In vivo footprint of a picornavirus internal ribosome entry site reveals differences in accessibility to specific RNA structural elements.
Internal ribosome entry site (IRES) elements were described in picornaviruses as an essential region of the viral RNA. Understanding of IRES function requires a detailed knowledge of each step involved in the internal initiation process, from RNA folding and IRES-protein interaction to ribosome recruitment. Thus, deciphering IRES accessibility to external agents due to RNA structural features, as well as RNA-protein protection within living cells, is of primary importance. In this study, two chemical reagents, dimethylsulfate (DMS) and aminomethylpsoralen, have been used to footprint the entire IRES of foot-and-mouth disease virus (FMDV) in living cells; these reagents enter the cell membrane and interact with nucleic acids in a structure-dependent manner. For FMDV, as in other picornaviruses, viral infection is dependent on the correct function of the IRES; therefore, the IRES region itself constitutes a useful target of antiviral drugs. Here, the in vivo footprint of a picornavirus IRES element in the context of a biologically active mRNA is shown for the first time. The accessibility of unpaired adenosine and cytosine nucleotides in the entire FMDV IRES was first obtained in vitro by DMS probing; subsequently, this information was used to interpret the footprint data obtained in vivo for the mRNA encompassing the IRES element in the intercistronic space. The results of DMS accessibility and UV-psoralen cross-linking studies in the competitive cellular environment provided evidence for differences in RNA structure from data obtained in vitro, and provided essential information to identify appropriate targets within the FMDV IRES aimed at combating this important pathogen.
Fernández-Miragall
Olga
O
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Martínez-Salas
Encarnación
E
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D029544:Foot-and-Mouth Disease Virus / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology
D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D012367:RNA, Viral; D013463:Sulfuric Acid Esters; C018822:aminomethyltrioxsalen; C007482:dimethyl sulfate; D014307:Trioxsalen