Revealing the mechanism of nucleotide selection, addition and proofreading of the SARS-coronavirus replication transcription complex at the single molecule level

• Applicant: Dr. David Dulin, Ph.D.
• Subject Area: Virology ; Biophysics
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 447835095

RNA viruses are a very diverse group of pathogens. This is particularly underlined by the large variety of factors involved in their replication transcription complex (RTC) and their artfulness in modes of messenger and genomic RNA synthesis. Viral RNA synthesis is ensured by the RTC, which also ensures evolution through nucleotide mismatch incorporations and by promoting viral genome recombination. However, the mutation rate of the RTC is too high to generate infectious virions for long genome single stranded positive RNA viruses, such as coronavirus (CoV) (~30 kb). Therefore, CoV have solved this problem by encoding an RNA proofreading enzyme, i.e. a 3’ to 5’ exonuclease to correct the excessive numbers of mutations, which also protects human pathogenic CoV’s, e.g. the Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and new coronavirus 2019 (COVID-19) from antiviral nucleotide analogues targeting the RTC. Understanding how CoV exonuclease senses nucleotide mismatch and analogues after incorporation remains an important research topic to develop more efficient antiviral drugs. To characterize the kinetics of nucleotide mismatch and analogues incorporation and derive a kinetic model, standard bulk biochemistry assays use short templates (~10 nt) and no competing nucleotides, which are not realistic conditions. Using high throughput magnetic tweezers and kilobases long templates, I will be able to monitor the kinetic signature of SARS-CoV RTC nucleotide mismatch and analogue incorporation at the single molecule level with near single base resolution and in competition with the four natural nucleotides at saturating concentrations. SARS-CoV RTC is arguably the best-known coronavirus RTC, and the high degree of conservation of CoV RTC will make the future results of my proposal extendable to other CoVs, such as MERS and COVID-19. I will derive a complete kinetic model describing the kinetics of nucleotide mismatch incorporation and characterize the mechanism of action of commercially available nucleotide analogues using SARS-CoV RTC.

• DFG Programme: Research Grants
• International Connection: France, Netherlands
• Cooperation Partner: Privatdozent Dr. Bruno Canard