Role and mechanism of protein kinase D in the replication of human coronavirus 229E / 中国人兽共患病学报

ABSTRACT

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 lev-els of HCoV-229E-infected cells.Cell viability was assessed with CCK8,viral titration was determined with the TCID50,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)P2)was measured to reflect phosphatidylinositol 4-kinaseⅢβ(PI4KⅢβ)activity.PI4KⅢβ activity was inhibited with a PI4KⅢβ inhibitor(BQR695)to observe changes in TGN struc-ture 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).Im-munofluorescence staining indicated that CRT0066101 inhibited TGN fission caused by HCoV-229E infection.Inhibition of PI4KⅢβ activity significantly decreased PI(4,5)P2 levels,and BQR695 rescued the elevated PI(4,5)P2 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).This study revealed that PKD regulates TGN fission through modu-latingPI4KⅢβ and is involved in HCoV-229E replication.PKD inhibitors and PI4KⅢβ inhibitors significantly decreased HCoV-229E replication,thus providing important insights for future research onthe treatment of coronavirus infections.