Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5 ' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new viru s variants containing identical substitutions at three sites within the vir al 5' nontranslated RNA (5'NTR): G(107)-->A, C-204-->A, and G(243)-->A (N, Nakajima, hi. Hijikata, II, 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 mor e efficient capindependent translation, since these nucleotide substitution s reside within the viral internal ribosome entry site (IRES), To test this hypothesis, we examined the translation of dicistronic RNAs containing ups tream and downstream reporter sequences (Renilla and firefly luciferases, r espectively) separated by IRES sequences containing different combinations of these substitutions, The activity of the IRES was assessed by determinin g the relative firefly and Renilla luciferase activities expressed in trans fected cells. Compared with the IRES present in the dominant H77 quasispeci es, 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 contras t, 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 increas ed translational activity in the lymphoblastoid cells. The 2- to 2.5-fold i ncrease in translation observed with the modified IRES sequence may facilit ate the replication of HCV: possibly accounting for differences in quasispe cies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.