Calvin J. Gordon, University of Alberta

Calvin J. Gordon1,2*, Mizar Oliva3*, Simon M. Walker1*, Dana Kocincova1, Emma Woolner1, Egor P. Tchesnokov1, Ross Edwards1,2, Matthias Gotte1, Kalyan Das1,2,3 1Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, AB, Canada 2Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada 3Molecular Structural and Translational Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

Fighting & Responding

The viral RNA-dependent RNA polymerase (RdRp) is essential for RNA virus replication and is a primary target for antiviral drug development. Structural characterization of RdRps at different functional states is critical for guiding rational drug design. Here, we present structural biology platforms for two distinct viral RdRps; the experimental steps include expression, purification, biochemical validation, and single-particle cryogenic electron microscopy (cryo-EM) studies. The antiviral remdesivir is used to treat SARS-CoV-2 and targets the RdRp. Remdesivir triphosphate (RTP) is a better substrate than its natural counterpart, ATP. The S759A substitution in the SARS-CoV-2 RdRp confers resistance to remdesivir. We show that the mutation switches the RdRp preference from RTP to ATP; RTP is incorporated only at 10-fold excess to ATP. The structures of S759A RdRp reveal that the primer 3′-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. Paramyxoviruses such as measles virus, Nipah virus, and Hendra virus pose serious threats to global human health. An avian paramyxovirus type 1 (APMV-1) was recently associated with a fatal case of neurological disease in a 2-year-old child. We obtained a single-particle cryo-EM structure of the APMV-1 RdRp complex at ~2.60 Å resolution. Like measles and other paramyxoviruses, APMV-1 RdRp can efficiently incorporate broad-spectrum nucleotide analogs, such as RTP. Our structural interrogation of the viral replication machinery provides a foundation for capturing polymerases bound to antivirals. Ultimately, this pipeline will accelerate mechanistic understanding of viral RNA synthesis and support structure-guided antiviral discovery targeting current and emerging RNA viruses.