 4,5-bisphosphate(PI(4,5)P 2)was measured to reflect phosphatidylinositol 4-kinase IIIβ(PI4KIIIβ)activity.PI4KIIIβactivity was inhibited with a PI4KIIIβinhibitor(BQR695)to observe changes in TGN structure and HCoV-229E replication after cell infection.CRT0066101 was used to inhibit PKD activity in Vero-E6 cells,and HCoV-229E replication levels were examined post-infection.The expression levels of PKD family genes and proteins,including Prkd1,Prkd2 and Prkd3,significantly increased during HCoV-229E infection.Knockdown of PKD3 significantly inhibited HCoV-229E mRNA levels and titers with respect to those in the control group(t=8.999,P<0.001;t=6.920,P<0.001),whereas overexpression of PKD3 increased HCoV-229E mRNA levels and titers(t=6.630,P<0.001;t=5.794,P<0.001).CRT0066101 also significantly inhibited HCoV-229E mRNA levels and titers(t=6.931,P<0.001;t=4.055,P<0.01).Immunofluorescence staining indicated that CRT0066101 inhibited TGN fission caused by HCoV-229E infection.Inhibition of PI4KIIIβactivity significantly decreased PI(4,5)P 2 levels,and BQR695 rescued the elevated PI(4,5)P 2 levels caused by PKD overexpression(t=6.671,P<0.01).BQR695 decreased TGN fission in HCoV-229E-infected cells,and HCoV-229E mRNA levels and titers(t=5.151,P<0.001;t=7.744,P<0.001).CRT0066101 inhibited HCoV-229E mRNA levels and titers in Vero-E6 cells(t=7.480,P<0.001;t=7.228,P<0.01).T
作者
韩慧娟
刘欢
赵志军
HAN Hui-juan;LIU Huan;ZHAO Zhi-jun(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750001,China;Key Laboratory of Clinical Pathogenic Microorganism,General Hospital of Ningxia Medical University,Yinchuan 750001,China;Cardiovascular Institute,Department of Medicine,University of Rochester,Rochester 14642,USA)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2023年第11期1044-1052,共9页
Chinese Journal of Zoonoses
基金
美国国立卫生院NIH(No.HL141171)资助。
