title: Translation elongation factor-1alpha, La, and PTB interact with the 3' untranslated region of dengue 4 virus RNA.
abstract: The 384-nt long 3' untranslated region (3'UTR) of dengue 4 virus (DEN4) is not polyadenylated, but contains the adjacent thermodynamically stable conserved short and long stem-loop structures (L-SL) and the conserved sequences CS1 and CS2. The latter are duplicated (CS2A and CS2B) in DEN4. Dengue virus replication, like that of other RNA viruses, might involve the cis-elements located within the 3'UTR and the trans-acting factors that could interact with the viral replicase to function as a replicase complex. The identification and characterization of viral and cellular proteins involved in the interaction with the 3'UTR of dengue virus will help us to understand the cellular requirements for viral replication. To determine these requirements, mobility shift and cross-linking assays were performed with uninfected and DEN4-infected C6/36 cell extracts as well as the different segments of the 3'UTR. Our results revealed that RNA-protein complexes were formed with the RNAs which involved the domains CS2A, CS2B, CS1, and L-SL. The minimum RNA sequence that was able to form specific and stable complexes with cellular proteins was the CS1-L-SL region. Using UV-induced cross-linking we identified eight proteins with molecular weights of 34, 39, 51, 52, 56, 62, 72, and 84 kDa that bound to the complete 3'UTR. The translation elongation factor-1alpha (EF-1alpha) bound to the complete 3'UTR and to the CS1-L-SL region. In addition, the recombinant GST-human La autoantigen bound to the 3'UTR and to the CS1-L-SL region as demonstrated by mobility shift and cross-linking assays. Although different antibodies against PTB were unable to react with any of the cellular proteins from C6/36, the recombinant His-PTB protein did bind to the complete 3'UTR and to the CS1-L-SL region. The specific binding of La and PTB to the sequences considered essential for viral RNA replication may suggest that these proteins could function as RNA chaperones to maintain RNA structure in a conformation that favors viral replication, while EF-1alpha may function as an RNA helicase.
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City 07360, Mexico.
authors.forename: Mónica
authors.initials: M
authors.lastname: De Nova-Ocampo
authors.forename: Nicolás
authors.initials: N
authors.lastname: Villegas-Sepúlveda
authors.forename: Rosa M
authors.initials: RM
authors.lastname: del Angel
mesh_terms: D020413:3' Untranslated Regions / Q000378:metabolism*; D000330:Aedes / Q000821:virology; D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003716:Dengue Virus / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D051379:Mice; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D000097585:SS-B Antigen
chemical_list: D020413:3' Untranslated Regions; D001324:Autoantigens; D003432:Cross-Linking Reagents; D020648:Peptide Elongation Factor 1; D038941:Polypyrimidine Tract-Binding Protein; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen