APOBEC3G对HIV-1及其Vif缺失株的抑制作用

Restriction of HIV-1 and the Vif-deficiency strain by APOBEC3G

目的研究细胞内在抗病毒因子APOBEC3G对HIV-1及其Vif缺失株的抑制作用。方法 HIV-1野生株病毒(BH10WT)和Vif缺失株病毒(BH10ΔVif)分别由转染293T细胞获得,通过感染MT4和H9细胞,分别检测其反转录酶活性。用分子克隆技术构建APOBEC3G与绿色荧光蛋白融合表达的质粒pEGFP-3G。HIV-1野生株和Vif缺失株质粒与不同剂量的pEGFP-3G共转染293T细胞,APOBEC3G-GFP在细胞内融合表达定位通过荧光显微镜观察。所生成的子代病毒粒子的感染力分别由MAGI检测和反转录酶活性检测方法定量。结果 BH10 WT病毒在H9细胞和MT4细胞中均有所复制,在感染MT4细胞后4 d已经具有较高的反转录酶活性。BH10ΔVif病毒感染H9细胞产生的子代病毒的反转录酶活性接近细胞对照水平,而其在MT4细胞中产生的子代病毒的反转录酶活性在感染后12 d有所显现,即非允许性H9细胞内APOBEC3G对ΔVif病毒株具有很强的抑制作用。与绿色荧光蛋白融合表达的APOBEC3G蛋白定位于细胞质。BH10ΔVif转染293T细胞后生成的病毒滴度为2.75×104 U/mL,随着pEGFP-3G共转染量的升高,所产生的子代病毒滴度从1.48×103 U/mL显著降至0.33×103 U/mL。与0.2μg质粒pEGFP-3G共转染产生的BH10ΔVif病毒的感染力比不表达APOBEC3G的相同病毒的感染力降低近20倍。结论 APOBEC3G具有抗HIV活性,同时HIV Vif在病毒复制过程中发挥关键作用,两者之间相互作用的量效关系为我们构建抗病毒药物筛选平台奠定了可靠的实验基础。

Objective To study the restriction of HIV-1 and the Vif-deficient strain. Methods Viruses of HIV-1 wild type（BH10 WT） or the Vif-deficient strain（BH10 ΔVif） produced from transfected 293T cells were used to infect various cell lines,including MT4 and H9.The results were determined by reverse transcriptase（RTase） assay.Molecular-cloning technique was used to construct expression plasmid pEGFP-3G which express APOBEC3G with a C-terminal GFP tag.The virus strains of BH10 WT or BH10 ΔVif co-transfection with different dose of pEGFP-3G in 293T cells,and the expression of APOBEC3G-GFP was observed by live cell fluorescence microscopy.The infectivity of virus was determined by multinuclear activation of galactosidase indicator（MAGI） assay and RTase assay. Results BH10 WT replication in MT4 cells showed much faster replication kinetics than that in H9 cells,with peak RT values being observed as early as 4 days post-infection.RT activity of BH10 ΔVif in H9 cells was suppressed almost to the level of negative control and that in MT4 cells was observed on the 12th day.GFP-APOBEC3G and a GFP-only control localized to the cytoplasmic and cell-wide,respectively.The titer of BH10 ΔVif by MAGI assay is about 2.75×104 U/mL.The titer of viruses,produced in 293T cells by cotransfection with increasing amounts of APOBEC3G,was significantly reduced from 1.48×103 U/mL to 0.33×103 U/mL.The infectivity of BH10 ΔVif produced in the presence of 0.2 μg of co-transfected pEGFP-3G was twenty fold less infectious than BH10 ΔVif produced in the absence of APOBEC3G. Conclusion The anti-HIV activity of APOBEC3G was dose-dependent,and HIV-1 Vif has the essential role in the virus replication.Based on the results we would construct a new platform to accurately and promptly select efficacious drugs.