Table 3.
Analyses of the recovered J6_LSG∆33U full-length viruses
| HCV | Peak titer (mL, log10) |
Passage (day) | E2 | p7 | NS2 | NS2 | NS3 | NS3 | NS3 | NS3 | NS4A | NS4B | NS5A | NS5A | NS5B | |
| FFU | RNA | |||||||||||||||
| Nucleotide position | ||||||||||||||||
| Recombinant-specific | 1826 | 2667 | 2873 | 3119 | 3639 | 3953 | 3962 | 4742 | 5366 | 6132 | 6857 | 7375 | 9342 | |||
| H77 ref. (AF009606) | 1821 | 2656 | 2862 | 3108 | 3628 | 3942 | 3951 | 4731 | 5355 | 6121 | 6846 | 7376 | 9277 | |||
| J6CF nucleotide | T | T | C | A | C | C | G | T | G | A | G | A | A | |||
| J6CF with mutations | ||||||||||||||||
| J6_LSGΔ33U* | 3.8 | 6.9 | First (16) | C/T | C | T | A/g/c | G | ||||||||
| 4.2 | 7.8 | Second (9)† | C/t | T/c | C | T | A/g/c | G | ||||||||
| 4.3 | 8.1 | Fourth (7) | C/t | T/c | T/c | C | T | A/g/c | G | |||||||
| 3.9 | 7.5 | Fifth (12) ‡ | C/t | C/t | C/t | C | T | A/G/C | G | |||||||
| 4.8 | 7.6 | Sixth (13) | C | A/G | C/t | C | T | C/a | A/C | G | ||||||
| J6_LSGΔ33U mutations | ||||||||||||||||
| +F776S | 4.1 | 7.5 | First (12) | T/C§ | C | T | C | G | ||||||||
| +P1100L | 3.7 | 7.4 | First (12) | T/C | T | C | T | G | ||||||||
| +N1931S | 3.7 | 7.1 | First (12) | C/t | C | T | G | G | ||||||||
| +N1931T | 3.5 | 6.4 | First (12) | C | T | C | G | |||||||||
| +F776S/P1100L | 4.2 | 7.7 | First (12) | C | T | C | T | G | ||||||||
| +F776S/N1931S | 4.0 | 7.6 | First (12) | C | C | T | G | G | ||||||||
| +F776S/N1931T | 4.2 | 7.3 | First (12) | T/C | C | T | T | A | C | T | C | G | ||||
| +P1100L/N1931S | 4.5 | 7.7 | First (12) | T/C | T | C | T | G | G | |||||||
| +P1100L/N1931T | 4.4 | 7.7 | First (12) | T/C | T | C | T | C | G | |||||||
| +F776S/P1100L/N1931S | 4.3 | 8.1 | First (12) †‡ | C | T | C | T | G | A | G | ||||||
| +F776S/P1100L/N1931Ta | 4.3 | 7.7 | First (12) †‡ | C | T | C | T | C | G | |||||||
| Amino acid position | ||||||||||||||||
| Recombinant-specific | 496 | 776 | 845 | 927 | 1100 | 1205 | 1208 | 1468 | 1676 | 1931 | 2173 | 2345 | 3001 | |||
| H77 ref. (AF009606) | 494 | 772 | 841 | 923 | 1096 | 1201 | 1204 | 1464 | 1672 | 1927 | 2169 | 2345 | 2979 | |||
| Amino acid change | C–R | F–S | R–W | T–A | P–L | L–F | V–I | F–L | A–S | N–S/T | D–N | Q–H | D–G | |||
For each passaged J6_LSG∆33U (original transfection shown in Fig. 2B) and its derived viruses with additional mutations (transfection in Fig. 2C), a representative peak infectivity titer (FFU/mL) with associated HCV RNA titer (IU/mL) is shown. Viruses from the indicated passage day were sequenced for ORF. Coding changes are shown. Shading indicates engineered mutations. See legend of Table 1 for nucleotide annotations. a, This virus was named “J6cc” (for “J6 cell culture-derived”).
*In a separate transfection experiment, first passage J6_LSGΔ33U (103.8 FFU/mL) acquired mutations T2667C/t (amino acid F776S), T2876T/G (F846V), A3548A/G (T1070A), A6132A/g/c (N1931S/T).
†The 5′ UTR was determined by 5′ RACE; no change was identified in the 5′ UTR. However, the G inserted immediately before the 5′-terminal A for enhancing in vitro transcription was deleted, consistent with our previous observations in JFH1-based systems (30, 67).
‡The 3′ UTR was determined by 5′ RACE on HCV-negative-strand RNA; no consensus changes were found in variable and 3′-X regions. However, the polyU/UC tract was variable in length among sequenced clones, being on average four nucleotides (eight clones with one U insertion to six U deletions) and 10 nucleotides (eight clones with 3–23 U deletions) shorter than the original polyU/UC for F776S/P1100L/N1931S and F776S/P1100L/N1931T mutants, respectively. In addition, genome sequence analysis of first-passage J6_LSGFΔ33U, J6_LSGVmΔ33U, and J6_LSGFVmΔ33U (transfection in Fig. 2B) revealed that the engineered mutations were maintained; no additional coding mutations were found.
§Engineered C was partially reverted.