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.