丙型肝炎病毒F蛋白缺失对病毒复制及感染性的影响

Effect of Hepatitis C F Protein and Core Secondary Structure on Viral Replication and Infection

探索了F蛋白缺失及核心蛋白(Core)二级结构改变对丙型肝炎病毒(HCV)复制和感染性的影响.利用定点突变方法,将J6JFH1的核心基因引进5个终止密码子以中断F蛋白的表达,从而获得F蛋白缺失的病毒复制子J6JFH1/ΔF.体外制备RNA转录体,并电穿孔转染Huh7.5.1细胞,采用免疫荧光、实时荧光定量PCR方法以及病毒感染等方法,观察F蛋白缺失对病毒复制、蛋白质表达及转染细胞上清感染性病毒颗粒产生的影响.在此基础上,构建5个单一突变病毒体,对HCV核心蛋白进行二级结构分析,观察核心蛋白二级结构对HCV复制和翻译的影响.结果显示,转染48 h后,J6JFH1/ΔF与野生型J6JFH1相比,J6JFH1/ΔF转染阳性细胞数明显降低,细胞内HCV RNA水平降低约95%,J6JFH1/ΔF转染后不同时间点细胞上清中HCV RNA拷贝数和病毒颗粒也明显降低.5个单一突变体不影响核心基因二级结构,病毒在细胞内复制和感染性与野生型水平一致.J6JFH1/ΔF所产生的改变可能是由于5处突变导致核心基因二级结构改变而造成的.结果说明,HCV F蛋白缺失不影响病毒的复制翻译及病毒颗粒的包装释放,核心蛋白二级结构的改变对病毒复制和翻译则产生较大影响.

Hepatitis C virus（HCV） F protein has been identified for more than ten years,but its functions remain unclear.In order to understand its roles in viral replication and infection,in this study 5 stop codons（nt406T-A,nt433T-A,nt472G-A,nt479/481G-A,G-A,nt613C-A） were introduced in plasmid J6JFH1 to construct J6JFH1/ΔF which interrupt F protein expression,and then monitor the viral RNA replication and viral protein expression.Our data showed that the five mutations did not affect core gene replication and expression.The virus strain J6JFH1/ΔF significantly reduced in the expression of viral proteins in transfected cells compared with wild-type J6JFH1,and viral RNA levels dropped by nearly 95%（J6JFH1/ΔF（7.39×106±2.54×104） vs wild type（1.17×108±1.46×107）,P 0.001）.The infectivity of virus released into the supernatant significantly reduced similarly.Further study found that these five mutations changed secondary structure of HCV core gene.In order to further explore the underlying reasons for the weakening of J6JFH1/ΔF virus replication and infection,we constructed five separate mutant viruses based on J6JFH1,respectively,and form J6JFH1m1 nt406T-A,J6JFH1m2 nt433T-A,J6JFH1m3 nt472G-A,J6JFH1m4 nt479/481G-A,G-A,J6JFH1m5 nt613C-A.These 5 virus mutants do not influence the secondary structure of the core region,while J6JFH1m1,J6JFH1m2,J6JFH1m3,J6JFH1m4,J6JFH1m5 respectively stopped F protein expression shifting from nt374-383,nt417-419,nt436-445,nt474-477,nt597-605,in which J6JFH1m5 stopped all reported forms of F protein.By in vitro transcription and transfection into Huh7.5.1 cells,48 hours post-transfection,the detection of intracellular viral protein NS5A expression levels,viral RNA in transfected cells,and virus titers in supernatant were carried out.The results showed that no significant differences were found among groups five separate mutants and wild type.These results indicated that the lack of F protein itself did not affect HCV viral replication and virion assembly and infectivity.All the results were seemed to result from the secondary structure of the core gene,but the mechanism was yet to be further studied.We concluded that HCV F protein deficiency did not affect translation of viral replication and packaging and release of virus particles,the core gene secondary structure changes had a great impact on viral replication and protein expression.