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


CONCLUSIONS
"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.
Lin
Xu
X
Research Center of Molecular Medicine, Fujian Medical University, Fuzhou, Fujian Province, China. linxu70@hotmail.com
Xu
Xiao
X
Huang
Qing-Ling
QL
Liu
Yu-Qing
YQ
Zheng
Da-Li
DL
Chen
Wan-Nan
WN
Lin
Jian-Yin
JY
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D011994:Recombinant Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein