Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

Abstract

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

Keywords: RNA-dependent RNA polymerase; backtracking; coronavirus; cryo-electron microscopy; molecular dynamics.

Fig. 1.

SARS-CoV-2 backtrack complex. (A) RNA scaffolds: (Top) RTC scaffold (14); (Bottom) backtrack complex scaffolds (BTC₃ and BTC₅). (B) A native gel electrophoretic mobility shift assay reveals that holo-RdRp requires nsp13(ADP-AlF₃) to bind the BTC scaffolds efficiently. (C) Cryo-EM structures of SARS-CoV-2 BTCs. Shown is the transparent cryo-EM density [local-resolution-filtered (47)] with the refined models superimposed (SI Appendix, Table S1). The models and density are colored according to the key. Two major BTCs were observed (SI Appendix, Fig. S2), one containing one nsp13 protomer (nsp13₁-BTC₅), and one containing two nsp13 promoters (nsp13₂-BTC₅). We designate the nsp13 promoter common to both structures nsp13.1 and the other nsp13.2 (14). The cyan spheres denote the path of the single-stranded t-RNA 5′ segment, some of which is engaged with nsp13.1 in both structures.

Fig. 2.

Cryo-EM density maps. (A, Left) Overall view of nsp13₂-BTC₅. Nsp13.2 is removed (outline) for clarity. The boxed region is magnified on the right. (A, Right) Magnified view of the t-RNA segment (+14-5′-CCCAUGU-3′-+8) enclosed in the nsp13.1 helicase subunit. The cryo-EM density map (from the nsp13₂-BTC structure) for the RNA is shown (blue mesh). (B, Left) Overall view of the BTC₅(local) structure. The boxed region is magnified on the right. (B, Right) Magnified view of the region around the RdRp active site, showing the t-RNA (cyan) and p-RNA (red) with the backtracked RNA segment. The cryo-EM density map for the RNA [from BTC₅(local)] is shown (blue mesh). (C) BTC₅(local) cryo-EM density maps around nsp12 conserved motifs F, C, and E. Selected residues are labeled.

Fig. 3.

SARS-CoV-2 RdRp and DdRp BTCs. (A and B) SARS-CoV-2 RdRp (A) and DdRp (B) BTCs. (Top) Proteins are shown as transparent molecular surfaces and nucleic acids as atomic spheres. The boxed regions are magnified on the bottom. (Bottom) Magnified, cross-sectional view. Proteins are shown as molecular surfaces and nucleic acids in stick format with transparent molecular surface. (A) The SARS-CoV-2 BTC₅(local). Nsp8a and nsp12 are shown (nsp7 and nsp8b are removed for clarity). Nsp12 motif F is shown as a magenta backbone ribbon (Top). Backtracked RNA (+1C to +3C of the BTC₅-scaffold; Fig. 1A) extrudes out the NTP entry tunnel. (B) A DdRp (Saccharomyces cerevisiae Pol II) BTC [Protein Data Bank (PDB) ID code 3PO2 (29)]. The BH is shown as a magenta backbone ribbon. The backtracked RNA extrudes out the NTP entry tunnel/secondary channel/funnel. (C) Views from the outside into the NTP entry tunnels of the SARS-CoV-2 (Left) and an S. cerevisiae DdRp [PDB ID code 3GTP (27)] BTC. Protein surfaces are colored by the electrostatic surface potential [calculated using APBS (48)]. Backtracked RNA is shown as atomic spheres with yellow carbon atoms.

Fig. 4.

Protein–RNA interactions in the BTC. (A, Top) Overall view of BTC₅(local). Proteins are shown as transparent molecular surfaces and nucleic acids as atomic spheres. Nsp8a and nsp12 are shown (nsp7 and nsp8b are removed for clarity). Nsp12 motifs C, E, and F are shown as backbone ribbons (colored according to the key on the bottom). The boxed region is magnified below. (A, Bottom) RNA is shown from −2 to +3. Proteins are shown as transparent molecular surfaces. RdRp motifs C, E, and F are shown as transparent backbone ribbons (colored according to the key) with side chains of residues that approach the backtracked RNA (≤4.5 Å) shown. (B) Schematic illustrating the same protein–RNA interactions as A. Drawn using Nucplot (49).

Fig. 5.

Comparison of active-site proximal RNA in the RTC and BTC structures and from simulations of a mismatched nucleotide at the p-RNA 3′ end. (A and B) Comparison of the active-site proximal RNA in the RTC [A; PDB ID code 6XEZ (14)], BTC₅(local) (B), and from selected snapshots of molecular dynamics simulations of a −1U + 1C complex (C). The schematics denote the nucleotides shown in the context of the RTC (A) and BTC₅ scaffolds (B; full scaffold sequences shown in Fig. 1A) or generated from the BTC₅ scaffold for the simulations (C). Carbon atoms of the t-RNA are colored cyan and p-RNA are colored salmon except in the case of mismatched Cs at the 3′ end, which are colored dark red. Watson–Crick base-pairing hydrogen bonds are denoted as dark gray dashed lines; other hydrogen-bonds as red dashed lines. Nsp12 motif C is shown as a yellow-orange backbone ribbon, and the side chain of D760 is shown as atomic spheres. (A) The RTC is in a posttranslocated state, with the A–U base pair at the p-RNA 3′ end in the −1 position (14). (B) The BTC₅(local) RNA is translocated compared to the RTC; the base pair corresponding to A–U at the 3′ end of the RTC RNA in the −1 position is in the −2 position of the BTC RNA. A C–A mismatch occupies the BTC −1 site. The +1, +2, and +3 mismatched Cs trail into the RdRp NTP entry tunnel (denoted by black squiggly lines). The +4C (present in the BTC₅ scaffold; Fig. 1A) is exposed to solvent, disordered, and not modeled. (C) Molecular dynamics simulations of the nsp13₂–BTC_−1U+1C complex. The complex was simulated with three replicates (green, blue, and orange traces). Rmsd values plotted as a function of time represent the heavy-atom rmsd of the +1C of the p-RNA compared with the starting configuration (Materials and Methods). The rmsd histograms (plotted on the right) are an aggregate of all three replicates. Two structures taken from one of the simulations are shown, one showing the +1C of the p-RNA in the active site (t = 0 μs) and the other showing the +1C frayed into the NTP entry tunnel (t = 4.5 μs).

Fig. 6.

Role of backtracking in proofreading and template switching during subgenomic transcription. Schematic illustrating the proposed model for backtracking of the SARS-CoV-2 RTC and its potential role in proofreading and template switching during subgenomic transcription. The structural models are shown as cartoons (holo-RdRp, light blue; nsp13 helicase, orange shades; RNA strands, colored tubes as indicated). (Top) In the RTC, the elongating RdRp moves from left to right. The RdRp active site holds the p-RNA 3′ end. The NTP entry tunnel provides access from solution to the RdRp active site. The downstream (5′) single-stranded t-RNA is not engaged with nsp13. (Bottom) In the BTC, nsp13 translocates on the downstream (5′) single-stranded t-RNA, pushing the RdRp backward (right to left) on the RNA. This causes the p-RNA to reverse-thread through the complex, with the resulting single-stranded 3′ fragment extruding out the NTP entry tunnel. The exposure of the p-RNA 3′ end could facilitate proofreading (9, 10, 12, 50) and also template switching during subgenomic transcription (7, 34).