title: Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication.
abstract: The programmed -1 ribosomal frameshifting (-1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a -1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the - PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the -1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the -1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that -1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking -1 PRF signals.
authors.affiliation: Department of Biotechnology and Translational Research Center for Protein Function Control, Yonsei University, Seoul, Republic of Korea.
authors.forename: Dae-Gyun
authors.initials: DG
authors.lastname: Ahn
authors.forename: Wooseong
authors.initials: W
authors.lastname: Lee
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Choi
authors.forename: Seong-Jun
authors.initials: SJ
authors.lastname: Kim
authors.forename: Ewan P
authors.initials: EP
authors.lastname: Plant
authors.forename: Fernando
authors.initials: F
authors.lastname: Almazán
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Taylor
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Oh
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000235:genetics; D006224:Cricetinae; D024202:Electrophoretic Mobility Shift Assay; D018965:Frameshifting, Ribosomal; D005807:Genes, Overlapping / Q000235:genetics; D017930:Genes, Reporter; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis; D049409:Luciferases, Firefly; D020320:Oligoribonucleotides, Antisense / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D057846:Cell-Penetrating Peptides; D007370:Interferon Type I; D020320:Oligoribonucleotides, Antisense; D012367:RNA, Viral; D049409:Luciferases, Firefly; D012324:RNA-Dependent RNA Polymerase