Table 4.
Analyses of recovered J8 full-length viruses
| HCV | Peak titer (mL, log10) |
Experiment (day) * | p7 | NS2 | NS2 | NS3 | NS3 | NS3 | NS3 | NS4A | NS4B | NS5A | NS5B | NS5B | NS5B | NS5B | |
| FFU | RNA (IU) | ||||||||||||||||
| Nucleotide position | |||||||||||||||||
| Recombinant-specific | 2656 | 2931 | 3115 | 3963 | 4209 | 4413 | 4743 | 5367 | 6243 | 7129 | 7657 | 9031 | 9106 | 9343 | |||
| H77 ref. (AF009606) | 2644 | 2919 | 3103 | 3951 | 4297 | 4401 | 4731 | 5355 | 6231 | 7129 | 7591 | 8965 | 9040 | 9277 | |||
| J8CF nucleotide | T | T | T | G | A | A | T | G | A | A | T | A | A | A | |||
| J8CF with mutations | |||||||||||||||||
| J8_LSGa† | 3.3 | — | Transfection (63/65) | G | C | A | G | C | T | G | T | G | G | ||||
| 3.6 | 6.9 | First passage (14) | T/G | C | A | A/g | C | T | G | T | G | G | G | ||||
| J8_LSGF772C/I1968V/H2922R | 3.6 | 7.0 | First passage (25) | G | G | C | T | G | G | G | |||||||
| +W864R | 3.6 | 7.2 | First passage (13) | G | C | A/G | C | T | G | G | G | ||||||
| +A1208T | 3.1 | 6.1 | First passage (16) | G | C | A | a/G | C | T | G | C | G | G | ||||
| +W864R/A1208T | 3.4 | 7.0 | First passage (12) | G | C | A | C | T | G | G | G | ||||||
| +W864R/E2263V | 3.3 | 7.2 | first passage (13) | G | C | A/G | C | T | G | T | G | G | |||||
| +A1208T/E2263V | 3.2 | 7.1 | First passage (13) | G | A | C | T | G | T | G | G | ||||||
| +W864R/A1208T/E2263V‡ | 3.3 | 7.2 | First passage (12) | G | C | A | C | T | G | T | G | G | |||||
| Amino acid position | |||||||||||||||||
| Recombinant-specific | 772 | 864 | 925 | 1208 | 1290 | 1358 | 1468 | 1676 | 1968 | 2263 | 2439 | 2897 | 2922 | 3001 | |||
| H77 ref. (AF009606) | 768 | 860 | 921 | 1204 | 1319 | 1354 | 1464 | 1672 | 1964 | 2263 | 2417 | 2875 | 2900 | 2979 | |||
| Amino acid change | F–C | W–R | V–A | A–T | T–A | T–A | F–L | A–S | I–V | E–V | V-A | N–S | H–R | D–G | |||
For each passaged J8 virus a representative peak infectivity titer (FFU/mL) with associated HCV RNA titer (IU/mL) is shown. Viruses of transfection- and passage-derived J8_LSGa (J8 with F1468L/A1676S/D3001G, one of two transfections) and first-passage J8_LSG with additional mutations were sequenced for ORF. Nucleotide and amino acid positions of the specific recombinant with complete coding changes are listed; noncoding and quasispecies mutations are shown in Table S4. Shading indicates engineered mutations: J6-derived mutations are indicated by dark gray shading and J8-derived mutations by light gray shading. See Table 1 legend for nucleotide annotations. –, RNA titer not determined.
*Viruses from indicated experiment were sequenced.
†The 5′ UTR was determined, and the 5′-terminal G was changed to A, consistent with our previous observations in JFH1-based systems (30, 67). The 3′ UTR of third-passage viruses was determined; no consensus changes were observed in variable and 3′-X regions. However, the polyU/UC tract had 4–23 (on average 14 ) U deletions among four sequenced clones. Second-passage J8_LSGa sequence is shown in Table S4.
‡This virus was named “J8cc” (for “J8 cell culture-derived”); in another experiment, no mutation was found. In addition, sequence analysis of transfection- and first passage-derived J8_LSGb showed that this virus also acquired mutations T2655T/G (amino acid F772V), A6243A/G (I1968V), and A9106G (H2922R), representing three of the six coding mutations found in J8_LSGa transfection and first-passage viruses.