title: Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.
abstract: Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element.
authors: Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-73100 Chania, Crete, Greece; Department of Agricultural Botany, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypt.
authors: Toba A M
authors: TA
authors: Osman
authors: Department of Molecular Genetics, Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Electronic address: olsthoor@chem.leidenuniv.nl.
authors: René C L
authors: RC
authors: Olsthoorn
authors: Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepion, GR-73100 Chania, Crete, Greece. Electronic address: livieratos@maich.gr.
authors: Ioannis C
authors: IC
authors: Livieratos
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010935:Plant Diseases / Q000821:virology; D017863:Potexvirus / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral
keywords: 3′-Untranslated region; Pepino mosaic virus; Potexviruses; RNA replication; S. lycopersicum