摘要
目的:探索与流感病毒核蛋白(NP)相互作用的宿主因子,研究其对流感病毒复制的影响,以及栀子环烯醚萜苷(IGE)抑制流感病毒的作用机制。方法:利用酵母双杂交系统筛选与流感病毒NP相互作用的宿主因子;通过免疫共沉淀实验对异质核糖核蛋白(HNRNPD)、氨基葡萄糖6磷酸脱氨酶1(GNPDA1)、Poly/rC结合因子1(PCBP1)、激活信号转导和转录激活因子(STAT)蛋白1抑制因子(PIAS1)进行验证;通过双荧光素酶实验比较PIAS1和HNRNPD对流感病毒复制的影响,并检测在转染核糖核蛋白体复合物(RNP)及PIAS1敲低的情况下,给予栀子环烯醚萜苷对流感病毒复制的影响。将ICR小鼠随机分为正常组、模型组、达菲组和IGE高、中、低剂量组,每组10只。除正常组外各组滴鼻感染甲型流感病毒FM1株建立病毒性肺炎模型。IGE高、中、低剂量组分别给予50、25、12.5 mg∙kg^(-1)IGE灌胃,达菲组给予27.5 mg∙kg^(-1)达菲灌胃,正常组和模型组灌服等体积蒸馏水,连续4 d。实验结束后,蛋白免疫印迹法检测肺组织中PIAS1、NP、磷酸化(p)-STAT3、STAT3、p-STAT1、STAT1的蛋白表达。结果:在酵母双杂交实验中,发现了16个流感病毒NP的潜在互作宿主靶点;免疫共沉淀实验发现HNRNPD和PIAS1可以与流感病毒NP相互作用;双荧光素酶报告实验中PIAS1敲低和过表达都可明显影响IAV RNP活性(P<0.05,P<0.01),HNRNPD对IAV RNP的影响差异无统计学意义;IGE高、低剂量组均可使流感病毒复制减少(P<0.05),并可逆转PIAS1敲低导致的流感病毒复制增加(P<0.05,P<0.01)。IGE各剂量组不同程度地降低感染小鼠肺组织PIAS1、NP、p-STAT3、p-STAT1、STAT1的表达量(P<0.05,P<0.01)。结论:PIAS1和流感病毒NP相互作用,并能够抑制流感病毒的复制,栀子环烯醚萜苷可能通过PIAS1/STAT1通路抑制IAV RNP的活性,从而发挥抗病毒作用。
Objective:To explore host factors interacting with influenza virus nucleoprotein(NP)and study their effects on influenza virus replication,as well as the mechanism of gardenia jasminoides iridoid glycoside(IGE)in inhibiting influenza virus.Method:A yeast two-hybrid system was utilized to screen host factors that interacted with influenza virus NP.Heterogeneous nuclear ribonucleoprotein D0(HNRNPD),glucosamine-6-phosphate deaminase 1(GNPDA1),poly(rC)-binding protein 1(PCBP1),and protein inhibitor of activated signal transducer and activator of transcription(STAT)protein 1(PIAS1)were validated by immunoprecipitation assay.The effects of PIAS1 and HNRNPD on influenza virus replication were compared by a dual luciferase assay,and the effects of IGE on influenza virus replication were examined in the presence of transfected ribonucleoprotein(RNP)and knockdown of PIAS1.ICR mice were randomly divided into a normal group,model group,oseltamivir phosphate group,and high,medium,and low dose IGE groups,with 10 mice in each group.In addition to the normal group,each group was infected with the influenza A virus FM1 strain by nasal drip to establish a viral pneumonia model.The high,medium,and low dose IGE groups were given drugs of 50,25,and 12.5 mg∙kg^(-1)by gavage,and the oseltamivir phosphate group was given the drug of 27.5 mg∙kg^(-1)by gavage.Equal amounts of distilled water were instilled in the normal and model groups for four consecutive days.Later,protein expression of PIAS1,NP,phosphorylated(p)-STAT3,STAT3,p-STAT1,and STAT1 were detected in the lung tissue by Western blot.Result:In yeast two-hybrid assays,16 potential host targets interacting with influenza virus NP were identified.Immunoprecipitation experiments revealed that HNRNPD and PIAS1 could interact with influenza virus NP.The dual luciferase reporter assays found that both PIAS1 knockdown and overexpression significantly affected IAV RNP activity(P<0.05,P<0.01),and the effect of HNRNPD on IAV RNP was not significant.Both high and low dose IGE groups reduced influenza virus replication(P<0.05)and reversed the increase in influenza virus replication caused by the knockdown of PIAS1(P<0.05,P<0.01).The expressions of PIAS1,NP,p-STAT3,p-STAT1,and STAT1 in the lung tissue of infected mice were reduced to different degrees in each IGE group(P<0.05,P<0.01).Conclusion:PIAS1 interacts with influenza virus NP and is able to inhibit influenza virus replication.IGE may exert antiviral effects by inhibiting the activity of IAV RNP through the PIAS1/STAT1 pathway.
作者
杨晓微
包蕾
张宇
刘宪
耿子涵
李舒冉
张敬升
崔晓兰
郭姗姗
YANG Xiaowei;BAO Lei;ZHANG Yu;LIU Xian;GENG Zihan;LI Shuran;ZHANG Jingsheng;CUI Xiaolan;GUO Shanshan(China Academy of Chinese Medical Sciences,Institute of Chinese Material Medica,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第13期60-66,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(82151210)
北京市自然科学基金项目(L222130)
中国中医科学院科技创新工程项目(CI2021A04607)。
