TABLE 1.
Mutations selected by serial passage of HCV E2 mutant poolsa
| Position(s) ofb: |
Sites identified by colony sequencing |
||
|---|---|---|---|
| Mutation in E2 of P0 virus | P3 putative adaptive mutations identified by consensus sequencing | Amino acid substitution(s) (% of colonies bearing substitution) | Other mutation(s) |
| 404–413 | Q412 | R (33), A (11), D (3) | I411, L413 |
| 414–423 | T416 | R (16), K (10), N (5), V (5), A (5), G (3), D (3) | I414, N415, N417, G418 |
| 445–455 | S449 | No datac | |
| 562–573 | T563 | V (33), L (33), I (2) | T565 |
| 574–583 | A579 | R (17), L (10), M (7) | |
| 618–628 | L619 | T (34), E (9), I (2) | W620 |
| V626 | S (15), T (2), Q (2), E (2), R (2) | ||
| 629–638 | K632 | T (62) | I630 |
| 639–649 | L644 | I (32), V (14) | A646 |
a
Each pool of E2 mutants was passaged three times in tissue culture. Subsequently, viral RNA was harvested, cDNA generated, and E1-E2 amplified by PCR, and products were Sanger sequenced across the sites of engineered mutations within E2. Eight of the 32 pools had changes to the E2 consensus sequences 404 to 413, 414 to 423, 445 to 455, 563 to 573, 574 to 583, 618 to 628, 629 to 638, and 639 to 649. The E1-E2 fragments from these samples were cloned, and plasmids from individual colonies were sequenced to determine the specific amino acid substitutions selected from each pool.
b
P0, passage 0; P3, passage 3.
c
Consensus sequencing suggested only a proline substitution.