HIV-1 Rev蛋白和人DDX3解旋酶对HCV RNA复制影响  

Effect of HIV-1 Rev protein and DDX3 helicase on HCV RNA replication

作　　者：罗亚东[1] 洪国枯 贾鸣[1] 郭艳[1] 刘明[1] 毛青[1] 

机构地区：[1]第三军医大学西南医院全军感染病研究所,感染病研究重庆市重点实验室,重庆400038

出　　处：《第三军医大学学报》2015年第23期2329-2333,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81271814);十二五国家科技重大专项(2012ZX10001003-003-003)~~

摘　　要：目的研究人类免疫缺陷病毒Ⅰ型(HIV-1)病毒颗粒蛋白表达调节因子(regulator of virion protein expression,Rev)和DEAD-box蛋白家族ATP依赖的RNA解旋酶3(DDX3)对HCV RNA复制的影响。方法利用HCV表达质粒p CDNA3.1-JFH1转染Huh7细胞以构建稳定的HCV细胞复制模型,另构建p CDNA3.1-Rev-Flag-m Cherry和p CDNA3.1-DDX3-YFP-Flag的过表达载体,通过PCR、Western blot和核苷酸测序等方法鉴定载体构建成功后,将实验分为2组对照实验,对照组均只将HCV复制质粒转染Huh7细胞:1组实验组为DDX3和HCV RNA复制质粒共转染入Huh7细胞;2组的实验组为Rev、DDX3和HCV RNA复制质粒共转染入Huh7细胞。最后通过q PCR定量测定各组HCV RNA复制水平并进行比较。结果转染后48 h测各组HCV RNA的复制水平,DDX3+HCV RNA复制质粒组中HCV RNA的水平(1.09×107±0.18×107)明显高于单独转染HCV RNA复制质粒组(4.79×106±1.24×106)(P=0.03);而Rev+DDX3+HCV RNA复制质粒组中HCV RNA的水平(1.74×107±0.40×107)明显低于单独转染HCV RNA复制质粒组(4.17×107±0.46×107)(P<0.001)。结论成功构建Rev蛋白和DDX3的过表达载体,并初步证明人DDX3解旋酶可促进HCV RNA的复制,而Rev蛋白和DDX3共同作用可抑制HCV RNA的复制。Objective To determine the effect of the regulator of HIV-1 virion protein expression （Rev） and a DEAD box RNA helicase （DDX3） on HCV RNA replication. Methods pCDNA3.1-Re-Flag- mCherry and pCDNA3.1-DDX3-YFP-Flag were constructed and identified by PCR, Western blotting and DNA sequencing. Then the Huh7 cells were divided into 2 groups, the control group （only HCV RNA replication plasmid was transfected into Huh7 cells ） and the experimental group （DDX3 and HCV RNA replication plasmids were co-transfected into Huh7 cells）, qPCR was used to detect the level of HCV RNA for the effects of DDX3 and HCV RNA replication plasmids. Results In 48 h after transfection, the expression level of HCV RNA was significantly higher in DDX3 ＋ HCV RNA replication p]asmid group than HCV RNA plasmid group [ （ 1.09 ± 0.18 ） ×10^7 vs （4.79 ± 1.24） × 10^6, P = 0.03 ]. But the expression level of HCV RNA was significantly lower in Rev ＋ DDX3 ＋ HCV RNA replication plasmid group than that of HCV RNA replicationHCV RNA plasmid group [ （1.74 ±0.40） ×10^7 vs （4.17 ±0.46） ×10^6, P 〈0.001]. Conclusion The over-expression vector of DDX3 and Rev is successfully constructed, and DDX3 can accelerate the replication of HCV RNA, but Rev combined with DDX3 can inhibit the replication of HCV in vitro.

关 键 词：人类免疫缺陷病毒Ⅰ型 病毒颗粒蛋白表达调节因子 DEAD-box蛋白家族的ATP依赖的RNA解旋酶3 丙型肝炎病毒 

分 类 号：R345[医药卫生—基础医学] R373.9