Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated

Abstract

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 pCMVbeta to Chang cells, which were lysed 48 h post-transfection and the intra-cellular beta-galactosidase activities were monitored (increase of the beta-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 anti-proliferation 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.

Figure 1

There were 16 amino acid variations between genotype B and C HBx, which were located at amino acid positions 5, 30, 31, 34, 36, 39, 40, 42, 43, 44, 47, 87, 88, 116, 118, and 119, respectively. Four boxed amino acids, located at the positions of 127, 130, 131, 132, respectively, were to be substituted by site-directed mutagenesis.

Figure 2

Chang cells were transfected by genotype B X gene expression vectors and screened by hygromycin, the drug-resistant colonies formed 14 d post-transfection were fixed by cold methanol, stained with Giemsa (A) and counted (B). The results are shown as mean±SD of six separate experiments.

Figure 3

Chang cells were transfected by genotype C X gene expression vectors and screened by hygromycin, the drug-resistant colonies formed 14 d post-transfection were fixed by cold methanol, stained with Giemsa (A) and scored (B). The results are shown as mean±SD of six separate experiments.

Figure 4

Chang cells were co-transfected by pCMVβ together with the X gene or related mutants with genotype B (A) or genotype C (B). Forty-eight hours post-transfection, the cells were lysed and the intracellular β-galactosidase activities were monitored after protein normalization. The results are shown as mean±SD of six separate experiments.