Intracellular organization of Ebola virus RNA synthesis; formation, maturation and transport of viral RNP

Research area: Molecular Virology

Replication of viral genomic RNA results in the formation of progeny genomes that need to be packaged and transported to the sites of viral budding. Replication of negative-strand Ebola virus genomes takes place in inclusion bodies located close to the nucleus. During synthesis, genomes are encapsidated to form ribonucleoprotein (RNP) complexes which mature to transport-competent nucleocapsids (NCs) inside the inclusions. Although the virus-induced inclusions play an important role in the synthesis of filovirus RNA, their organization is not well understood. In the proposed project we will investigate the spatial and temporal organization of filoviral RNA synthesis, its packaging into RNPs, the molecular basis for RNP maturation to become transport-competent NCs, and which cellular and viral factors are involved in the recruitment of the actin-polymerizing machinery that drives the transport of NCs. Results gained with surrogate systems under BSL-2 conditions will be validated using recombinant filoviruses and CRISPR/Cas9 knock out cell lines under BSL-4 conditions. We will use proteomics to identify cellular proteins associated with encapsidated genomes, quantitative live cell imaging to understand the transport dynamics, super resolution microscopy (dSTORM), and correlative light and electron microscopy (CLEM) to visualize actin and actin-binding proteins at the NCs.

Project-related publications of the investigator:

  • Takamatsu Y, Kolesnikova L, Schauflinger M, Noda T, Becker S. 2020. The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis. J Virol 94.
  • Grikscheit K, Dolnik O, Takamatsu Y, Pereira AR, Becker S. 2020. Ebola Virus Nucleocapsid-Like Structures Utilize Arp2/3 Signaling for Intracellular Long-Distance Transport. Cells 9.
  • Takamatsu Y, Dolnik O, Noda T, Becker S. 2019. A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures. Virol J 16:159.
  • Takamatsu Y, Kolesnikova L, Becker S. 2018. Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport. Proc Natl Acad Sci U S A 115:1075-1080.
  • Mittler E, Schudt G, Halwe S, Rohde C, Becker S. 2018. A Fluorescently Labeled Marburg Virus Glycoprotein as a New Tool to Study Viral Transport and Assembly. J Infect Dis 218:S318-S326.
  • Wan W, Kolesnikova L, Clarke M, Koehler A, Noda T, Becker S, Briggs JAG. 2017. Structure and assembly of the Ebola virus nucleocapsid. Nature 551:394-397.
  • Schudt G, Dolnik O, Kolesnikova L, Biedenkopf N, Herwig A, Becker S. 2015. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells. J Infect Dis 212 Suppl 2:S160-6.
  • Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S. 2014. Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells. PLoS Pathog 10:e1004463.
  • Schudt G, Kolesnikova L, Dolnik O, Sodeik B, Becker S. 2013. Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances. Proc Natl Acad Sci U S A 110:14402-7.
  • Bharat TA, Noda T, Riches JD, Kraehling V, Kolesnikova L, Becker S, Kawaoka Y, Briggs JA. 2012. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci U S A 109:4275-80.

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.

Control of Hepatitis C Virus replication by viral and cellular factors

Research area: Molecular Virology

Replication of Hepatitis C Virus (HCV) genome RNA in cells requires both the establishment and specific activity of viral RNA replication complexes and also viral countermeasures against the cellular innate immune system. We found that HCV stimulates expression of a long non-coding RNA (Lnc-ITM2C-1 or GCSIR) that in turn stimulates expression of a cannabinoid receptor, GPR55. This in turn suppresses expression of interferon-stimulated genes (ISGs) in favour of HCV replication. In this project, we aim at gaining deeper insight into the molecular mechanisms of the Lnc-ITM2C-1 – GPR55 – ISG regulation axis. Complementary to the early steps of the cellular response, we aim at elucidating the early steps of HCV RNA synthesis in cells, using full-length and subgenomic replicon systems. Ribosome pausing at two positions at the NS5B replicase stop codon and directly upstream is caused by inefficient codons, indicating that a slow-down of NS5B translation may be important for the initiation of minus strand synthesis. We want to identify HCV RNA cis-signals and long-range interactions involved in the balance of initial RNA genome translation starting at the genome´s 5´-end versus the initiation of RNA minus strand synthesis starting at the genome´s 3´-end.

Project-related publications of the investigator:

  • Niepmann M, Gerresheim GK. 2020. Hepatitis C Virus Translation Regulation. Int J Mol Sci 21.
  • Gerresheim GK, Hess CS, Shalamova LA, Fricke M, Marz M, Andreev DE, Shatsky IN, Niepmann M. 2020. Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication. Int J Mol Sci 21.
  • Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. 2019. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses 11.
  • Gerresheim GK, Roeb E, Michel AM, Niepmann M. 2019. Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells. Cells 8.
  • Gerresheim GK, Bathke J, Michel AM, Andreev DE, Shalamova LA, Rossbach O, Hu P, Glebe D, Fricke M, Marz M, Goesmann A, Kiniry SJ, Baranov PV, Shatsky IN, Niepmann M. 2019. Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling. Int J Mol Sci 20.
  • Fricke M, Gerst R, Ibrahim B, Niepmann M, Marz M. 2019. Global importance of RNA secondary structures in protein-coding sequences. Bioinformatics 35:579-583.
  • Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. 2018. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol 9:395.
  • Jost I, Shalamova LA, Gerresheim GK, Niepmann M, Bindereif A, Rossbach O. 2018. Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges. RNA Biol 15:1032-1039.
  • Nieder-Rohrmann A, Dunnes N, Gerresheim GK, Shalamova LA, Herchenrother A, Niepmann M. 2017. Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the 5′ untranslated region of hepatitis C virus RNA. J Gen Virol 98:212-224.
  • Gerresheim GK, Dunnes N, Nieder-Rohrmann A, Shalamova LA, Fricke M, Hofacker I, Honer Zu Siederdissen C, Marz M, Niepmann M. 2017. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure. Cell Mol Life Sci 74:747-760.

Replication of large RNA virus genomes: key enzymes and mechanisms

Research area: Molecular Virology

The Nidovirales represent a monophyletic but highly diverged order of plus-strand RNA viruses that currently comprises the families Coronaviridae, Mesoniviridae, Arteriviridae, Tobaniviridae and 10 other families of vertebrate and invertebrate viruses that share common genome organization and expression strategies. Viruses in this order include many important animal and human pathogens, with SARS-CoV, SARS-CoV-2, and MERS-CoV being prominent examples. With genome sizes of up to 41 kilobases (kb), nidoviruses feature the largest RNA virus genomes known to date. Nidoviruses have evolved an unusually complex set of enzymes involved in viral RNA synthesis that is unparalleled in the RNA virus world and believed to be required for the expansion of RNA genomes to unprecedented sizes and efficient replication in special ecological niches. The nidovirus replication/transcription complex (RTC) consists of a large number of virally encoded enzymes. Some of these enzymes are unique to specific nidovirus (sub)families and not conserved in other RNA viruses. This also includes a 3′-5′ exoribonuclease (ExoN) presumed to be involved in increasing the fidelity of nidovirus RNA synthesis. In the previous funding period, we characterized the biochemical properties of this enzyme for representative viruses of the Corona– and Tobaniviridae and extended our studies to other coronavirus nonstructural proteins (nsp7, 8, 9, 10, 12, 14) known to be essential components of the coronavirus RTC. Second, we identified and characterized two novel enzymatic activities that we confirmed to be essential for coronavirus replication, an RNA 3’-polyadenylyltranferase activity associated with nsp8 and a protein-specific NMP transferase activity associated with the NiRAN domain in nsp12. Third, we identified and characterized essential cis-active RNA elements involved in alphacoronavirus replication and, fourth, we embarked on the characterization of replicative proteins of other nidovirus families, focusing on the Mesoniviridae. In this context, we characterized the replicase polyprotein processing by the mesonivirus main protease, determined the crystal structure of this enzyme and identified the structural basis for its unusual substrate specificity. In the next funding period, we plan to advance our studies on the characterization of in vitro reconstituted nidovirus RTCs, involving corona- and mesonivirus protein complexes. We will investigate the mechanistic roles of protein-primed, RNA-primed and de novo initiation of viral RNA synthesis, focusing on the roles of specific viral proteins and cis-active RNA structural elements in these processes. Hypotheses derived from the in vitro studies will be validated by using genetically engineered human coronaviruses (HCoV-229E, SARS-CoV-2) and mesoniviruses as model systems. The studies will be based on biochemical approaches using recombinantly expressed proteins and protein complexes, RNA structure probing, and reverse-genetic approaches that are available in the laboratory or will be developed in the course of the project.

Project-related publications of the investigator:

  • Slanina H, Madhugiri R, Bylapudi G, Schultheiss K, Karl N, Gulyaeva A, Gorbalenya AE, Linne U, Ziebuhr J. 2021. Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit. Proc Natl Acad Sci U S A 118.
  • Shaban MS, Muller C, Mayr-Buro C, Weiser H, Meier-Soelch J, Albert BV, Weber A, Linne U, Hain T, Babayev I, Karl N, Hofmann N, Becker S, Herold S, Schmitz ML, Ziebuhr J, Kracht M. 2021. Multi-level inhibition of coronavirus replication by chemical ER stress. Nat Commun 12:5536.
  • Pfafenrot C, Schneider T, Muller C, Hung LH, Schreiner S, Ziebuhr J, Bindereif A. 2021. Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs. Nucleic Acids Res 49:12502-12516.
  • Krichel B, Bylapudi G, Schmidt C, Blanchet C, Schubert R, Brings L, Koehler M, Zenobi R, Svergun D, Lorenzen K, Madhugiri R, Ziebuhr J, Uetrecht C. 2021. Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes. Sci Adv 7.
  • Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, Haagmans BL, Lauber C, Leontovich AM, Neuman BW, Penzar D, Poon LLM, Samborskiy DV, Sidorov IA, Sola I, Ziebuhr J. 2020. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5:536-544.
  • Tvarogova J, Madhugiri R, Bylapudi G, Ferguson LJ, Karl N, Ziebuhr J. 2019. Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3′-Terminal Adenylyltransferase Activity. J Virol 93.
  • Kanitz M, Blanck S, Heine A, Gulyaeva AA, Gorbalenya AE, Ziebuhr J, Diederich WE. 2019. Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus. Virology 533:21-33.
  • Madhugiri R, Karl N, Petersen D, Lamkiewicz K, Fricke M, Wend U, Scheuer R, Marz M, Ziebuhr J. 2018. Structural and functional conservation of cis-acting RNA elements in coronavirus 5′-terminal genome regions. Virology 517:44-55.
  • Durzynska I, Sauerwald M, Karl N, Madhugiri R, Ziebuhr J. 2018. Characterization of a bafinivirus exoribonuclease activity. J Gen Virol 99:1253-1260.
  • Snijder EJ, Decroly E, Ziebuhr J. 2016. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing. Adv Virus Res 96:59-126.
  • Madhugiri R, Fricke M, Marz M, Ziebuhr J. 2016. Coronavirus cis-Acting RNA Elements. Adv Virus Res 96:127-163.
  • Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, Canard B, Ziebuhr J. 2006. Discovery of an RNA virus 3′->5′ exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci U S A 103:5108-13.