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.

Author Summary

Filoviruses (Ebola and Marburg viruses) are emerging zoonotic pathogens that cause lethal hemorrhagic fever in humans and have the potential to be employed as bioterrorism agents. Currently, approved therapeutics to treat filovirus infections are not available and new treatment strategies could be facilitated by improved mechanistic insight into the virus replication cycle. Compared to other related viruses, filovirus messenger RNAs have unusually long 5′ untranslated regions (UTRs) with undefined functions. In the Zaire ebolavirus (EBOV) genome, four of its seven messenger RNAs have 5′-UTRs with a small upstream open reading frame (uORF). We found that a uORF present in the EBOV polymerase (L) 5′-UTR suppresses L protein production and established a reporter assay to demonstrate that this uORF maintains L translation following the induction of an innate immune response; a phenomenon observed with several uORF-containing cellular messenger RNAs. The presence of the uORF is important for optimal virus replication, because a mutant virus lacking the upstream reading frame replicates less efficiently than a wildtype virus, an attenuation which is more pronounced following the induction of cellular stress. These studies define a novel mechanism by which filovirus upstream open reading frames modulate virus protein translation in the face of an innate immune response and highlight their importance in filovirus replication.