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

Table 1 Sequences and related applications of the primers used for site-directed mutagenesis and nomenclature of HBx mutants

Primers	Sequences (5’→3’)	Applications		Nomenclature of the HBx mutants
		Nucleotide mutation2	Amino acid substitution3
R	CTCCTCCCCCAACTCTTCCCACTCAGTAAAC
F1	GAAGAGTTGGGGGAGGAGAC1	1753T→C	I127T	XB/XC-127
	TAGGTTAAAGGTC
F2	GAAGAGTTGGGGGAGGAGACTAGGTTA	1753T→C + 1762A→T + 1 764G→A	I127T + K130M + V131I	XB/XC-127-130-131
	ATGATCTTTGTACTAGG
F3	GAAGAGTTGGGGGAGGAGATTAGGTTA	1762A→T + 1764G→A	K130M + V131I	XB/XC-130-131
	ATGATCTTTGTACTAGG
F4	GAAGAGTTGGGGGAGGAGATTAGGTTA	1762A→T + 1764G→A + 1768T→A	K130M + V131I + F132Y	XB/XC-130-131-132
	ATGATCTATGTACTAGGAGGC
F5	GAAGAGTTGGGGGAGGAGATTAGGTTA	1768T→A	F132Y	XB/XC-132
	AAGGTCTATGTACTAGGAGGC

¹Mutated nucleotides are shown as underlined;

²1 753, 1 762, 1 764, 1 768 indicate the positions of mutated nucleotides in HBV genome;

³127, 130, 131, 132 are positions of substituted amino acids in HBx.

Table 2 Intracellular β-galactosidase activities of transfected Chang cells by pCMVβ and X gene expression vectors

DNA transfected	β-galactosidase activities1	P2	P3
pcDNA3.1/Hygro(–)	0.147 ± 0.039	-	-
XB	0.593 ± 0.051	< 0.01	-
XB-127	0.666 ± 0.054	< 0.01	< 0.05
XB-132	0.492 ± 0.052	< 0.01	< 0.01
XB-130-131	0.477 ± 0.065	< 0.01	< 0.01
XB-127-130-131	0.680 ± 0.090	< 0.01	< 0.05
XB-130-131-132	0.472 ± 0.081	< 0.01	< 0.05
XC	0.505 ± 0.064	< 0.01	-
XC-127	0.641 ± 0.044	< 0.01	< 0.01
XC-132	0.571 ± 0.104	< 0.01	> 0.05
XC-130-131	0.459 ± 0.065	< 0.01	> 0.05
XB-127-130-131	0.457 ± 0.067	< 0.01	> 0.05
XB-130-131-132	0.646 ± 0.177	< 0.01	> 0.05

¹All data are expressed as mean±SD of six separate experiments; ²Paired-samples t-test between pcDNA3.1/Hygro(–) and all HBx; ³Paired-samples t-test between genotype B(XB)/genotype C(XC) and the relative mutants.

Table 3 Summary of the biological impact of “hot-spot” mutations on genotype B and C HBx

HBx mutants	Genotype B1		Genotype C2
	Transactivation	Antiproliferation	Transactivation	Antiproliferation
I127T	↑3	↑	↑	-5
I127T+K130M+V131I	↑	↑	-	-
K130M+V131I	↓4	↓	-	↓
K130M+V131I+F132Y	↓	↓	-	↑
F132Y	↓	↓	-	↓

¹Changes of biological functions were compared with XB;

²Changes of biological functions were compared with XC. ^3,4,and⁵ accordingly indicate increased, decreased and equal activities of HBx mutants as compared with XB/XC.