新型冠状病毒非结构蛋白NSP13通过调控IκBα蛋白降解抑制NF-κB信号通路

SARS-CoV-2 NSP13 inhibits NF-κB signal pathway by regulating degradation of IκBα

目的研究新型冠状病毒非结构蛋白13(NSP13)调控NF-κB信号通路的作用机制。方法利用GV367-NSP13-FLAG慢病毒感染的方法制备高表达NSP13的A549细胞(A549-NSP13)和A549对照组,用实时荧光定量PCR(RT-qPCR)和ELISA检测NF-κB下游细胞因子白细胞介素6(IL-6)和趋化因子5(CCL-5)表达水平;Western印迹法检测NF-κB信号通路P65、磷酸化P65和NF-κB抑制因子α(IκBα)蛋白表达。放线菌酮10 mg·L^(-1)处理细胞0,15,30,45,90和120 min,Western印迹法检测IκBα蛋白半衰期。结果与A549对照组相比,A549-NSP13细胞检测到FLAG标签蛋白条带,且NSP13 mRNA表达显著上调(P<0.01),表明A549-NSP13细胞系构建成功。与A549对照组相比,A549-NSP13细胞IL-6 mRNA和蛋白表达水平(P<0.01)及CCL-5 mRNA和蛋白表达水平(P<0.05,P<0.01)均显著降低;磷酸化P65蛋白表达下降(P<0.01),而IκBα蛋白表达上调(P<0.01);且IκBα蛋白半衰期增加(P<0.01)。结论NSP13蛋白可通过抑制IκBα降解进而抑制NF-κB信号通路的活化并降低其下游IL-6和CCL-5等炎症相关分子产生和分泌。

OBJECTIVE To study the mechanism by which non-structure protein 13(NSP13)of severe acute respiratory syndrome coronavirus(SARS-CoV-2)regulates the NF-κB signal pathway.METHODS A549 cells with a high expression of NSP13(A549-NSP13 cells)and A549 control were constructed via GV367-NSP13-FLAG lentivirus infection in order to gear up for subsequent experi⁃ments.RT-qPCR and ELISA were used to detect downstream inflammatory factors of NF-κB signal pathway such as interleukin 6(IL-6)and C-C motif chemokine 5(CCL-5).Western blotting was used to test the protein levels of P65,phosphorylated P65(p-P65)and NF-κB inhibitor alpha(IκBα).After being treated with cycloheximide(10 mg∙L^(-1))for 0,15,30,45,90 and 120 min,t_(1/2) of IκBαwas detected by Western blotting.RESULTS Compared with the control group,flag tag was detected in A549-NSP13 and the mRNA level of NSP13 in the A549-NSP13 group was significantly increased(P<0.01),suggesting that A549-NSP13 was constructed.Compared with the A549 control group,the mRNA levels and protein expression levels of IL-6(P<0.01,P<0.01)and CCL-5(P<0.05,P<0.01)in the A549-NSP13 cell group decreased significantly.The protein level of p-P65 was decreased(P<0.01)while that of IκBαwas increased(P<0.01)in A549-NSP13 cells.A longer t_(1/2) of IκBαwas detected in A549-NSP13 cells(P<0.01).CONCLU⁃SION SARS-CoV-2 NSP13 can inhibit IκBαdownregulation before suppressing the NF-κB signal pathway activation,which results in the reduction of inflammatory factors like IL-6 and CCL-5.