Ebola Virus VP30-Mediated Transcription Is Regulated by RNA Secondary Structure Formation

Abstract

The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.Ebola virus (EBOV) and the closely related Marburg virus (MBGV) are the only members of the family Filoviridae, which belongs to the order Mononegavirales.

Filovirus Replication and Transcription

“…These mRNAs are capped and polyadenylated [51,52]. It is believed that for all NNS RNA viruses, the polymerase gains access to the viral genes through a single polymerase binding site at the 3 end of the genome.…”

“…When the formation of the RNA secondary structure was impaired, transcription took place independently of VP30. Although each EBOV-specific transcription start signal is predicted to form a RNA hairpin loop, VP30 is not required for transcription reinitiation at the internally located gene start sites [51]. A hypothetical model of VP30-dependent transcription is depicted in Figure 2.…”

“…It may be speculated that VP30 resolves the RNA structure or directs additional cofactors to the folded RNA. (3) Elongation and transcription reinitiation: transcription elongation and reinitiation at the following genes takes place independently of VP30, although each transcription start signal is involved in the formation of an RNA stem-loop structure [51]. Please note that the transcription start signal of MARV NP mRNA may also be involved in secondary structure formation.…”

Predictive and comparative analysis of Ebolavirus proteins

“…NP encapsidates the genome and protects it from nucleases. 17,18 VP30 is a transcription anti-terminator 19,20 and regulates the switch between transcription and replication. 21,22 VP35 acts as a cofactor of the polymerase, 23,24 and VP40 may also play a role in genome replication and transcription.…”

Structure of theReston ebolavirusVP30 C-terminal domain

“…VP30 appears unique to the filoviruses and is essential for rescuing recombinant ebolavirus (Enterlein et al, 2006;Theriault et al, 2004). VP30 allows the polymerase to read beyond a cis-RNA element in the NP mRNA 5 0 untranslated region (Weik et al, 2002) and to re-initiate transcription at gene junctions (Martínez et al, 2008). Furthermore, VP30 phosphorylation modulates the composition and function of the RNA synthesis machinery (Biedenkopf et al, 2013;Martinez et al, 2011).…”