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丙型肝炎病毒高变区1中介导感染的关键氨基酸残基的鉴定
解冰; 劳文光; 刘文宇; 关默; 刘小青; 陶清源; 王岩; 赵平; 戚中田
第二军医大学微生物学教研室,上海 200433
Identification of the key amino acid residues in hypervariable region 1 for hepatitis C virus infection
XIE Bing; LAO Wen-Guang; LIU Wen-Yu; GUAN Mo; LIU Xiao-Qing; TAO Qing-Yuan; WANG Yan; ZHAO Ping; QI Zhong-Tian
Department of Microbiology, Second Military Medical University, Shanghai 200433, China
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摘要: 丙型肝炎病毒(HCV)包膜E2蛋白氨基末端的高变区1(HVR1)由27个氨基酸组成,是HCV蛋白中变异频率最高的肽段。HVR1含中和抗体表位,同时对HCV细胞侵入起重要作用,其结构与功能的关系目前尚不清楚。本研究对H77株包膜蛋白基因中的HVR1进行了一系列缺失突变,然后将突变体表达质粒与假病毒包装质粒共转染人胚肾293T细胞,用蛋白免疫印迹法检测细胞中HCV包膜E2蛋白的表达,用荧光定量聚合酶链反应(PCR)检测培养上清液中的HCV假病毒(HCVpp)含量,以人肝癌细胞Huh7.5为靶细胞检测HCVpp的感染力。结果显示,缺失整个HVR1或HVR1中部分氨基酸残基对HCV包膜蛋白表达及HCVpp产量均无明显影响,但对HCVpp感染力产生不同影响。缺失第16~24位氨基酸残基导致HCVpp感染力增强,缺失第1~8或1~12位氨基酸残基仅部分降低HCVpp的感染力,缺失第13~15和25~27位中的任意一个氨基酸残基均导致HCVpp感染力为原型的5%。结果提示,HVR1中第13~15和25~27位的6个氨基酸是介导HCV感染的关键氨基酸残基。

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解冰
劳文光
刘文宇
关默
刘小青
赵平
戚中田
关键词 丙型肝炎病毒高变区1侵入细胞关键残基    
Abstract:The hypervariable region 1 (HVR1) of hepatitis C virus (HCV), consisting of 27 amino acids at the amino terminus of envelope protein 2 (E2), is the most variable region in HCV polyprotein. HVR1 contains dominant neutralizing epitopes and plays an important role in HCV cell entry. Despite these observations, the relationship between the structure and the function of HVR1 remains unknown. In this study, a serial of deletion mutants in HVR1 of envelope gene of H77 isolate were prepared, and the respective expression plasmids were co-transfected with pseudoparticles package plasmids into human embryo kidney (HEK) 293T cells. The expression of E2 in HEK 293T cells were assayed by using Western blot, and the level of HCV pseudoviral particle (HCVpp) in supernatants were detected by using fluorescence quantitative polymerase chain reaction (PCR). Human hepatoma Huh7.5 cells were used as the target cells to assay the infectivity of HCVpp. The results showed that deletion of the complete HVR1 or partial amino acid residues in HVR1 did not affect either the expression of E2 protein or the production of HCVpp. Deletion of the amino acids at positions 16-24 increased HCVpp infectivity, and deletion of the amino acids at positions 1-8 or 1-12 only partially decreased HCVpp infectivity. Importantly, deletion of any residue at positions 13-15 or 25-27 decreased HCVpp infectivity to below 5% of the prototype pseudoparticles. The results indicate that the six amino acids at position 13-15 and 25-27 are the key residues mediating HCV cell entry.

Key wordsHepatitis C virus    Hypervariable region 1    Cell entry    Key amino acid residue
通讯作者: 赵平;戚中田   
引用本文:   
解冰; 劳文光; 刘文宇; 关默; 刘小青; 陶清源; 王岩; 赵平; 戚中田. 丙型肝炎病毒高变区1中介导感染的关键氨基酸残基的鉴定[J]. 微生物与感染 , 2011, 6(4): 225-230.
XIE Bing; LAO Wen-Guang; LIU Wen-Yu; GUAN Mo; LIU Xiao-Qing; TAO Qing-Yuan; WANG Yan; ZHAO Ping; QI Zhong-Tian . Identification of the key amino acid residues in hypervariable region 1 for hepatitis C virus infection. Journal of Microbes and Infections, 2011, 6(4): 225-230.
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