title: The Rhopalosiphum padi virus 5' internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system.
abstract: Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5' UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.
authors.affiliation: School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Royall
authors.affiliation: School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Woolaway
authors.affiliation: RiNA GmbH, Takustraße 3, 14195 Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Schacherl
authors.affiliation: RiNA GmbH, Takustraße 3, 14195 Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
authors.affiliation: BBSRC Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.affiliation: School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
authors.forename: Lisa O
authors.initials: LO
authors.lastname: Roberts
mesh_terms: D020121:5' Untranslated Regions / Q000502:physiology*; D000818:Animals; D001042:Aphids / Q000821:virology*; D016367:Baculoviridae / Q000235:genetics*; D002474:Cell-Free System; D012270:Ribosomes / Q000502:physiology*; D018411:Spodoptera
chemical_list: D020121:5' Untranslated Regions