Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

Table 1 Mutations in the HCV NS3 region

Sample	Month 12	Month 28	Month 63	Total
No. of clones tested	30	30	30	90
Clones with mutations	11	14	15	40
Total mutations	13	20	18	51
SYN mutations	2	4	6	12
NSY mutations	11	16	12	39

SYN: Synonymous; NSY: Nonsynonymous; HCV: Hepatitis C virus; NS3: Non-structural 3.

Table 2 Nonsynonymous and synonymous mutations in different regions of HCV NS3

Region (aa)	Length (bp)	PCR cycles	No. of clones	Mutation		P	Mutation
				Expected sporadic	NSY		SYN	NSY/ SYN
1060-1069	30	70	90	0.25	1	> 0.050	0	1/0
1070-1079	30	70	90	0.25	5	< 0.001	3	5/3
1080-1089	30	70	90	0.25	4	< 0.001	0	4/0
1090-1099	30	70	90	0.25	3	< 0.001	5	3/5
1100-1109	30	70	90	0.25	5	< 0.001	0	5/0
1110-1119	30	70	90	0.25	3	< 0.001	1	3/1
1120-1129	30	70	90	0.25	1	> 0.050	1	1/1
1130-1139	30	70	90	0.25	4	< 0.001	0	4/0
1140-1149	30	70	90	0.25	0	NS	0	0
1150-1159	30	70	90	0.25	4	< 0.001	0	4/0
1160-1169	30	70	90	0.25	0	NS	0	0
1170-1179	30	70	90	0.25	3	< 0.001	2	3/2
1180-1189	30	70	90	0.25	6	< 0.001	0	6/0
1190-1198	27	70	90	0.23	0	NS	0	0
1073-10811	27	70	90	0.23	4	< 0.001	2	4/2
1131-11391	27	70	90	0.23	4	< 0.001	0	4/0
1169-11771	27	70	90	0.23	3	< 0.001	2	3/2

¹Epitope region. Expected sporadic mutations = ER × L × Nc × N × P/Ns, where ER = error rate of polymerases (2.02 × 10^-6), L: Nucleotide length of compared region; Nc: Number of PCR cycles; N: Number of clones sequenced; P: Proportion of sporadic mutations expected to produce amino acid substitutions (2/3), Ns: Number of sample (1) according to Smith et al[37] (1997). PCR: Polymerase chain reaction; NS: No significant.

Table 3 Binding capacity, cytotoxicity and antagonicity of HCV NS3 peptides

Sequence	Name	Peptide origin	Position	Peptide length (aa)	IC50 (nmol/L)	R2	Cytotoxicity 10 nmol/L (%)1	Inhibition1:1 (%)
CINGVCWTV	Wt1073	HCV NS3	1073-1081	9	54 640	0.994	86.74	-
CINGVSWTV	C1078S	HCV NS3	wt1073 (C6S)	9	1271	0.993	24.57	59.30
YINGVCWTV	C1073Y	HCV NS3	wt1073 (C1Y)	9	1627	0.999	45.21	44.70
CINGVCRTV	W1079R	HCV NS3	wt1073 (W7R)	9	297 500	0.991	21.02	54.50
YLVTRHADV	Wt1131	HCV NS3	1131-1139	9	4800	0.995	52.03	-
YLVMRHADV	T1134M	HCV NS3	wt1131 (T4M)	9	7200	0.991	44.53	36.75
YLVTRYADV	H1136Y	HCV NS3	wt1131 (H6Y)	9	7401	0.993	35.56	92.54
CLVTRHADV	Y1131C	HCV NS3	wt1131 (Y1C)	9	8345	0.995	2.01	67.89
YLVTRRADV	H1136R	HCV NS3	wt1131 (H6R)	9	27 700	0.993	14.53	47.46
LLCPAGHAV	Wt1169	HCV NS3	1169-1177	9	35 440	0.999	84.56	-
LLCPAGHTV	A1176T	HCV NS3	wt1169 (A8T)	9	27 640	0.998	27.75	61.42
LLRPAGHAV	C1171R	HCV NS3	wt1169 (C3R)	9	82 100	0.997	59.98	44.39
FLPSDFFPSV	HBV-Core	HBV Core	(18-27)[99]	10	481.1	0.999	NS	NS
SLYNTVATL	HIV-p17	HIV-1 p17	(77-85)[100]	9	4248	0.984	NS	NS
ILKEPVHGV	HIV-RT	HIV-1 Pol	(476-484)[47]	9	7082	0.997	NS	NS

¹Means of two experiments. Variant substitutions are expressed in underlined letters.