Table 2.

Sequence analysis of J6 full-length genome with mutations recovered from long-term cultures

HCV	Experiment (day)	E1	p7	NS3	NS4A	NS5A	NS5B	NS5B	NS5B	NS5B	NS5B	VR
Nucleotide position
Recombinant-specific		1325	2667	4742	5366	7656	9086	9102	9216	9342	9348	9458
H77 ref. (AF009606)		1326	2656	4731	5355	7591	9021	9037	9151	9277	9283	9397
J6CF nucleotide		A	T	T	G	T	C	C	G	A	A	C
J6CF with mutations
J6_7mVmΔ33U*	Transfection (27)		C	C	T		A	A	A	G/a	T	G
J6_4mVmΔ33U	Fourth passage (5)†	T/a	C	C	T	C/T				G	T	G
J6_3mVmΔ33U	First passage (18)			C	T					G	T	G
Amino acid position
Recombinant-specific		329	776	1468	1676	2439	2916	2921	2959	3001	3003
H77 ref. (AF009606)		329	772	1464	1672	2417	2894	2899	2937	2979	2981
Amino acid change		T–S	F–S	F–L	A–S	V–A	L–M	P–H	R–K	D–G	Y–F

Transfection- or first passage-recovered viable J6 recombinants (transfection in Fig. 2A) with different engineered mutations were subjected to ORF (coding changes are shown) and 3′ UTR sequence analysis. See legend of Table 1 for nucleotide annotations. Shading indicates engineered mutations. Mutations introduced into J6CF: 7m, F776S/F1468L/A1676S/L2916M/P2921H/R2959K/Y3003F; 4m, F776S/F1468L/A1676S/Y3003F; 3m, F1468L/A1676S/Y3003F; Vm, nucleotide change C9458G in 3′ UTR variable region (VR); Δ33U, 33 U in polyU tract deleted. The 3′ UTR of recovered virus was determined by 5′ RACE on an HCV-negative-strand RNA. No consensus changes were observed in the variable and 3′-X regions. The length of the polyU/UC tract was found to be variable among analyzed clones: Recombinants with 7m, 4m, and 3m mutations were, on average, six nucleotides (seven clones with three U insertions to 15 U deletions), eight nucleotides (six clones with seven U insertions to 19 U deletions) shorter, and four nucleotides (seven clones with 19 U insertions to six U deletions) longer than the original polyU/UC tract, respectively.

*Six U were deleted in the polyU/UC tract of the final construct plasmid.

^†The first-, second-, and third-passage viruses were not sequenced.