Mechanisms and regulation of Ebola virus RNA synthesis
Research areas: Molecular Virology, RNA Biochemistry
Ebola virus (EBOV) RNA synthesis is a highly regulated process that involves the interplay of cis-acting elements in the viral genome with viral RNA binding proteins. While replication of the EBOV genome is driven by the viral polymerase L, its cofactor VP35 and the nucleoprotein NP, viral transcription additionally requires VP30, an EBOV-specific transcription factor whose activity is regulated via phosphorylation. In the second funding period, we were able to unveil the mechanism of VP30’s dynamic phosphorylation, identifying the serine/arginine-rich protein kinase 1 (SRPK1), and demonstrating that NP acts as recruitment factor for subunit B56 of the protein phosphatase 2A to dephosphorylate VP30 if simultaneously bound to NP. Based on the study of mutant minigenomes (MGs), we further demonstrated that hexamer phasing in the 3’-leader promoter is not only key to replication, but also to efficient transcription initiation. The genomic EBOV replication promotor is bipartite, consisting of promoter elements 1 (PE1) and 2 (PE2), that are separated by the transcription start sequence/site (TSS) for the first gene (NP) and a spacer sequence. We found that spacer extensions of up to ~54 nt are tolerated, while minor incremental stabilization of hairpin (HP) structures at the TSS rapidly abolished viral polymerase activity. Balanced viral transcription and replication can still occur when any potential RNA structure formation at the TSS is eliminated, and transcription remains VP30-dependent also in this case. In addition, the HP structure at the TSS of the native 3’-leader was demonstrated to be optimized for tight regulation by VP30 and to enable the switch from transcription to replication when VP30 is not part of the polymerase complex. Increasing stability of the HP impaired viral transcription. Short leader RNAs of 60-80 nt length are synthesized from the EBOV genome. Like replicative antigenomic RNA, leader RNAs are initiated opposite to the penultimate C residue at the genomic 3’-end and their amount is reduced in the presence of VP30, suggesting that leader RNAs are products of abortive antigenome synthesis.
For the next funding period, we would like to intensify our efforts regarding the interplay between cis-acting regulatory elements in the EBOV genome and viral proteins that contribute to RNA synthesis. We will address the following work packages: (i) Advanced investigations on the mechanistic role of structural elements and hexamer phasing in the 3’-leader promotor and at internal TSS by mutational analysis in the context of mono-, bi-, or tetracistronic MGs. Selected mutations will be introduced into the EBOV genome and recombinant viruses will be generated and characterized. (ii) Characterization of constraints for leader RNA synthesis by engineering the 3’-leader in terms of length, sequence and structure. (iii) To investigate the accessibility of the encapsidated RNA genome by the polymerase complex, we want to analyze protein-RNA interactions by iCLIP or PAR-CLIP, either using reconstituted complexes of recombinantly expressed polymerase complex and leader/trailer RNAs or nucleocapsids derived from tetracistronic MGs. (iv) We aim at establishing an in vitro transcription/replication system based on the expression of the viral polymerase complex in insect cells. Moreover, we will investigate (v) the functional role of VP35 helicase activity and (vi) will search for host factors interacting with the viral polymerase complex.
The herein proposed work packages will contribute to a deeper understanding of regulatory mechanisms involved in EBOV RNA synthesis.
Project-related publications of the investigators:
- Bach S, Demper JC, Biedenkopf N, Becker S, Hartmann RK. 2021. RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner. RNA Biol 18:523-536.
- Takamatsu Y, Krahling V, Kolesnikova L, Halwe S, Lier C, Baumeister S, Noda T, Biedenkopf N, Becker S. 2020. Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription. mBio 11.
- Bach S, Demper JC, Grunweller A, Becker S, Biedenkopf N, Hartmann RK. 2020. Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter. J Virol doi:10.1128/JVI.02215-20.
- Bach S, Biedenkopf N, Grunweller A, Becker S, Hartmann RK. 2020. Hexamer phasing governs transcription initiation in the 3′-leader of Ebola virus. RNA 26:439-453.
- Kruse T, Biedenkopf N, Hertz EPT, Dietzel E, Stalmann G, Lopez-Mendez B, Davey NE, Nilsson J, Becker S. 2018. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30. Mol Cell 69:136-145 e6.
- Lier C, Becker S, Biedenkopf N. 2017. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies. Virology 512:39-47.
- Biedenkopf N, Lange-Grunweller K, Schulte FW, Weisser A, Muller C, Becker D, Becker S, Hartmann RK, Grunweller A. 2017. The natural compound silvestrol is a potent inhibitor of Ebola virus replication. Antiviral Res 137:76-81.
- Biedenkopf N, Hoenen T. 2017. Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Viruslike Particle Assays. Methods Mol Biol 1628:119-131.
- Schlereth J, Grunweller A, Biedenkopf N, Becker S, Hartmann RK. 2016. RNA binding specificity of Ebola virus transcription factor VP30. RNA Biol 13:783-98.
- Biedenkopf N, Schlereth J, Grunweller A, Becker S, Hartmann RK. 2016. RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription. J Virol 90:7481-7496.