title: Ribosomal binding to the internal ribosomal entry site of classical swine fever virus.
abstract: Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.
authors.affiliation: Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, 11203, USA.
authors.forename: V G
authors.initials: VG
authors.lastname: Kolupaeva
authors.forename: T V
authors.initials: TV
authors.lastname: Pestova
authors.forename: C U
authors.initials: CU
authors.lastname: Hellen
mesh_terms: D020121:5' Untranslated Regions / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D006692:Classical Swine Fever Virus / Q000235:genetics*; D003062:Codon / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
chemical_list: D020121:5' Untranslated Regions; D003062:Codon; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012367:RNA, Viral