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

doi:10.3877/cma.j.issn.1674-1358.2016.03.028

中华实验和临床感染病杂志(电子版) ›› 2016, Vol. 10 ›› Issue (03) : 380 -384. doi: 10.3877/cma.j.issn.1674-1358.2016.03.028

基础论著

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

梁顺涛¹, 戴国瑞¹, 李蕊¹, 蒋栋¹, 曾辉¹^,^†(

)

^1. 100015　北京，首都医科大学附属北京地坛医院传染病研究所；新发突发传染病研究北京市重点实验室

收稿日期:2015-08-27 出版日期:2016-06-15
通信作者: 曾辉
基金资助:
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)

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

Shuntao Liang¹, Guorui Dai¹, Rui Li¹, Dong Jiang¹, Hui Zeng¹^,^†()

^1. Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University; Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China

Received:2015-08-27 Published:2016-06-15
Corresponding author: Hui Zeng

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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pcDNA5，构建融合蛋白Flag-CNP2的表达质粒pcDNA5-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pLP1、pLP2、pLP/VSVG、pLenti6-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。

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。

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 pcDNA5 to get pcDNA5-Flag-CNP2. CNP1 and CNP2 mutants CNP2-S9/22A, CNP-2HM was constructed by PCR based mutagenesis and inserted into the same vector. CNP constructs was respectively transfected into 293T cell with pseudotyped HIV-1 particle assemble vectors pLP1, pLP2, pLP/VSVG, pLenti6-EGFP. Pseudotyped HIV-1 particle titer was tested by foci formation after infection of 293T 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-S9/22A 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.

Key words: 2’, 3’-cyclic nucleotide phosphodiesterase (CNP), HIV-1 particle assembly, Mitochondrial targeting signal, Phosphodiesterase activity.

表1 扩增CNP2、CNP2-S9/22A、CNP1和CNP2-2HM等片段所需引物

引物名称	序列
Bam H Ⅰ-CNP2+	5'-CACGGATCCATGAACAGAGGCTTCTCCCG-3'
Bam HⅠ-CNP2-S9/22A+	5'-CACGGATCCATGAACAGAGGCTTCTCCCGAAAAGCCCACACATTCCTGCCCAAGATCTTCTTCCGCAAGATGGCATCCTCAGGGGCCAAGGACAAG-3'
Bam HⅠ-CNP1+	5'-CACGGATCCATGTCATCCTCAGGGGCCAAG-3'
CNP2-2HM+	5'-GGAAAGAGACCCCCAGGCGTGCTGGCTGCCGCAACCAAGTTTTGTGACTACGGG-3'
CNP2-2HM-	5'-CTACGTCAGCTGCACAGCCGAGGGCGGCGGCGGCGCGGCTCCCCCGCGGCAGG3'
Not Ⅰ-CNP2-	5'- CACGCGGCCGCTCATATGATGGTGCAGGACT -3'

表1 扩增CNP2、CNP2-S9/22A、CNP1和CNP2-2HM等片段所需引物

引物名称	序列
Bam H Ⅰ-CNP2+	5'-CACGGATCCATGAACAGAGGCTTCTCCCG-3'
Bam HⅠ-CNP2-S9/22A+	5'-CACGGATCCATGAACAGAGGCTTCTCCCGAAAAGCCCACACATTCCTGCCCAAGATCTTCTTCCGCAAGATGGCATCCTCAGGGGCCAAGGACAAG-3'
Bam HⅠ-CNP1+	5'-CACGGATCCATGTCATCCTCAGGGGCCAAG-3'
CNP2-2HM+	5'-GGAAAGAGACCCCCAGGCGTGCTGGCTGCCGCAACCAAGTTTTGTGACTACGGG-3'
CNP2-2HM-	5'-CTACGTCAGCTGCACAGCCGAGGGCGGCGGCGGCGCGGCTCCCCCGCGGCAGG3'
Not Ⅰ-CNP2-	5'- CACGCGGCCGCTCATATGATGGTGCAGGACT -3'

图1 CNP 2 MTS和2HXT结构域与突变型载体模式图

图2 阳性克隆质粒酶切鉴定结果

图3 阳性克隆质粒代表性测序结果

图4 CNP2及突变型载体对细胞上清中病毒滴度的影响

图5 转染CNP2及其突变载体对抑制Gag蛋白组装的影响

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