Genetic analysis of the internal ribosomal entry site of hepatitis C virus

Hepatitis C Virus (HCV), the causing agent of Hepatitis C is an enveloped positive-strand RNA virus that belongs to the family of Flaviviridae . Translation of the open reading frame (ORF) for its polyprotein is directed by an Internal Ribosomal Entry Site (IRES) element at the 5 ′ end of the genome which encompasses most of the 5′ Non-translated region (5′NTR) and part of the core encoding sequences. Genetic and functional analyses of the HCV IRES element have been impeded by the lack of a robust and efficient tissue culture system and the limited host range of the virus. In an effort to clearly understand the structure and function of the HCV IRES, a poliovirus/HCV chimera P/H710-d40 was constructed in which the poliovirus IRES element was replaced by the HCV IRES. The fusion protein between the core of HCV and the P1 of poliovirus was cleaved by the viral 2Apro.; In order to determine whether the truncated HCV core protein is required for the HCV IRES function, a new chimeric virus P/H710-SH2-2A with a frameshift in the HCV encoding region, was constructed. Plaque assays and in vitro translation results clearly suggest that the RNA sequences, rather than the HCV core protein itself, is required for the HCV IRES to function normally in the chimera.; A point mutation in the HCV specific sequence of P/H710-d17-2A was discovered to be responsible for the enhanced plaque phenotype of the chimeric virus after one passage on HeLa cells. Interestingly, the release from growth restriction was due to the formation of an alternative stem-loop structure in the HCV specific sequence that was possibly a result of the mutation. The original attenuation of the progenitor strain resulted from base-pair interaction between the HCV specific sequence and the PV cloverleaf-like structure, the latter being involved in the replication of the PV genome. Our hypothesis was confirmed by the improved plaque phenotype of the chimeric virus as a result of either the introduction of the mutation back into the parental plasmid or the deletion of those HCV-specific sequences that were presumably involved in the interference.; The 5′ boundary of the HCV IRES was confirmed to be around nt 40, the bottom of the Domain II. A chimera, P/H710-d40, which can grow to the level of wildtype PV1 (mahoney), was created. A new domain II structure was proposed based our genetic analyses.