Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication

Abstract

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Keywords: COVID-19 treatment; RNA-dependent RNA polymerase; SARS-CoV-2; remdesivir.

Graphical abstract

Figure 1

RdRp stalls after incorporation of 4 remdesivirs (A) The RNA substrate used in experiments consists of a 20-nt, 5′-[6-FAM]-labeled primer annealed to a 40-nt template. The sequence is from the 3′ end of the SARS-CoV-2 genome, preceding the poly-A tail. (B) Time course of UTP + RTP incorporation by the RdRp complex. A solution of 3.33 μM nsp12/7/8, 4.7 μM nsp8, and 200 nM FAM-20/40 RNA was mixed with 14 μM RTP and 8 μM UTP to start the reaction. Time points were quenched by the addition of EDTA, and samples were analyzed by capillary electrophoresis. Lengths of various RNA products are given at the top of the panel. The solid lines through the data points are from the fit by simulation in KinTek Explorer. (C) UTP + RTP product formation and extension. The chromatogram for the zero-time point is given at the top (ER₂₀). Initially, RTP and UTP were added for 20 s (ER₂₆) at the concentrations given in the figure. Then, 250 μM of all NTPs were added for 15 s to allow extension to the full-length product (ER₄₀). On the timescale of the experiment, only a fraction of ER₂₆ is converted to the full-length product (ER₄₀). (D) UTP + ATP product formation and extension. The experiment was performed as in (C), but with ATP instead of RTP in the first mixing step, allowing extension to the ER₂₇ product. After addition of all of the nucleotides for 15 s in the second mixing step, the ER₂₇ complex is completely converted to the full-length ER₄₀ product.

Figure 2

Structural basis of SARS-CoV-2 RdRp complex stalling by remdesivir (A) Chemical structures of adenosine triphosphate. (B) Chemical structure of remdesivir triphosphate. (C) Cryo-EM reconstruction of remdesivir-stalled RdRp complex. (D) Corresponding atomic model of remdesivir-stalled RdRp complex. (E) Close-up view of polymerase active site. Four copies of remdesivir monophosphate (RMP) occupy positions +1 (active site), −1, −2, and −3. Uracil monophosphate (UMP) is in position −4 (gray). C1 carbon of UMP and side chain of nsp12(Ser861) are shown as spheres. Cryo-EM density corresponding to template RNA strand (blue) and primer strand positions +1 through −5 is shown (transparent gray). The active site loop is also shown. (F) Hypothetical model of post-translocated state, whereby the first copy of RMP is translocated into the −4 polymerase position. The RMP(−4) cyano group and nsp12(Ser861) are shown as spheres and show a steric clash. (G) Spatial relationship between nucleotide at the −4 position and side chain of nsp12(Ser861) in pre-translocated state. (H) Spatial relationship between nucleotide at −4 position and side chain of nsp12(Ser861), illustrating the clash between the cyano group of RMP that prevents translocation. See Table 1 and Figure S1 for cryo-EM data collection, model validation, and analysis.