云南松松塔粗提物体外抗拉沙病毒活性及机制的研究

Antiviral activity and mechanism of pinecone extract of pinus yunnanensis against Lassa virus infection

目的研究云南松松塔粗提物抗拉沙病毒(LASV)活性及机制。方法利用拉沙假病毒系统及拉沙细胞-细胞融合检测松塔粗提物抗LASV活性。其中,构建拉沙假病毒系统以慢病毒骨架质粒PNL-4-3.Luc.R-E.与含有拉沙包膜蛋白质粒转染于293T细胞,生成了一种含有荧光素酶报告基因的假性拉沙病毒。可以在BSL-2级生物安全实验室中检测松塔粗提物抗LASV活性的研究。其次,拉沙细胞-细胞融合模型的构建是通过对已有的质粒进行酶切,连接,鉴定,获得重组质粒,编码带有荧光报告基因及拉沙包膜蛋白的质粒(pAAV-EGFP-Ⅰ,Ⅱ,Ⅲ,Ⅳ)将其转染至293T细胞,获得表面表达有拉沙包膜蛋白的荧光细胞,与靶细胞Huh-7细胞混合铺于96孔板,在37℃,酸性件下发生膜融合,后续用膜融合系统来检测松塔粗提物抗LASV活性研究;对松塔粗提物作用于LASV假病毒感染的具体时期进行分析,通过time of addition及time of remove试验分析松塔粗提物抗病毒作用靶点;采用Western-blot方法检测p24蛋白量,判断松塔粗提物对病毒的裂解作用。结果松塔粗提物可有效抑制LASV感染,用4个谱系的LASV假病毒(LASV lineagesⅠ、Ⅱ、Ⅲ、Ⅳ)感染靶细胞,测得IC 50值分别为:42.06μg/mL、22.26μg/mL、80.57μg/mL及19.32μg/mL,抑制LASV包膜蛋白(GPC)介导的细胞-细胞融合活性的检测,测得IC 50值分别为:122.9μg/mL、64.43μg/mL、90.05μg/mL及195.6μg/mL;松塔粗提物具有广谱抗病毒作用,可有效抑制沙粒病毒属:淋巴细胞脉络丛脑膜炎病毒、卢霍病毒及胡宁病毒假病毒感染,IC 50值分别为:2.72μg/mL、12.02μg/mL、29.95μg/mL;松塔粗提物作用于LASV假病毒感染早期,于感染后2 h内加入粗提物的抑制率明显高于4 h后加入药物,且以阻断LASV假病毒的吸附及融合为主;松塔粗提物对LASV假病毒无裂解作用,提取物处理组测得p24蛋白含量与未处理组无统计学差异。结论本研究证实云南松松塔粗提物在体外环境下可有效抑制LASV感染,且具有广谱抗病毒作用;经试验结果证实松塔粗提物主要作用于病毒感染早期,以阻断病毒的吸附及融合为主,为松塔提取物抗LASV的深入研究提供重要的参考依据。

A Lassa pseudovirus system and Lassa cell-cell fusion were used to detect the anti-LASV activity of Pineta crude extract.The Lassa pseudovirus system was constructed with the lentivirus skeleton plasmid PNL-4-3.Luc.R-E.After transfection into 293T cells with Lassa-coated protein granules,a fake Lassa virus containing luciferase reporter gene was generated.The anti-LASV activity of pine tar crude extract was detected in a BSL-2 biosafety laboratory.Second,a Lassa cell-cell fusion model was constructed by enzymatically cutting,linking,and identifying existing plasmids to obtain recombinant plasmids,which were transfected into 293T cells to obtain fluorescent cells with Lassa envelope protein on the surface.These cells were mixed with Huh-7 target cells in 96-well plates,and membrane fusion occurred at 37℃under acidic conditions.Subsequently,a membrane fusion system was used to detect the anti-LASV activity of pine tar crude extract.The specific period of action of pine tar crude extract on LASV pseudovirus infection was analyzed,and the antiviral target of pine tar crude extract was analyzed with time of addition and time of removal tests.The protein content of p24 was detected by western blotting to determine the viral cleavage effect of pine tar crude extract.The crude extract of pine tar effectively inhibited LASV infection.Four lineages of LASV pseudoviruses(LASV lineagesⅠ,Ⅱ,Ⅲ,andⅣ)were used to infect target cells,and the IC 50 values were as follows:42.06μg/mL,22.26μg/mL,80.57μg/mL,and 19.32μg/mL inhibited the cell-cell fusion activity mediated by LASV envelope protein(GPC),and the IC 50 values were as follows:122.9μg/mL,64.43μg/mL,90.05μg/mL and 195.6μg/mL.The crude extract of Songta had broad-spectrum antiviral effects,and effectively inhibited infection with arenavirus,including lymphocytic choriomeningitis virus,Luho virus,and Junin virus pseudovirus.The IC 50 values were 2.72μg/mL,12.02μg/mL,and 29.95μg/mL,respectively.The crude extract of pine tar acted in early stages of LASV infection.The inhibitory rate after was significantly higher with addition of crude extract within 2 h rather than 4 h after infection,and the adsorption and fusion of LASV pseudovirus were largely blocked.The crude extract of pine tar had no cleavage effect on LASV pseudovirus,and no significant difference in p24 protein content was observed between the treated group and the untreated group.This study confirmed that crude Yunnan pine tree extract effectively inhibits LASV infection in vitro,and has broad-spectrum antiviral effects.The experimental results showed that crude pine tar extract acts mainly in early stages of viral infection,by blocking viral adsorption and fusion.These findings provide a reference for further research on the inhibitory effects of pinecone extract toward LASV.