Evolutionary characteristics and neutralization sensitivity of HIV-1 envelope genes in a broad neutralizer
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摘要:
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pcDNATM3.1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。 Abstract: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 pcDNATM3.1 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. Conclusions Theviral 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. -
Key words:
- Human immunodeficiency virus /
- Envelope gene /
- Pseudovirus /
- Glycosylation site /
- Immune escape
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图 1 HIV-1膜基因在两个时间点的Neighbor-Joining系统进化树
注：▲. 代表2008年的膜基因序列，●. 代表2012年的膜基因序列；橙色方框部分表示单独的进化簇
Figure 1. Neighbor-joining phylogenetic tree of HIV-1 envelope sequences in two time points
图 2 HIV-1膜基因在两个时间点的基因距离及碱基替换率
注：A. 两个时间点gp120及gp41的基因距离；B. 两个时间点gp120及gp41的碱基替换率（gp120, gp41是HIV-1膜蛋白的两个组成部分）
Figure 2. Gene distance and dn/ds of HIV-1 envelop sequences at two time points
图 3 HIV-1膜基因可变区序列在两个时间点的氨基酸长度和糖基化数目比较
注：A. 两个时间点膜基因可变区氨基酸长度；B. 两个时间点膜基因可变区糖基化数目；V1, V2, V3, V4, V5为HIV-1膜蛋白上的5个可变区
Figure 3. Comparison of amino acid length and glycosylation number of HIV-1 env gene variable region sequences at two time points
图 4 HIV-1膜基因V1区部分氨基酸序列及相应假病毒对V3环bNAbs的中和敏感性
注：A. 进化树簇2的膜基因V1区氨基酸序列（蓝色标记的为半胱氨酸） ；B. 两株假病毒对V3环bNAbs（PGT121, PGT135）的中和敏感性（IC50为抗体的半数抑制浓度）；橙色标记为挑选出构建假病毒的克隆；蓝色标记的为半胱氨酸
Figure 4. Amino acid of partial sequence in V1 region of HIV-1 env gene and neutralization sensitivity of two pseudoviruses to V3-glycan-dependent bNAbs
图 5 HIV-1膜基因CD4bs氨基酸分布及假病毒对CD4bs类抗体的中和敏感性
注：A. Inner domain区氨基酸比例分布；B. Loop D区氨基酸比例分布；C. V5区氨基酸比例分布；（Inner domain, Loop D, V5为CD4结合位点，纵坐标bits表示氨基酸的保守程度）；D. 两株假病毒对CD4bs类bNAbs（VRC01, 3BNC117）的中和敏感性（红色标记的氨基酸为糖基化位点，IC50为抗体的半数抑制浓度）
Figure 5. Distribution of amino acids in CD4 binding sites and neutralization sensitivity of two pseudoviruses to CD4bs-specific bNAbs
表 1 感染者CBJC451的背景信息
Table 1. Background information of donor CBJC451
采样年份 CD4＋T
（个/μL）病毒载量
（拷贝/mL）中和
宽度(%)中和
强度2008 658 21 600 82.6 49.4 2012 805 NA NA NA 注： NA. 未评价 
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