In the core protein-coding region of hepatitis C virus (HCV), evidence exists for both phylogenetically conserved
RNA structures and a +1 alternative reading frame (ARF). To investigate its role in HCV
infection, we introduced four
stop codons into the ARF of a genotype 1a H77 molecular clone. The changes did not alter the core
protein sequence, but were predicted to disrupt
RNA secondary structures. An attenuated
infection was established after inoculation of the mutant HCV
RNA into an HCV naïve chimpanzee. The acute
infection was atypical with low peak
viremia, minimal
alanine aminotransferase elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop
codon. In cell culture, RNA replication of a genome with four
stop codons was severely impaired. In contrast, the revertant genome exhibited only a 5-fold reduction in replication. Genomes harboring only the first two
stop codons replicated to WT levels. Similarly, reversions at
stop codons 3 and 4, which improved replication, were selected with recombinant, infectious HCV in cell culture. We conclude that ARF-encoded
proteins initiating at the
polyprotein AUG are not essential for HCV replication in cell culture or in vivo. Rather, our results provide evidence for a functionally important
RNA element in the ARF region.