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HIV-1Rev蛋白和人DDX3解旋酶对HCVRNA复制影响

, PP. 2329-2333

Keywords: 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,丙型肝炎病毒

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Abstract:

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(regulatorofvirionproteinexpression,Rev)和DEAD-box蛋白家族ATP依赖的RNA解旋酶3(DDX3)对HCVRNA复制的影响。方法利用HCV表达质粒pCDNA3.1-JFH1转染Huh7细胞以构建稳定的HCV细胞复制模型,另构建pCDNA3.1-Rev-Flag-mCherry和pCDNA3.1-DDX3-YFP-Flag的过表达载体,通过PCR、Westernblot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HCVRNA复制质粒共转染入Huh7细胞;2组的实验组为Rev、DDX3和HCVRNA复制质粒共转染入Huh7细胞。最后通过qPCR定量测定各组HCVRNA复制水平并进行比较。结果转染后48h测各组HCVRNA的复制水平,DDX3+HCVRNA复制质粒组中HCVRNA的水平(1.09×107±0.18×107)明显高于单独转染HCVRNA复制质粒组(4.79×106±1.24×106)(P=0.03);而Rev+DDX3+HCVRNA复制质粒组中HCVRNA的水平(1.74×107±0.40×107)明显低于单独转染HCVRNA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HCVRNA的复制,而Rev蛋白和DDX3共同作用可抑制HCVRNA的复制。

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