HIV-1 Vpr蛋白对小鼠神经瘤母细胞N2a活性及细胞周期的影响

Effects of HIV-1 Vpr protein on cell viability and cell cycle of mouse neuroblastoma N2a cells

目的探讨HIV-1 Vpr蛋白对小鼠神经细胞的毒性作用。方法巢式PCR扩增了1例HIV相关痴呆(HIV associated dementia,HAD)和1例非HAD的艾滋病患者脾(spleen,SPL)、脑膜(meninges,MG)共4个部位的HIV-1 vpr基因测序,对基因序列和氨基酸位点进行分析。构建pEGFP-N1-vpr真核表达载体,脂质体法转染N2a细胞,Western blot检测Vpr蛋白在N2a细胞中的表达,流式细胞术研究了不同来源的Vpr蛋白对N2a细胞活性和细胞周期的影响。结果PCR扩增出HIV-1 vpr基因,序列分析显示vpr基因序列属于HIV-1B亚型,HAD和非HAD患者中枢来源的Vpr蛋白在C末端84、86和87位存在氨基酸位点的突变;Vpr蛋白可抑制神经细胞活性,导致G2周期阻滞,不同来源的Vpr作用存在差异,HAD患者MG来源的Vpr蛋白,表现出更强的抑制细胞活性和G2周期阻滞能力。结论Vpr蛋白关键氨基酸位点的变异可致其生物学功能的明显改变,其在HAD发病机制中的意义仍待进一步研究。

Objective To investigate the toxic effect of HIV-1 Vpr protein on neurons.Methods HIV-1 vpr gene was amplified by nested PCR in four parts of peripheral spleen(SPL)and central nervous tissue meninges(MG)of HIV-associated dementia(HAD)patients and non-HAD patients.Eukaryotic expression vector pEGFP-N1-vpr was constructed.The gene sequence and key amino acid sites were analyzed by BLAST and MEGA6.The expression of Vpr protein in N2a cells was detected by Western-blotting.The effects of Vpr proteins from different sources on the activity and cell cycle of N2a cells were studied by flow cytometry.Results HIV-1 vpr gene was successfully amplified by PCR.Sequence analysis showed that the vpr gene sequence belonged to HIV-1B subtype.There were amino acid mutations at C-terminal 84,86 and 87 sites of central Vpr protein from HAD and non-HAD patients.Vpr protein could inhibit the activity of nerve cells,leading to G2 phase arrest.Different sources of Vpr had different intensity of action.Compared with other groups,Vpr protein from the meninges of HAD patients showed stronger inhibition of cell activity and G2 phase arrest ability.Conclusions Variations in key amino acid sites of Vpr protein could cause significant changes in its biological functions,and its significance in the pathogenesis of HAD remains to be further studied.