Zheng Yuhuang, Zhou Huaying, Zhang Chunying, He Yan, Li Hui, Chen Zi, Liu Meng
AIDS Research Laboratory, Department of Infectious Diseases, The Second Xiangya Hospital, Central-South University, 410011 Changsha, China.
Curr HIV Res. 2009 Sep;7(5):519-25. doi: 10.2174/157016209789346291.
The vpr gene of human immunodeficiency virus type 1 (HIV-1) plays an important role in the pathogenicity of viruses. Previous studies showed that mutation of certain sites in HIV-1 Vpr amino acid sequences might influence the clinical process of infected individuals and attenuate its apoptosis-inducing capacity on infected cells. The present study was designed to transfect hela cells with several HIV-1 vpr fragments carrying specific mutation sites, and to observe the distinction of apoptosis-inducing effects of different HIV-1 vpr variant fragments on host cells and explore the possible mechanisms. According to the previous results, 14 typical vpr variant fragments were chosen from Chinese HIV-1 infected individuals. After PCR amplification of vpr gene, the products were purified and double digested with Hind and BamHI. Then pcDNA3.1 (+) eukaryotic expression plasmids were used for the ligation and transduction experiments. The recombinant plasmids were transiently transfected into Hela cells with liposomes, and meanwhile the blank cell and empty vector cell were established as control. RT-PCR was used to detect the mRNA expression of target genes; fluorescent microscope for observing apoptotic cells with Hoechst staining; DNA agarose electrophoresis for detecting apoptotic gradient band; Annexin assay for detecting cell apoptosis; and Caspase activity test for exploring the pathway of cell apoptosis. After transfected with 14 vpr gene segments, the Hela cells exhibited various apoptosis-inducing capabilities. We found that the HIV-1 Vpr segments with mutation at the 70(th), 85(th), 86(th), or 94(th) site showed lower apoptosis-inducing capabilities than other segments on Hela cells, and the apoptosis-inducing ability of HIV-1 vpr gene on host cells might be related to its subtype. Meanwhile, we found that the Caspase3 activity of transfected cell carrying these mutated fragment sites was decreased as compared to the cell with other fragments. This study firstly found that the HIV-1 Vpr fragments with the 70(th), 85(th), 86(th), or 94(th) site mutation might attenuate their apoptosis-inducing abilities on Hela cells. One of the mechanisms might be the attenuation of Caspase-3 activity.
人类免疫缺陷病毒1型（HIV-1）的vpr基因在病毒致病性中发挥重要作用。先前的研究表明，HIV-1 Vpr氨基酸序列中某些位点的突变可能会影响受感染个体的临床进程，并减弱其对受感染细胞的凋亡诱导能力。本研究旨在用携带特定突变位点的几个HIV-1 vpr片段转染HeLa细胞，观察不同HIV-1 vpr变异片段对宿主细胞凋亡诱导作用的差异，并探讨其可能的机制。根据先前的结果，从中国HIV-1感染个体中选取了14个典型的vpr变异片段。对vpr基因进行PCR扩增后，将产物纯化并用Hind和BamHI进行双酶切。然后使用pcDNA3.1(+)真核表达质粒进行连接和转导实验。重组质粒用脂质体瞬时转染到HeLa细胞中，同时设立空白细胞和空载体细胞作为对照。用RT-PCR检测靶基因的mRNA表达；用荧光显微镜通过Hoechst染色观察凋亡细胞；用DNA琼脂糖电泳检测凋亡梯度条带；用膜联蛋白检测法检测细胞凋亡；用半胱天冬酶活性试验探索细胞凋亡途径。用14个vpr基因片段转染后，HeLa细胞表现出不同的凋亡诱导能力。我们发现，在第70、85、86或94位点发生突变的HIV-1 Vpr片段对HeLa细胞的凋亡诱导能力低于其他片段，并且HIV-1 vpr基因对宿主细胞的凋亡诱导能力可能与其亚型有关。同时，我们发现携带这些突变片段位点的转染细胞的半胱天冬酶3活性与携带其他片段的细胞相比有所降低。本研究首次发现，在第70、85、86或94位点发生突变的HIV-1 Vpr片段可能会减弱其对HeLa细胞的凋亡诱导能力。其中一个机制可能是半胱天冬酶-3活性的减弱。
