Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.