Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

Abstract

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

Figure 1.. SARS-CoV-2 nsp13 helicase forms a stable complex with the holo-RdRp and an RNA scaffold.

a. RNA scaffold used for biochemistry, native mass spectrometry (nMS), and cryo-EM. b. Native gel electrophoretic mobility shift assay reveals that nsp13 forms a stable complex with holo-RdRp:RNA. The 4.5% polyacrylamide gel was visualized with Gel Red to stain the RNA. c. nMS spectra and the corresponding deconvolved spectra for the holo-RdRp containing the RNA scaffold (a) with and without nsp13. The measured mass for the holo-RdRp:RNA complex corroborates the established stoichiometry of 1:2:1:1 for nsp7:nsp8:nsp12:RNA (Hillen et al., 2020; Kirchdoerfer and Ward, 2019; Wang et al., 2020; Yin et al., 2020), respectively (bottom). Addition of the 67.5-kDa nsp13 helicase to the RNA-bound holo-RdRp holo sample forms a transcription complex/helicase assembly with 1:1 stoichiometry (top). See also Figure S1 and S2.

Figure 2.. Overall structure of the SARS-CoV2 nsp13 helicase with the holo-RdRp:RNA replication/transcription complex (RTC).

a. Schematic illustrating domain structure of SARS-CoV-2 holo-RdRp (nsp7, nsp8, nsp12) and nsp13. Structural domains discussed in the text are labeled. The color-coding corresponds to the figures throughout this manuscript unless otherwise specified. b-e. Orthogonal views showing the overall architecture of the nsp13₂-RTC. Proteins are shown as molecular surfaces (except nsp13.1 in panel d), RNA as atomic spheres. Adventitious CHAPSO detergent molecules are shown as atomic spheres and colored dark grey. The path of downstream t-RNA through the nsp13.1 helicase, shown as cyan spheres, is revealed with low-pass filtered (6 Å) difference density (shown in panel d). b. Two copies of the nsp13 helicase bind to the RTC. Nsp13.1 forms a tripartite interaction with the nsp8b-extension and the nsp12-thumb via the nsp13.1-ZBD. The 5’-end of the tRNA extrudes through the nucleic acid binding channel of nsp13.1. The two helicases interact via the nsp13.1–1B domain and the nsp13.2-RecA1 domain. c. In addition to the nsp13.1-ZBD:nsp8b-extension:nsp12-thumb tripartite interaction, nsp13.1-RecA1 interacts with nsp7 and the nsp8b-head. d. ADP-AlF₃ is modeled in the NTP binding site of each helicase. the low-pass filtered (6 Å) cryo-EM difference density revealing the path of the downstream t-RNA is shown (cyan surface). e. The nsp13.2-ZBD interacts with the nsp8a-extension. ADP-Mg²⁺ is bound to the NiRAN domain. See also Figure S3–S5 and Table S1.

Figure 3.. Interactions of the nsp13-ZBDs with the RTC.

a.-b. Views of the nsp13-ABD:RTC interactions. Proteins are shown as α-carbon backbone worms. Side chains that make protein:protein interactions are shown. Polar interactions are shown as dashed grey lines. Sequence logos (Schneider and Stephens, 1990) from alignments of α- and β-CoV clades for the interacting regions are shown. The residues involved in protein:protein interactions are labeled underneath the logos. Dots above the logos denote conserved interacting residues. a. View of the tripartite nsp13.1-ZBD:nsp8b-extension:nsp12-thumb interaction. The adventitiously-bound CHAPSO molecule is shown in dark grey. b. View of the nsp13.2-ZBD:nsp8a-extension interaction. See also Figure S6.

Figure 4.. SARS-CoV-2 nsp12-NiRAN domain, pseudokinase SelO, and ADP binding.

a. The colored histograms denote identity in a sequence alignment of 45 α- and β-CoV nsp12 sequences (red bar, 100% identity; dark blue bar, ≤ 20%) in the N-terminal signature motifs A_N, B_N, and C_N (Lehmann et al., 2015) of the NiRAN domain. The consensus sequence is shown just below. The SARS-CoV-2 nsp12 and the pseudokinase P. syringae (Psy) SelO (Sreelatha et al., 2018) are aligned below. Residues that are 100% identical in the nsp12 alignment and conserved in SelO are highlighted by a red dot underneath. b. (left) Structures of the SARS-CoV-2 NiRAN domain (cyan ribbon) with ADP-Mg²⁺ (spheres) and Psy SelO (orange) with AMP-PNP-Mg²⁺ (6EAC; Sreelatha et al., 2018). The A_N, B_N, and C_N regions are highlighted. (right) Structure-based alignment via α-carbons of the A_N, B_N, and C_N regions, with side chains of conserved residues shown. The α- and β-phosphates of the NiRAN-domain ADP-Mg²⁺ (limon carbon atoms, yellow sphere, respectively) superimpose almost exactly with the β- and γ-phosphates of the SelO AMP-PNP-Mg²⁺ (dark gray), whereas the nucleoside moieties diverge. c. The ADP-Mg²⁺-bound pocket of the SARS-CoV-2 NiRAN domain is shown. Side chains interacting with the ADP-Mg²⁺ are shown (polar interactions are denoted by gray dashed lines). D208 likely makes a water-mediated interaction with the Mg²⁺ (Sreelatha et al., 2018). The cryo-EM difference density for the ADP-Mg²⁺ is shown (light gray mesh).

Figure 5.. Correspondence of structural determinants for backtracking between cellular multi-subunit DdRp and SARS-CoV-2 RdRp.

a.-b. (left) Overall views of complexes. Proteins are shown as cartoon ribbons with transparent molecular surfaces. Nucleic acids are shown as atomic spheres. Color-coding is shown in the keys. (right) Cross-sectional view of the active site region (at the 3’-end of the RNA product). a. (left) Structure of the E. coli DdRp transcription elongation complex [EC; (Kang et al., 2017)] viewed down the secondary channel. The DdRp active site Mg²⁺-ion is shown as a yellow sphere. The secondary channel is highlighted inside the red circle. The thin dashed line illustrated the cut and viewing direction of the cross-section on the right. (right) Cross-sectional view showing the RNA/DNA hybrid. The bridge helix (viewed end on in cross-section) is denoted. The bridge helix directs the downstream template duplex DNA to the top (dark grey arrow). Underneath the bridge helix, the secondary channel allows NTP substrates to diffuse into the active site (Westover et al., 2004; Zhang et al., 1999) and accomodates the single-strand RNA transcript 3’-end in backtracked complexes (Abdelkareem et al., 2019; Cheung and Cramer, 2012; Wang et al., 2009). b. (left) View down the newly described secondary channel of the SARS-CoV-2 holo-RdRp. The secondary channel is highlighted inside the red circle. (right) Cross-sectional view showing the t-RNA/p-RNA hybrid. Motif F (viewed end on in cross-section) is denoted. Motif F directs the downstream t-RNA to the top (cyan arrow). Underneath motif F, the secondary channel could accomodate the single-strand p-RNA 3’-end in the event of backtracking.

Figure 6.. Structural basis for possible nsp13 helicase functions during viral genome replication/transcription.

Structural models are shown as cartoons (holo-RdRp, light blue; nsp13.1 helicase, orange shades; RNA strands, colored tubes). The nsp13.2 helicase is not shown for clarity (all of the models are compatible with the presence of nsp13.2). With each structural diagram is a schematic cartoon illustrating the arrangement of RNA strands. Additional proteins involved in these processes are omitted. The product RNA (p-RNA) being elongated by the RdRp is shown in red. a. The SARS-CoV-2 nsp13-RdRp cryo-EM structure likely represents an equilibrium between two states. b. During RNA synthesis on a single-stranded RNA template (cyan), nsp13 could function distributively to clear downstream RNA secondary structure (or RNA binding proteins). Similarly, on a duplex RNA template (cyan and green), nsp13 could processively unwind downstream duplex RNA. c. Proposed helicase function during template-switching associated with sub-genomic (sg) transcription [see (Enjuanes et al., 2006; Lehmann et al., 2015b; Pasternak et al., 2001; Snijder et al., 2016; Sola et al., 2015)]. i) Negative-strand RNA synthesis proceeds from the genomic 3’-poly(A)-tail until a Transcription-Regulating Sequence [TRS-R, orange; (Alonso et al., 2002)] is transcribed (cTRS, yellow). ii) The TRS causes transcription complex stalling. iii) Helicase function acting on the +-strand RNA (cyan) causes backtracking of the transcription complex, freeing the pRNA 3’-end. iv) The p-RNA 3’-end cTRS (yellow) hybridizes with the complementary TRS-L (orange) following the genomic 5’-leader sequence [magenta; (Alonso et al., 2002; Pasternak et al., 2001; Zúñiga et al., 2004)]. v) Processive helicase function backtracks the RdRp complex and unwinds the p-RNA from the genomic 3’-end. A second RdRp complex can load into the p-RNA 3’-end and continue transcription using the 5’-leader as template.