SARS-CoV-2 RNA: Investigation of the Architecture of SARS-CoV-2 RNA

• Applicant: Professor Dr. Harald Schwalbe
• Subject Area: Structural Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 575062633

During cyclization of the SARS coronavirus-2 genome, the 5'- and 3'-terminal ends of the 30,000 nucleotide (+) single-stranded viral mRNA interact. Cyclization competes with other functions of the viral mRNA, such as its role as genetic storage material, replication template, or messenger RNA. To coordinate and regulate these functions in time and space, RNA viruses use interactions between highly conserved cis-RNA elements. Interestingly, conformational rearrangements of the mRNA are a central and conserved aspect of how they dictate the evolution of the virus. For betacoronaviruses, genome cyclization is thought to be relevant for the stages of replication and transcription. During cyclization, two mRNA segments must interact transiently, but these can also interact with other segments of viral mRNA and with viral and host cell proteins to adopt alternative structures with alternative functions. We want to investigate the interaction of the stem loop structure 5'_SL3 localized at the 5'-terminus with the nucleotide sequence at the 3'-terminus. Both mRNA elements enter into further competing essential interactions. 5'SL3 contains also the leader transcriptional regulatory sequence (TRS-L) for the discontinuous transcription of subgenomic mRNA (sgmRNA), while the 3'-terminal mRNA is the template for (-)-strand synthesis. As an alternative to genome cyclization, the 3'-terminal sequence can enter into a 3'SL3base interaction that inhibits the initiation of (-)-strand synthesis. As an additional level of regulation, the nucleotide A74 in 5'SL3 is modified to m6A74 by the host cell protein complex METTL3/14. In our DFG proposal, we will therefore use NMR methods both for the structural elucidation of the RNA elements described above and for the determination of the thermodynamics and kinetics of their transient RNA-RNA interactions. These detailed, atomically resolved studies will be validated by complementary methods to obtain a comprehensive picture of genome cycling in the context of viral replication and transcription. These include structural probing experiments to observe cyclization inside infected cells and gain insights on biological function using antisense oligonucleotides to impede cyclization specifically. For characterizing mRNA-protein interactions, we will first focus on already known interaction partners of individual elements such as the viral proteins nucleocapsid and Nsp7/Nsp8 in order to investigate the RNA-binding domain of the human m6A “reader” protein YTHDF3 and its potential interaction with the m6A-modified version of the 5'-strand in the further course of the project. Our aim is to investigate how the RNA elements, both in isolation and in complexes with their respective interaction partners, control the function of genome cycling.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Dr. Redmond Smyth, Ph.D.