title: An upstream open reading frame modulates ebola virus polymerase translation and virus replication.
abstract: Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5' and 3' untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5'-UTRs lack internal ribosomal entry site function. However, the 5'-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5'-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5'-UTR. The L 5'-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5'-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a "weak" Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10-100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target.
authors: Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America.
authors: Reed S
authors: RS
authors: Shabman
authors: Thomas
authors: T
authors: Hoenen
authors: Allison
authors: A
authors: Groseth
authors: Omar
authors: O
authors: Jabado
authors: Jennifer M
authors: JM
authors: Binning
authors: Gaya K
authors: GK
authors: Amarasinghe
authors: Heinz
authors: H
authors: Feldmann
authors: Christopher F
authors: CF
authors: Basler
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D029043:Ebolavirus / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D019284:Thapsigargin / Q000494:pharmacology; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D014764:Viral Proteins; D019284:Thapsigargin; D012321:DNA-Directed RNA Polymerases
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