title: Evidence for a functional RNA element in the hepatitis C virus core gene.
abstract: 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.
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10021, USA.
authors.forename: Laura K
authors.initials: LK
authors.lastname: McMullan
authors.forename: Arash
authors.initials: A
authors.lastname: Grakoui
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Evans
authors.forename: Kathleen
authors.initials: K
authors.lastname: Mihalik
authors.forename: Montserrat
authors.initials: M
authors.lastname: Puig
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Branch
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Feinstone
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D019143:Evolution, Molecular; D017353:Gene Deletion; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D002679:Pan troglodytes / Q000821:virology; D010641:Phenotype; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D014758:Viral Core Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus