柯萨奇病毒B组3型和肠道病毒71型入胞机制比较研究

Comparative studies on viral entry mechanism of coxsackievirus B3 and enterovirus 71

目的比较柯萨奇病毒B组3型(coxsackievirus B3,CV-B3)和肠道病毒71型(enterovirus 71,EV-A71)的入胞机制。方法分别用CV-B3和EV-A71感染经不同小分子入胞抑制剂处理后的HeLa细胞,通过细胞免疫染色及图像分析,量化比较感染结果。结果细胞松弛素D、长春碱、巴弗洛霉素A1及布雷菲德菌素A均能抑制CV-B3和EV-A71对HeLa细胞的早期感染,表明两种病毒的入胞过程均需要细胞骨架、内吞小体酸化及膜泡运输作用;肝素能抑制EV-A71的感染并呈浓度依赖关系,但对CV-B3无显著作用,表明细胞膜表面的硫酸乙酰肝素参与EV-A71但可能不参与CV-B3的入胞过程;巴弗洛霉素A1对EV-A71的抑制更明显,说明在病毒脱衣壳过程中,EV-A71较CV-B3依赖更低的pH;细胞骨架抑制剂处理结果表明CV-B3的入胞过程更依赖于微管蛋白而非微丝蛋白的作用。结论 CV-B3和EV-A71具有相似的入胞机制,但却各具特点,为研究两者的抗病毒药物提供了依据。

Objective To compare the viral entry mechanisms of coxsackievirus B3 （CV-B3） and enterovirus 71 （EV-A71） . Methods After blocked with well characterized small molecular inhibitors for viral entry in different stages, HeLa cells were then infected separately with CV-B3 and EV-A71. Immunostaining and image analysis software were used to observe and compare the viral infection. Results Cytochalasin D, vinblastine, bafilomycin A1, brefeldin A showed inhibitory effects on the early infection of CV-B3 and EV-A71 in HeLa cells, indicating both viruses required cytoskeleton, endosome acidification, dynamics of ER-Golgi membrane traffic for the entry. Heparin could block EV-A71 entry in a dose-dependent manner, but not CV-B3, suggesting that heparan sulfate on cell surface was only involved in EV-A71 entry, not CV-B3. Bafilomycin A1 had more inhibitory effect on EV-A71 entry, implying lower pH was more required during virus uncoating process of EV-A71 then CV-B3. The result of cytoskeleton inhibitor showed that tubulin rather than actin was more involved in CV-B3 entry. Conclusions Although CV-B3 and EV-A71 shared the similar ceil entry mechanism, but each has its unique entry pathway, which provides scientific evidence for the research of anti-viral therapy.