摘要
AIM: To investigate the biological impacts of 'hot-spot'mutations on genotype B and C HBV X proteins (HBx).METHODS: Five types of'hot-spot' mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I,I127T+K130M+V131I, or K130M+V131I+F132Y, respectively,were generated by means of site-directed mutagenesis.To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity,and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test.RESULTS: As compared to standard genotype B HBx,mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity,compared to standard genotype C HBx, F132Y and K130M+V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity,while the mutants of I127T and I127T+K130M+V131I showed no differences.CONCLUSION: 'Hot-spot' mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most 'hot-spot' mutations on HBx are genotype B and C differentiated.
AIM： To investigate the biological impacts of “hot-spot” mutations on genotype B and C HBV X proteins （HBx）. METHODS： Five types of “hot-spot” mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M＋V131I, I127T＋K130M＋V131I, or K130M＋V131I＋F132Y, respectively, were generated by means of site-directed mutagenesis. To evaluate the anti-proliferative effects, HBx or related mutants＇ expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drugresistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored （increase of the colonies indicated the reduction of the anti-proliferation activity, and vice versa）. Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h posttransfection and the intra-cellular β-galactosidase activities were monitored （increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa）. All data obtained were calculated by paired-samples t-test. RESULTS： As compared to standard genotype B HBx, mutants of I127T and I127T＋K130M＋V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M＋V131I, and K130M＋V131I＋F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity, compared to standard genotype C HBx, F132Y and K130M＋V131I mutants showed lower activities, and K130M＋V131I ＋F132Y mutant, on the other hand, showed higher activity, while the mutants of I127T and I127T＋K130M＋V131I showed no differences. CONCLUSION： “Hot-spot” mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most “hot-spot” mutations on HBx are genotype B and C differentiated.
基金
Supported by the Foundation for the Author of National Excellent Doctoral Dissertation of PR China, No. 200359Fujian Natural Science Key Foundation, No, 2002F005Fujian Science and Technology Innovation Foundation for Young Scientists, No.2001J058
