Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus

Table 1 Oligonucleotides used to amplify the NS3 region*

	Primer sequences	Strand	Nucleotide positionin HCV genome
First round primers (5’-3’)	CCCCATCAC(A/G)TACTC(C/T)ACCTA	+	4 197
	ACA(C/T)GT(A/G)TT(G/A)CAGTC(T/G)ATCAC	_	4 681
Second round primers (5’-3’)	CGAGGATCCGTCCT(T/G)GACCAAGC(A/G)GAGAC	+	4 315
	GCAACTGCAGCT(G/A)G(T/A)(C/T)GG(G/T)ATGAC(A/G)GACAC	_	4 597

*Degenerate primers were used to cover the variable HCV genomes.

Table 2 Assigned accession number for each nucleotide se-quence of three patients

Time point measured	Patient W	Patient C	Patient Z
0 mo	AF051270	AF051261	AF051270
	AF051270	AF051261	AF051270
	AF051271	AF051261	AF051270
	AF051272	AF051260	AF051270
	AF051273	AF051259	AF051270
32th mo	NA	AF051254	AF051265
		AF051255	AF051266
		AF051256	AF051267
		AF051257	AF051268
		AF051258	AF051269
60th mo	NA	AF051253	AF051262
		AF051253	AF051262
		AF051253	AF051263
		AF051253	AF051263
		AF051253	AF051264

NA: not applicable as the sample was PCR negative for HCV RNA.

Table 3 Predicted CTL epitopes by MHC binding peptide motifs while incorporating persistent substitutions

Patient	HLA sutype	MHC binding peptide motif[38,39]	Genome position	Predicted epitope	Time point measured
C	A11	X[MLIVSATGNCDF]XXXXXX[KRHY]	1349	ATPPGSITVPH*	0 mo
				ATPPGSVTVPH*	32th, 60th mo
			1378	KAIPIEAIR	0 mo
				KAIPIEAIK	32th, 60th mo
Z	A11	X[MLIVSATGNCDF]XXXXXX[KRHY]	1381	PIEAIKGGR	0 mo
				PIEAIRGGR	32th, 60th mo
			1347	ATATPPGSV	0 mo
				ATATPPGSI	32th, 60th mo
	A 2	X[LMIVAT]XXXXXX[LVIAMT]	1348	TATPPGSVT	0 mo
				TATPPGSIT	32th, 60th mo
			1349	ATPPGSVTV	0 mo
				ATPPGSITV	32th, 60th mo

*These two peptides fitted the MHC binding motif because proline and glycine residues allowed flexibility which made the peptides accommodate to the motif by kinking in the peptide backbone[40].