| ObjectiveTo investigate the functional differences between hepatitis B virus X proteins (HBx) and related mutants with genotype B and C, and elucidate preliminarily the mechanism of different clinical manifestations and pathological outcomes between genotype B and C hepatitis B virus (HBV) related liver diseases/ hepatocellular carcinoma (HCC).MethodsX genes of genotype B and C HBV cited from GenBank were converted into related amino acids, and amino acids alignment were carried out to identify the conserved and the genotype-specfic amino acids of genotype B and C HBx. Recombinant eukaryotic expression vectors haboring the conserved genotype B and C HBV X gene were construct separately and used to transfect the Chang cells, and the transactivation and antiproliferation activities were monitored post-transfection. Full-length HBV genomes were amplified from the sera of HCC patients by means of PCR, and the genotypes of obtained HBV DNA were categorized by phylogenetic analysis. The variations of HBx from HCC patients were identified as compared with consensus genotype B and C HBx sequences. Recombinant eukaryotic expression vectors haboring different types of mutated genotype B and C HBV X gene were constructed and used to transfect the Chang cells, and the transactivation and antiproliferation activities of X mutants were detected to evaluate the effects of amino acids variations on genotype B or C HBx. ResultsThere were 17 amino acids difference between genotype B and C HBx which located in amino acids position 5, 30, 31, 34, 36, 39, 40, 42, 43, 44, 47, 87, 88, 116, 118, 119 and 127, respectively. In addition, the 100% conserved and genotype B-specific amino acids located in position 34, 39, 40, 42, 88 and 119, while the 100% conserved and genotype C-specific amino acids located in position 34, 39, 43, 47, 88, 118 and 119. Constructed separately the recombinant eukaryotic expression vector harboring either the conserved genotype B or C HBV X gene (nomenclatured the vector as BS and CS accordingly) and transfected to the Chang cells, the results showed that the transactivation activity of conserved genotype B HBx was higher than that of genotype C HBx,while the antiproliferation activity was lower than that of genotype C HBx.Amplified the HBV complete genomes by means of PCR from the sera of HCC patients and cloned into the vector pUC18, obtained 22 full-length HBV recombinant. Phylogenetic analysis revealed that all 22 HBV strains could be categorized into genotype B and C. Comparison of the HBx sequence revealed that HBx from HCC patients with genotype B and C showed common variations in amino acid position 127, 130, 131, 132 and displayed as five types as V/I127T, F132Y, K130M+V131I, V/I127T+K130M+V131I and K130M+ V131I+F132Y, respectively. In addition, the variation rate of HBx originated from HCC patients were higher than that from GenBank.Constructed five types of recombinant eukaryotic expression vectors by means of site-directed mutagenesis , each of them harbored one type of mutated X genes which sequentially lead to the amino acid variation as V/I127T, F132Y, K130M+V131I, I127T+K130M+V131I or K130M+V131I+F132Y, respectively.. (nomenclatured the mutated vectors according to the mutated amino acids of genotype B or C HBx, ie. BT, BY, BMI,BTMI,BMIY for genotype B , and CT, CY, CMI, CTMI,CMIY for genotype C ). Transfected the recombinant vectors to Chang cells. With regard to transactivation activity, BT was higher than BS, and BY,BMI,BMIY were lower than BS, while there was no difference between BTMI and BS, in addition, CT was higher than CS, and there was no difference between CY,CMI,CTMI,CMIY and CS. With regard to the antiproliferation activity, BT and BTMI were higher than BS, while BY, BMI ,BMIY were lower than BS, in addition, CY, CMI were lower than CS, and CMIY was much higher than CS, while there was no difference between CT, CTMI and CS.ConclusionsThe transactivation and antiproliferation activities were different between genotype B and C HBx , and these dif... |
PDF Full Text Request