title: Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication.
abstract: Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5'- and 3'-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3'-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is reduced by ∼60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation.
authors.affiliation: Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, D C 20057, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Manzano
authors.forename: Erin D
authors.initials: ED
authors.lastname: Reichert
authors.forename: Stephanie
authors.initials: S
authors.lastname: Polo
authors.forename: Barry
authors.initials: B
authors.lastname: Falgout
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kasprzak
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Shapiro
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Padmanabhan
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000465:Algorithms; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D003716:Dengue Virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D049410:Luciferases, Renilla / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D012093:Replicon / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D049410:Luciferases, Renilla