急性感染期内恒河猴外周血中人/猴嵌合免疫缺陷病毒包膜蛋白的N糖基化位点分析  

Characterization of N-linked glycosylation sites on envelope proteins of simian/human immunodeficiency virus in peripheral blood of Chinese rhesus macaques during acute infection

作　　者：石银[1] 万延民[2] 陈健[2] 王婧[2] 任艳琴[2] 魏强 从喆 徐建青[1] 

机构地区：[1]温州医科大学,325035 [2]上海市公共卫生临床中心 [3]中国医学科学院医学实验动物研究所

出　　处：《中华预防医学杂志》2016年第10期869-873,共5页Chinese Journal of Preventive Medicine

基　　金：国家科技重大专项（2013ZX10001-002）;国家自然科学基金（81302605）

摘　　要：目的：分析人/猴嵌合免疫缺陷病毒（SHIVSF162P3）包膜蛋白N糖基化位点的数量和分布及病毒传播能力。方法两只雌性成年（4周岁）中国恒河猴，编号Rh1和Rh2，体重分别为4和5 kg。通过静脉攻毒的方式向Rh1和Rh2接种SHIVSF162P3，每只剂量为300半数组织培养感染剂量，在攻毒后第3、7、10、14、17、21、24、28、35、42、49、56、63、70和77天采集血液样本。对样本进行病毒RNA抽提和cDNA合成，采用实时荧光定量PCR测定病毒RNA水平。对攻毒后第7、14、28和77天的血浆样本进行单基因组扩增，使用Bio-edit中的Clustal W软件进行包膜蛋白全长多序列比对，用MEGA6.06软件计算配对差异距离，采用HIV Databases中的软件计算包膜蛋白N糖基化位点和可变区氨基酸数目，比较序列多样性、N糖基化位点数量的差异。结果从接种的病毒中共扩增得到55条包膜蛋白全长序列，其核苷酸序列平均配对差异距离为（0.1666±0.0963）%。病毒载量在攻毒后第10天达到峰值，lg转换值分别为7.68和7.49拷贝/ml；在攻毒后第42天，病毒载量达到调定点，lg转换值分别为4.27和4.81拷贝/ml。攻毒后第7天，Rh1和Rh2血样中包含25个N糖基化位点的病毒包膜蛋白序列的比例分别达到83%（20/24）和94%（29/31），高于接种病毒中的比例[49%（27/55）]。随着感染时间的延长，Rh1和Rh2血浆中SHIVSF162P3病毒包膜序列的多样性逐渐增大，包含25个N糖基化位点的包膜蛋白序列比例逐渐降低，包含27个N糖基化位点病毒所占比例逐渐升高；在第28天Rh1体内包含27个N糖基化位点的序列比例达到29%（6/21），Rh2在第77天达到35%（13/37）。V1~V5区糖基化位点分析发现，N糖基化位点数目的变化主要来自于V4区。与包含27个糖基化位点的包膜蛋白序列相比，包含25个糖基化位点的病毒包膜蛋白序列在V4区缺失7个氨基酸（TWNNTIG），导致V4区在392位和39Objective To investigate the number and distribution of N-linked glycosylation sites of simian/human immunodeficiency virus envelope proteins （SHIVSF162P3） and SHIV transmission. Methods Two female adult Chinese rhesus macaques （4 years old） were intravenously inoculated with 300 TCID50 SHIVSF162P3. The macaques weighed 4 and 5 kg and were identified as Rh1 and Rh2. We collected plasma samples at days 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, 56, 63, 70 and 77 post-challenge. Subsequently, we monitored plasma viral load by real-time PCR after viral RNA isolation and cDNA synthesis. We amplified the full-length envelope gene by single genome amplification （SGA） at days 7, 14, 28 and 77. BioEdit, MEGA, and the HIV Databases were used to analyze envelope sequences. Sequence diversity and N-linked glycosylation sites were compared between virus stock and plasma viruses of the two macaques. Results A total of 55 env sequences were obtained from virus stock and their average pairwise distances were （0.166 6±＆amp;nbsp;0.096 3）%. Viral loads peaked at 7.68 and 7.49 log10 copies/ml at day 10 and reached the set point at day 42 （4.27 and 4.81 log10 copies/ml）. The percentages of envelope sequences containing 25 potential N-linked glycosylation sites （PNGSs） were 83%（20/24） and 94%（29/31） in Rh1 and Rh2, respectively, at day 7;these were significantly higher than the proportion in SHIVSF162P3 stock （49%（27/55））. Viral diversity after infection increased with time whereas the proportion of sequences containing 25 PNGSs decreased and sequences containing 27 PNGSs gradually increased. In Rh1, the percentage of sequences containing 27 PNGSs increased to 29%at day 28 and reached 35%at day 77 in Rh2. By analyzing the number of PNGSs in the V1-V5 regions, we found that PNGS variation mainly occurred in the V4 loop. Compared with sequences containing 27 PNGSs, a seven amino acid （TWNNTIG） deletion was found in the V4 loop, which resulted in a loss of two PNGSs at positions 392 and 396.

关 键 词：猴免疫缺陷病毒 感染 病毒包膜蛋白质类 糖基化 

分 类 号：R512.91[医药卫生—内科学]