CNP2线粒体定位信号以及磷酸酯酶活性对抑制HIV-1 Gag蛋白组装作用的影响  

Effect of mitochondrial targeting signal and phosphodiesterase activity of CNP2 on its inhibitory property against HIV-1 assembly

作　　者：梁顺涛[1,2] 戴国瑞[1,2] 李蕊 蒋栋[1,2] 曾辉 

机构地区：[1]首都医科大学附属北京地坛医院传染病研究所 [2]新发突发传染病研究北京市重点实验室,北京100015

出　　处：《中华实验和临床感染病杂志（电子版）》2016年第3期380-384,共5页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：国家自然科学基金(No.31170853);教育部留学回国启动基金(No.jwsl453);北京市自然科学基金(No.5162011)

摘　　要：目的研究CNP2在线粒体上的定位以及磷酸酯酶活性是否参与抑制HIV-1病毒颗粒组装。方法采用RT-PCR扩增野生型CNP2编码区,插入到真核表达载体pc DNA5,构建融合蛋白FlagCNP2的表达质粒pc DNA5-Flag-CNP2。应用融合PCR诱导突变法构建CNP1及CNP2突变表达载体。CNP表达载体分别与HIV-1假病毒包装质粒p LP1、p LP2、p LP/VSVG、p Lenti6-EGFP共同瞬时转染293T细胞。集落形成实验检测上清病毒滴度,Western blot检测细胞中Gag蛋白p55、p24以及细胞培养上清中p24的表达情况。结果与对照组比较,CNP2、CNP1、CNP2-S9/22A可以显著抑制细胞中病毒的释放,培养上清中病毒滴度显著降低(滴度分别为5.66、4.20、5.75、6.63 Log10IU/ml,NC组为7.65 Log10 IU/ml;t=58.23、17.24、12.77、6.131,P=0.0035、0.0066、0.0004、0.0039)。CNP1抑制HIV-1病毒颗粒组装作用更强,Western blot检测培养上清中病毒蛋白完全p24消失。磷酸酯酶结构域(2HM)突变后CNP2抑制HIV-1病毒颗粒组装的作用显著降低。结论 CNP2抑制HIV-1病毒颗粒组装需要磷酸酯酶结构域(2HM)的参与,CNP2的线粒体定位信号可能会影响其抑制HIV病毒颗粒组装的作用。Objective To confirm whether the mitochondrial targeting and phosphodiesterase activity of CNP2 affect its inhibitory ability against HIV-1 assembly. Methods Vector expressing wild type CNP2 with N-terminal FLAG tag was constructed by RT-PCR of CNP2 coding region from Huh7 cell and subsequently inserted into eukaryotic expressing vector pc DNA5 to get pc DNA5-Flag-CNP2. CNP1 and CNP2 mutants CNP2-S9/22 A, CNP-2HM was constructed by PCR based mutagenesis and inserted into the same vector. CNP constructs was respectively transfected into 293 T cell with pseudotyped HIV-1 particle assemble vectors p LP1, p LP2, p LP/VSVG, p Lenti6-EGFP. Pseudotyped HIV-1 particle titer was tested by foci formation after infection of 293 T cell and also estimated particle p24 by Western blot. TP55 gag precursor and p24 in transfected cell were tested by Western blot. Results CNP2, CNP1 and CNP2-S9/22 A could potently inhibit HIV titers in transfection supernant（t = 58.23, 17.24, 12.77, 6.131; P = 0.0035, 0.0066, 0.0004, 0.0039）. The inhibitory property of CNP1 without mitochondrial targeting signal was more potently than CNP2. 2HM mutant losing phosphodiesterase activity could only minimally inhibit HIV-1 assembly. Conclusions CNP2 inhibition of HIV-1 assembly needs the phosphodiesterase activity, and mitochondrial targeting signal could modulate the inhibitory function.

关 键 词：2’ 3’-环核苷酸磷酸二酯酶 HIV-1病毒颗粒组装 线粒体定位信号 磷酸二酯酶活性. 

分 类 号：R512.91[医药卫生—内科学]