Abstract
The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.
Highlights
The COVID-19 coronavirus pandemic has spurred research into novel and existing antiviral treatments
While the current COVID-19 pandemic continues, the US Food and Drug Administration (FDA) has approved only one drug against the virus—remdesivir. It is a nucleotide analogue inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase; favipiravir is another member of the same class
Proteins were captured with Co2+-conjugated resin (Cytiva) for immobilized-metal affinity chromatography (IMAC), eluted in a buffer containing 300 mM NaCl, 20 mM Hepes pH 7.5, 300 mM imidazole, and 1 mM TCEP, followed by overnight cleavage of the 6×-His-Sumo tag by Ulp1, and subjected to subtractive IMAC to remove cleaved tags, uncleaved nsp8, and Ulp1
Summary
While the current COVID-19 pandemic continues, the US Food and Drug Administration (FDA) has approved only one drug against the virus—remdesivir It is a nucleotide analogue inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase; favipiravir is another member of the same class. PNAS 2021 Vol 118 No 7 e2021946118 exclusion chromatography on a Superdex 200 16/60 size-exclusion column (Cytiva) in a buffer of 20 mM Hepes (pH 7.5), 200 mM NaCl, 2 mM MgCl2, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Proteins were captured with Co2+-conjugated resin (Cytiva) for immobilized-metal affinity chromatography (IMAC), eluted in a buffer containing 300 mM NaCl, 20 mM Hepes pH 7.5, 300 mM imidazole, and 1 mM TCEP, followed by overnight cleavage of the 6×-His-Sumo tag by Ulp, and subjected to subtractive IMAC to remove cleaved tags, uncleaved nsp, and Ulp. Fractions containing nsp7–nsp were concentrated, flash frozen in liquid nitrogen, and stored at −80 ◦C as single-use aliquots
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
