Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP

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.

Keywords: COVID-19; T-705; cryoEM; drug design; structural biology.

Fig. 1.

Reconstitution of SARS-CoV-2 RdRp activity and inhibition by favipiravir-RTP (FTP). (A) Sequence of the annealed primer (Upper):template (Lower) dsRNA duplex employed in biochemical assays. (B) Reconstituted SARS-CoV-2 RdRp extends the 24mer primer in the presence (lanes 4 to 6), but not the absence (lanes 1 to 3), of ribonucleotides (rNTPs). A 31mer product is present in lane 6, due to addition of a nontemplated base to the primer strand. (C) Favipiravir is weakly incorporated into the 24mer primer strand (lanes 7 to 15), and suppresses RNA replication by the SARS-CoV-2 RdRp (lanes 16 to 24). Replication, nucleotide incorporation, and replication inhibition assays are representative results of four, six, and six technical replicates, respectively.

Fig. 2.

Electron cryomicroscopy of RdRp complexes in the presence of RNA and favipiravir-RTP. (A) Electron cryomicrographs of the reconstituted complexes in unsupported ice (HexAuFoil grid, Upper), and on hydrogenated graphene (Lower) were used for structure determination. (Scale bar, 500 Å.) (B) This 2D class average from the images of the complex, containing RNA, corresponds to the most frequent orientation of the particles in the thin film of vitreous water. (C) The orientation distribution, with efficiency, $E_{od}$ (23), of the particles used in the reconstruction is plotted on a Mollweide projection, with the most common views on each type of grids marked with gray lines. (D) The Fourier shell correlation (FSC) between the two independent masked half maps, and between the final map and the atomic model, is plotted versus resolution. (E) An overview of the EM map of the polymerase complex is colored by subunit: green, nsp12; blue, nsp7; pink and purple, two copies of nsp8; yellow, template:primer RNA.

Fig. 3.

Structure of the RdRp complex bound to dsRNA and favipiravir-RTP. (A) The overall structure of the complex is shown, colored by subunit, in the same way as in Fig. 2E. The black lines point to the approximate positions of the catalytic sites, displayed in B and C, where the coloring is by heteroatom, and the EM map is contoured in gray. (B) A pyrophosphate is present at the NiRAN catalytic site of the enzyme, and is coordinated by key conserved residues in this domain. (C) The template:primer RNA duplex is shown, along with favipiravir at the catalytic site ($+$1) of the polymerase. Hydrogen bonds are indicated.

Fig. 4.

Coordination of favipiravir-RTP in the active site of the SARS-CoV-2 RdRp. (A) A nonproductive conformation of favipiravir-RTP (F-RTP), base paired to the P+1 nucleotide of the template strand, as shown, was observed in the cryoEM density map. For clarity, the primer strand is omitted, and only the P$+$1 and P$-$1 nucleotides of the template strand are shown. (B) The nonproductive conformation may be favored in the presence of pyrophosphate (PPi), which is a possible by-product from the incorporation of rNTPs into the RNA. (C) A hypothetical productive conformation of favipiravir-RTP, which may lead to its incorporation into the primer RNA strand, is modeled. (D) For comparison, the position of an incoming nonhydrolyzable UTP analog in the bat influenza polymerase elongation complex is shown (PDB ID code 6SZV) (30).