EDⅢK307是西尼罗病毒感染的重要位点

EDⅢK307 is an important site for West Nile virus infection

目的探讨西尼罗病毒EDⅢK307位点对西尼罗病毒包装以及入侵的影响。方法对西尼罗病毒EDⅢ进行点突变——K307A,再将突变后的表达载体pcDNA3.1-CME K307A与prWNV-Rluc共转染293T细胞,72 h后收集细胞培养上清,通过电镜观察上清中西尼罗假病毒颗粒形态,并利用PCR法扩增病毒基因组NS1片段序列以确证突变假病毒是否包装成功;继而利用上清感染BHK21细胞,通过检测感染后BHK21细胞胞内荧光素酶值来判断突变假病毒对靶细胞的感染性。结果细胞培养上清中可见比较均一的病毒颗粒,呈圆形、有包膜,直径在40~50 nm;PCR扩增可见基因组NS1特异条带。上述结果均证实成功包装出突变型假病毒颗粒。感染实验显示,与未突变假病毒颗粒相比,K307A位点突变后的假病毒颗粒丧失了感染靶细胞的能力。结论K307位点很可能参与了西尼罗病毒的感染,是西尼罗病毒与受体结合的关键位点,从而可能成为阻断西尼罗病毒感染药物的重要靶点。

Objective To investigate the effect of the E protein DⅢK307 site on the packaging and invasion of West Nile virus.Methods 293T cells were cotransfected with pCDNA3.1-CME K307A and prWNV-RLuc.The supernatant of cell culture was collected 72 h later.To find out whether the pseudovirus was packaged properly,the morphology of pseudotype virions in the supernatant was observed by electron microscopy,and the NS1 fragment sequence of viral genome was amplified by PCR.Then the infectivity of the mutated pseudovirus to target cells was determined by infecting BHK21 cells with the supernatant.Results Viral particles were uniform in the cell culture supernatant,round and enveloped,with a diameter of about 40-50 mm.Genomic NS1 specific bands were observed by PCR amplification.All the above results confirmed the successful packaging of mutated pseudoviral particles.However,infection experiments showed that the mutated pseudovirions at the EDⅢK307 site lost the ability to infect target cells compared with the wild type pseudovirions.Conclusion West Nile virus EDⅢK307 site is probably involved in infection,possible the key site for West Nile virus to bind to receptors,with may be an important target for anti-West Nile virus drugs.