Zheng Yu-huang, Zhang Chun-ying, He Yan, Gong Guo-zhong, Li Hui, Chen Zi, Liu Meng, Zhou Hua-ying, Li Ying, Liu Chun, Li Jing, Zhou Guo-qiang, Yin Wei, Yuan Hong-li
AIDS Research Laboratory, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhonghua Yi Xue Za Zhi. 2009 Mar 10;89(9):614-9.
To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host cells, and the possible mechanism thereof.
Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3.1 (+) plasmid was extracted, the PCR purified product was double-cut by HindIII and BamH, and the cut products were ligated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after 24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument.
The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspase-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 cells were all lower than the cells transfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1-7 recombinant plasmids (all of the Vpr AE subtype) were all lower than those of the No 8-14 plasmids (of Vpr B, AB, C, and C/BC subtypes).
The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.
探讨人类免疫缺陷病毒（HIV）vpr基因不同突变位点对宿主细胞凋亡的影响及其可能机制。
从HIV感染者体内获取14个HIV-1 vpr片段。提取真核表达载体pcDNA3.1(+)质粒，将PCR纯化产物用HindIII和BamH进行双酶切，酶切产物与载体连接，构建JM109感受态细胞。通过测序确认pcDNA-vpr真核表达载体构建成功，随后将其转染至HeLa细胞。转染空载体作为对照组。24小时后收集细胞，进行Hoechst 33258染色，在荧光显微镜下观察。采用Annexin-FITC-PI染色和流式细胞术观察细胞凋亡率。用酶标仪检测caspase-3活性。
用Hoechst和Annexin-FITC-PI染色法显示，转染含70、85、86和94位点突变基因片段的HeLa细胞凋亡率及caspase-3活性水平均低于转染不含这些突变位点基因片段的细胞。1-7号重组质粒（均为Vpr AE亚型）诱导细胞凋亡的能力均低于8-14号质粒（Vpr B、AB、C及C/BC亚型）。
含70、85、86、94位点突变vpr序列的HIV诱导细胞凋亡的能力明显低于不含这些位点的HIV。AE亚型在宿主细胞中诱导较低的凋亡行为，caspase-3途径激活降低可能是其中一个机制。
