INVOLVEMENT OF THE 5'-PROXIMAL CODING SEQUENCES OF HEPATITIS-C VIRUS WITH INTERNAL INITIATION OF VIRAL TRANSLATION

The 5' nontranslated region (NTR) of hepatitis C virus (HCV) consists of 341 nucleotides (nt). This region comprises the majority of the int ernal ribosome entry site (IRES) which controls the efficiency of vira l translation. Previous studies of the 3' boundary of the HCV IRES yie lded conflicting data regarding the involvement of viral coding sequen ces in IRES activity. We therefore studied the functional significance of the 5' proximal coding sequences of the HCV core gene on IRES acti vity. We constructed monocistronic and bicistronic DNAs that contained either a chloramphenicol acetyl transferase (CAT) gene or a luciferas e (Luc) gene as the reporter. Results from both in vitro and in vivo e xperiments indicated that the optimal IRES ranged within nt 1-371. Fur ther mutational analyses of sequences surrounding the initiation codon revealed that primary sequences downstream of the AUG initiator rathe r than the secondary structure are important in regulating optimal IRE S function. We are also able to demonstrate that a non-AUG codon could be used to initiate the synthesis of a reporter protein, albeit with lower efficiency. These findings bear important implications for the H CV IRES secondary structures. (C) 1998 Academic Press.