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

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SARS-Cov-2 Nsp16 belongs to a conserved family of viral 2’-O-methyltransferases (2’OMTs) that are still poorly characterized. The SARS-CoV and MERS Nsp16 2’OMTs were shown to be necessary for interferon resistance and viral pathogenesis {Menachery, 2014 #14}. However, the general mechanisms of coronavirus 2’OMTs as well as their full RNA target spectrum beyond capped RNA remain largely elusive. In case of SARS-Cov Nsp16, longer capped RNA substrates were methylated more efficiently suggesting specific sequence requirements or/and methylation of residues distant from the m7G-cap. It is therefore the general aim of our joined project to characterize the molecular mechanism, structure and function of the SARS-CoV-2 Nsp16 2’OMT in modulating gene expression in detail and to potentially develop Nsp16 inhibitors. Structures of Nsp16 and its accessory protein Nsp10 in complex with a m7GpppA-RNA have only recently been reported {Krafcikova, 2020 #1; Lin, 2020 #3; Rosas-Lemus, 2020 #4; Viswanathan, 2020 #17}. However, the specific recognition of longer RNA substrates by SARS-CoV-2 and critical residues of Nsp16 and Nsp10 remain unclear. Using recombinant Nsp16 and Nsp10 proteins, our initial experiments indicate that the viral RNA-processing mechanism is re-organized depending on the RNA sequence that is modified. In vitro SELEX experiments are aimed to identify non-capped RNA substrates. We will investigate Nsp16/Nsp10 in a complex with diverse capped and uncapped RNA fragments using biochemical experiments and NMR and will attempt to provide structural details of the complex from X-ray, cryo-EM and NMR of the complex. The Landthaler and Popowicz/Sattler labs are synergizing here to use their expertise in protein expression, structural and biochemical characterization of RNA-modifying enzymes and RNA binding proteins and high-throughput analysis of RNA-interacting proteins. SIGNIFICANCE: The Nsp16/Nsp10 complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance. The proposed work will provide the first comprehensive analysis of the chemistry, structure and RNA substrate specificity of SARS-CoV-2 Nsp16 and the 2’O-methylation status of viral RNA. Assays and strategies developed as part of this project can readily be adapted to 2’OMTs of other viruses that replicate in the cytoplasm including Ebola viruses, Measles virus, Sindbis virus, Zika virus, Dengue virus and Vaccinia virus to provide a path for novel antiviral therapies.