一例 HIV-1 广谱中和者体内病毒膜蛋白基因 进化特征及中和敏感性研究

Evolutionary characteristics and neutralization sensitivity of HlV-l envelope genes in a broad neutralizer

目的分析一例HIV-1广谱中和者体内病毒膜蛋白基因(env)进化特征及其中和敏感性。方法从感染者两个随访时间点的全血样本中提取前病毒DNA,使用单拷贝基因组扩增(SGA)方法扩增全长env基因,测序后进行序列比对和进化树构建,分析env基因可变区特征及CD4结合位点(CD4bs)的氨基酸特征。将有代表性的env基因克隆到pcDNA^(TM)3.1载体上,与骨架质粒pSG3△env共转染293T细胞制备假病毒,检验其感染能力,筛选出功能性膜蛋白,再将有功能性的假病毒与不同的广谱中和抗体进行中和试验,分析膜蛋白的中和特征。结果从两个时间点全血样本中共获得43条全长env基因,进化树分析显示两个时间点的env序列交叉聚集,膜蛋白序列的基因距离随时间推移而增加。对病毒膜蛋白基因可变区的分析显示,V5区的糖基化位点数目随时间的推移而减少,后一时间点有16%的序列在V1区进化出两个额外的半胱氨酸;中和实验显示V1区半胱氨酸插入不会影响病毒针对V3环中和抗体的敏感性,V5区N460糖基化位点缺失导致假病毒对CD4bs类抗体的敏感性略有下降。结论病毒膜蛋白基因在体液免疫的压力下持续进化;关键中和表位中氨基酸变异导致病毒发生中和逃逸。

Objective To analyze the sequence characteristics of HIV-1 envelope genes(env)and their neutralization sensitivity from a HIV-1 broad neutralizer.Methods The env genes were amplified from the blood samples at two different follow-up time points by the single genome amplification method.The sequence alignment and phylogenetic tree construction were performed after sequencing.The characteristics of env genes and the amino acid of CD4 binding site(CD4bs)were analyzed.The representative env genes were cloned into peDNA^(TM)3.1 vector,and the pseudovirus were prepared by transfecting it with the skeleton plasmid pSG3△env into 293T cells.Screening out the functional membrane protein by testing its infectivity,and then neutralizing the functional membrane protein pseudovirus with four different broadly neutralizing antibodies to analyze the neutralization characteristics of the Env protein.Results A total of 43 full-length env genes were obtained from the blood samples at two time points.Evolutionary analysis showed that the sequences at the two time points were clustered,the divergence of env gene sequences increased with time.The analysis of the variable region of the virus membrane protein gene showed that the number of glycosylation sites in V5 region decreased over time,and 16%of the sequences in the latter time point evolved two additional cysteine in V1 region.Neutralization test showed that whether extra cysteines were inserted into V1 region was not related to the sensitivity of pseudoviruses to V3-glycan-dependent class antibody,the deletion of N460 glycosylation site in V5 region leads to a slight decrease in the sensitivity of pseudoviruses to CD4bs-directed neutralizing antibody.ConclusionssTheviral envelope genes continue to evolve under the pressure of humoral immunity.Amino acid mutations in the key neutralizing epitopes lead to the viral neutralization escape.