FUNCTIONAL ROBES OF HEPATITIS A VIRUS NONSTRUCTURAL PROTEINS IN SIGNAL TRANSDUCTION

甲型肝炎病毒(HAV)感染仍然是一个世界性的主要问题。感染不会引起任何明显的细胞病变效应，也没有证据表明甲型肝炎病毒干扰其宿主细胞的大分子合成。最近，在乙型和丙型肝炎病毒感染中，有报道称细胞内信号被诱导。为了阐明甲型肝炎病毒感染的影响，我们检测了9种标志甲型肝炎病毒(VP2、VP3、VP1-2A、2B、2C、3A、3BC、3C、3D)蛋白对多种细胞内信号通路的影响。将病毒蛋白表达载体与报告载体共转化入HeLa细胞，由合成启动子控制，该启动子分别含有4个直接循环AMP反应元件(CRE)、5个血清反应因子(SRF)、7个激活蛋白1(AP 1)、5个核因子kB(NF-kB)和5个血清反应元件(SRE)。42h后收集细胞，进行荧光素酶活性测定。检测至少进行一式三份，病毒蛋白活性高于对照组的两倍被定义为显著。HAV FLAG-VP3蛋白可显著激活HeLa细胞中SRE相关信号，激活幅度为对照的2.2±0.3倍。而FLAG-VP3蛋白不激活CRE-(1.1±0.1)、SRF-(1.6±0.2)、AP-1-(1.1±0.1)、NF-kB-(0.7±0.1)相关通路。其他甲型肝炎病毒蛋白在Cre-、SRF-、AP-1、NF-kB和SRE相关途径中没有信号激活。我们发现甲型肝炎病毒VP3蛋白在HeLa细胞中激活了SRE相关信号，这是与细胞增殖、分化相关的细胞内信号通路。

Hepatitis A virus (HAV) infection is still a major problem worldwide. The infection does not induce any visible cytopathic effects, and there is no evidence that HAV interferes with the macromolecular synthesis of its host cells. Recently, in the hepatitis B and C virus infection, intracellular signals have been reported to be induced. To Clarify the effects of HAV infection, we examined the influence of 9 FLAG-HAV (VP2, VP3, VP1-2A, 2B, 2C, 3A, 3BC, 3C, 3D) proteins on various intracellular signaling pathways. Viral protein expression vectors were cotransfacted into HeLa cells with the reporter plasmids, controlled by a synthetic promoter that contains direct 4 repeats of the cyclic AMP response element (CRE), 5 repeats of the serum response factor (SRF), 7 repeats of the activator protein 1 (AP 1), 5 repeats of the nuclear factor kB (NF-kB), and 5 repeats of the serum response element (SRE), respectively. Cells were harvested 42 hours after transfaction, and luciferase assays were performed. Assays were conducted at least in triplicate, and a viral protein activation twice greater than that of the control was defined as significant. HAV FLAG-VP3 protein significantly activated the SRE-associated signal in HeLa cells at a value 2.2± 0.3 times higher than control. However, FLAG-VP3 protein did not activate the CRE-(1.1±0.1), SRF-(1.6± 0.2), AP-1-(1.1±0.1), NF-kB-(0.7±0.1) associated pathways. The other HAV proteins showed no signal activation in the CRE-, SRF-, AP-1-, NF-kB-, and SRE-associated pathways. We found that HAV VP3 protein activated the SRE-associated signal, intracellular signaling pathways associated with cell proliferation, differentiation, in HeLa cells.