Ebola Virus RNA Editing Depends on the Primary Editing Site Sequence and an Upstream Secondary Structure

Abstract

Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

Subtype-specific structural constraints in the evolution of influenza A virus hemagglutinin genes

“…The location of insertion sites in the hairpin loops is suggestive for a recombination mechanism with hairpin-mediated template switching, resembling the copy choice mechanism in retroviruses60. On the other hand, transcriptional slippage of a DNA-dependent RNA polymerase was shown to be stimulated by nascent RNA hairpins located near the slippage site61, and a similar topology was suggested to explain RNA editing by the RNA-dependent RNA polymerase of Ebola virus62. Stabilization of the H5 HA structure in the gs/GD/96 lineage years after the slippage has occurred and the covariation in the homologous fold in human H3N2 viruses (Fig.…”

Filovirus proteins for antiviral drug discovery: A structure/function analysis of surface glycoproteins and virus entry

“…The RNA editing of ebolaviruses involves a slippage region composed of seven consecutive template uridines where viral polymerase stuttering results in a frameshift in the middle of the GP sequence (Volchkov et al, 1995;Sanchez et al, 1996;Mehedi et al, 2011). Indeed, this editing mechanism has been recently shown to be regulated by neighboring sequences of the uridine template, probably in synergy with VP30 as a transacting factor (Mehedi et al, 2013). Thus, the ebolavirus GP gene is able to generate three different mRNAs coding for protein precursors pre-sGP, pre-GP and ssGP in a ratio of approximately 14:5:1, respectively, albeit the exact ratio is cell type dependent ( Fig.…”

The emerging role of RNA editing in plasticity

“…Recent evidence shows that many viruses, upon entering a cell, do in fact undergo RNA editing (see Tomaselli et al, 2014 for an excellent review). The list includes some notorious members such as HIV-1, the Epstein-Barr virus, herpes virus 8, dengue virus, hepatitis C virus, influenza A virus and the measles virus (Mehedi et al, 2013). Interestingly, the relationship between RNA editing and infectivity is not always clear-cut: in some cases, editing appears to combat it and in others it promotes it (Pfaller et al, 2011;Samuel, 2011).…”