Biochemical and structural characterization of the SARS-CoV-2 non-structural protein 16 (Nsp16), a cap ribose 2’O-methyltransferase

• Applicants: Professor Dr. Markus Landthaler; Dr. Grzegorz Popowicz; Professor Dr. Michael Sattler
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2021 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458682959

• Final Report Year: 2023

Final Report Abstract

Nsp10 and nsp16 are two non-structural proteins encoded by SARS-CoV-2, the virus that causes COVID-19. They form a heterodimeric complex that acts as a 2'-O-methyltransferase (MTase), catalyzing the methylation of the 2'-OH group of the first nucleotide (usually an adenine) of the viral RNA cap. The methylation converts the cap structure from Cap-0 (me7GpppA) to Cap-1 (me7GpppAm), which mimics cellular mRNAs and helps the virus evade immune recognition by host factors such as MDA5 and IFIT1. Crystal structures of nsp10/nsp16 complexes in the presence or absence of SAM (the methyl donor) had been solved previously and revealed molecular details of the enzyme-substrate interaction and the conformational changes upon product release. The structures also provide insights into the effect of clinical variants and previous SARS-CoV outbreak strains on the enzymatic activity of nsp10/nsp16. nsp10/nsp16 complex is a potential target for antiviral drug design, as inhibitors that block its activity could impair viral replication and immune evasion. The goals of this project were to characterize RNA substrates, and the methylation specificity using biochemical assays, NMR and structural biology. MTase Glo assay were established and revealed the RNA substrate requirements and specificity of the nsp10/nsp16 complex. Structural biology, biophysical and biochemical experiments revealed a dynamic lid domain in nsp14, which, unexpectedly, also enables the formation of a ternary nsp10,nsp14,nsp16 complex. Our findings form the basis for future experiments aimed at identifying inhibitors of the nsp16 methyltransferase activity.

Publications

• Characterization of SARS-CoV-2 replication complex elongation and proofreading activity. Scientific Reports, 12(1).
  Jones, Alisha N.; Mourão, André; Czarna, Anna; Matsuda, Alex; Fino, Roberto; Pyrc, Krzysztof; Sattler, Michael & Popowicz, Grzegorz M.
  
• Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity. Structure, 30(8), 1050-1054.e2.
  Czarna, Anna; Plewka, Jacek; Kresik, Leanid; Matsuda, Alex; Karim, Abdulkarim; Robinson, Colin; O.’Byrne, Sean; Cunningham, Fraser; Georgiou, Irene; Wilk, Piotr; Pachota, Magdalena; Popowicz, Grzegorz; Wyatt, Paul Graham; Dubin, Grzegorz & Pyrć, Krzysztof
  
• Despite the odds: formation of the SARS-CoV-2 methylation complex. Nucleic Acids Research, 52(11), 6441-6458.
  Matsuda, Alex; Plewka, Jacek; Rawski, Michał; Mourão, André; Zajko, Weronika; Siebenmorgen, Till; Kresik, Leanid; Lis, Kinga; Jones, Alisha N.; Pachota, Magdalena; Karim, Abdulkarim; Hartman, Kinga; Nirwal, Shivlee; Sonani, Ravi; Chykunova, Yuliya; Minia, Igor; Mak, Paweł; Landthaler, Markus; Nowotny, Marcin ... & Czarna, Anna