灵长类动物限制性因子SAMHD1的生物学功能分析

doi:10.13481/j.1671-587x.20190304

摘要/Abstract

摘要： 目的：探讨不同灵长类动物限制因子不育-α-基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋白1（SAMHD1）的抗病毒、抑制逆转录转座子LINE-1和减弱干扰素（IFN）产生信号通路等作用，为灵长类动物SAMHD1的研究提供依据。方法：构建稳定表达不同灵长类动物SAMHD1蛋白的U937细胞系，以空载体构建的细胞作为阴性对照组，以稳定表达不同灵长类动物SAMHD1蛋白的U937细胞系为实验组。经佛波酯（PMA）刺激分化成巨噬样细胞后，流式细胞术分析各组HIV-1病毒感染率。以单独转染SAMHD1表达质粒的HEK293T细胞为对照组，共同转染SAMHD1与HIV-2/SIV Vpx表达质粒的细胞为实验组，转染后48 h收获细胞，采用Western blotting法检测SAMHD1蛋白表达水平，免疫荧光检测法观察不同SAMHD1蛋白的细胞内定位。以单独转染LINE-1-GFP报告质粒的HEK293T细胞作为对照组，以共同转染LINE-1-GFP与SAMHD1表达质粒的细胞为实验组，采用流式细胞术检测GFP阳性细胞率，代表SAMHD1对LINE-1转座子的活性。以单独转染IFN-luc报告质粒的HEK293T细胞为对照组，共同转染IFN-luc和SAMHD1表达质粒的细胞为实验组，采用化学发光仪检测HEK293T细胞中荧光素酶的表达水平。结果：与阴性对照组比较，灵长类动物SAMHD1稳定表达的实验组HIV-1病毒感染率明显降低（P<0.01）。与对照组比较，实验组灵长类动物SAMHD1蛋白表达水平降低（P<0.05或P<0.01）。免疫荧光检测，灵长类动物SAMHD1蛋白均定位于细胞核中。与对照组比较，实验组GFP阳性细胞率明显降低，即SAMHD1对LINE-1转座子活性降低（P<0.05或P<0.01）。与对照组比较，实验组灵长类动物HEK293T细胞中荧光素酶表达水平明显降低（P<0.05）。结论：不同灵长类动物SAMHD1蛋白具有抵抗HIV-1病毒感染、抑制逆转录转座子LINE-1以及拮抗天然免疫系统产生IFN的作用。

关键词: 灵长类动物, 限制因子, 不育-α-基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋白1, 逆转录病毒, 逆转录转座子, Vpx

Abstract: Objective:To discuss the antiviral effect, the inhibitory effect on LINE-1 retrotransposon activity and the redection of interferon production signal pathway of restriction factor SAMHD1 of the primates,and to provide the basis for further study of the SAMHD1 of the primates. Methods:The U937 cells stably expressing the SAMHD1 of primates were established; the U937-control cells established with pLVX-puro were used as negative control group,and the U937-SAMHD1 cells stably expressing the SAMHD1 protein of the different primates were used as experimental groups; the cells were treated with PMA to induce cell differentiation. The virus infection rates of HIV-1 in the cells in various groups were determined by flow cytometry. The HEK293T cells transfected with the expression plasmid of SAMDH1 were used as control group, and the cells co-transfected with the SAMHD1 and HIV-2/SIV Vpx expression plasmids were used as experimental groups.The cells were obtained 48 h after transfection,and the expression levels of SAMHD1 protein were determined by Western blotting method. The intracellular location of SAMHD1 protein was determined by immunofluorescence.The HEK293T cells transfected with LINE-1-GFP report plasmid were used as control group, and the cells co-transfected with LINE-1-GFP and SAMHD1 expression plasmids were used as experimental groups. The rates of GFP positive cells(activity of SAMHD1 to LINE-1 transposon) were determined by flow cytometry. The HEK293T cells transfected with IFN-Luc report plasmid were used as control group,and the cells co-transfected with IFN-Luc and pSAMHD1 expression plasmids were used as experimental groups. The expression levels of luciferase in HEK293T cells were determined by chemiluminescence instrument. Results:Compared with negative control group, the virus infection rates of HIV-1 in experimental groups with stable expression of SAMHD1 in the primates were significantly decreased (P<0.01). Compared with control group, the expression levels of SAMHD1 protein of the primates in experimental groups were decreased(P<0.05 or P<0.01).The immunofluorescence results showed that the SAMHD1 protein of the primates was localized in the nucleus. Compared with control group, the rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) in experimental groups were significantly decreased (P<0.05 or P<0.01). Compared with control group, the expression levels of luciferase in the HEK293T cells in experimental groups were significantly decreased(P<0.05). Conclusion:The SAMHD1 protein of the different primates can resist the HIV-1 infection, inhibit the LINE-1 retrotransposon and antagonize the IFN production by natural immune system.

Key words: primate, restriction factor, sterile alpha motif and HD domain containing protein-1, retrovirus, retrotransposon, Vpx

中图分类号:

李爽, 郭浩然, 魏伟. 灵长类动物限制性因子SAMHD1的生物学功能分析[J]. 吉林大学学报(医学版), 2019, 45(03): 484-490.

LI Shuang, GUO Haoran, WEI Wei. Analysis on biological functions of restriction factor SAMHD1 of primates[J]. Journal of Jilin University(Medicine Edition), 2019, 45(03): 484-490.

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