title: A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.
abstract: A wide range of RNA viruses use programmed -1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed -1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
authors.forename: Ewan P
authors.initials: EP
authors.lastname: Plant
authors.forename: Gabriela C
authors.initials: GC
authors.lastname: Pérez-Alvarado
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Jacobs
authors.forename: Bani
authors.initials: B
authors.lastname: Mukhopadhyay
authors.forename: Mirko
authors.initials: M
authors.lastname: Hennig
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Dinman
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D014709:Vero Cells
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral