Translational control of the hepatitis C virus internal ribosomal entry site (IRES)

Hepatitis C is a major public health threat causing 40000 new infections in the United States every year. The Centers of Disease Control and Prevention recently estimated 8000--10000 annual deaths by Hepatitis C. No vaccine is available and the combination therapy of pegylated interferon alpha-2b and ribavirin still has a low sustained response rate in HCV patients.; Hepatitis C virus (HCV) is an enveloped virus with a positive sense, single-stranded RNA genome belonging to the family Flaviviridae. HCV like pestiviruses in this family has an internal ribosomal entry site (IRES) in the 5' nontranslated region (NTR). A sequence alignment study of HCV and pestiviruses showed that four highly conserved regions are in domain II of the IRESes; a five nucleotide sequence in the apical loop, two regions in the loop E motif and three base pairs in domain IIa. Several mutations in the sequences of all four conserved regions caused a severe translation phenotype and abolished replication in the context of the P/H chimera and subgenomic HCV replicon. The mutations in the apical loop, loop E motif, and the three base pairs most likely affected the interaction between domain II and the 40S ribosome and the subsequent 80S complex assembly for translation initiation.; The 3' end of the HCV IRES extends downstream of the initiating AUG codon into an adenosine(A)-rich region in the core-coding sequence. Deletion and substitution of this A-rich region influenced the HCV IRES activity, as assayed by replication assays in the context of the P/H chimera and the subgenomic HCV replicon. The minimum requirement of core length for efficient translation initiation seems to be around the nucleotide 356 in the core coding sequence. However, in order to allow efficient replication of the P/H chimera and the subgenomic HCV replicon, the core sequence up to nt 374 is needed to allow sufficient translation. The A-rich region is required for the binding of a cellular factor called NSAP1 which enhanced HCV IRES mediated translation. It is likely that this translation factor facilitates the 48S ribosome complex formation by binding near the initiation AUG codon.; We conclude that the four conserved regions in domain II of the HCV IRES have a significant effect on HCV IRES mediated translation and are most likely important for the 48S complex formation in the 80S ribosome assembly pathway. Immediately downstream of the initiating AUG codon exist an A-rich region which is required for efficient HCV IRES mediated translation and is important for the replication of the RNA genome of HCV.