title: Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X-RNA.
abstract: The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.
authors.affiliation: Department of Biotechnology and Translational Research Center for Protein Function Control, Yonsei University, Seoul, Korea.
authors.forename: D G
authors.initials: DG
authors.lastname: Ahn
authors.forename: S B
authors.initials: SB
authors.lastname: Shim
authors.forename: J E
authors.initials: JE
authors.lastname: Moon
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
authors.forename: S J
authors.initials: SJ
authors.lastname: Kim
authors.forename: J W
authors.initials: JW
authors.lastname: Oh
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D055029:Inverted Repeat Sequences / Q000187:drug effects; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D016372:RNA, Antisense / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D020135:Peptide Nucleic Acids; D016372:RNA, Antisense; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase