Sequence requirements for the development of a chimeric HCV replicon system

The hepatitis C virus (HCV) 3′nontranslated region (3′NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3′NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3′NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3′NTR supported stable replication suggesting that the interaction between the NS proteins and the 3′NTR may represent a critical determinant. Lastly, the type 1a 3′NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3′NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3′NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3′NTR sequences from relevant clinical samples.