Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis

Abstract

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the r eplication and t ranscription c omplex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transition towards cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp8 2 -nsp12-nsp13 2 -RNA and a single RNA binding protein nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N-terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5’ extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC towards mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

“…Previous X-ray structures of NSP13 have been solved for MERS-CoV and the highly related SARS-CoV to 3.0 Å and 2.8 Å respectively (10,15). More recently, cryo-electron microscopy studies have revealed the architecture of the NSP13 containing replication and transcription complex, which contains two copies of NSP13 that interact with NSP8 via the N-terminal ZBD (12,16,17) (7CXM). One of the NSP13 protomers makes additional interactions with NSP12 and is located with its RNA binding site in the path of downstream RNA (12).…”

SARS-CoV-2 Nsp8 N-terminal domain dimerizes and harbors autonomously folded elements

“…and Nsp7 could really combine to form a hexameric ring, which would afford PCNA-like progressivity to the RTC as proposed by Zhai et al 1 (Fig 2A). By contrast, if the physiological RTC were like the recent cryo-EM structures 2,3,[8][9][10][11][12][13][14][15] (Fig. 2B), then the dimerization of the Nsp8 NTD might initially serve to self-chaperone hydrophobic patches which evolve to form interactions with Nsp12, Nsp13 and RNA.…”

“…The blue trace corresponds to absorbance at 280 nm, the green trace marks the imidazole gradient, which ranged from 10 mM to 500 mM, the red lines mark the collected fractions, the magenta line indicates the injection point and the light blue line represents the conductivity. B. Gradient (4 -20% arcylamide) PAGE-SDS gel of the final purified 13 C, 15 N Nsp8 NTD. The numbers on the right indicate the size, in kDa, of the molecular weight markers.…”

“…Nsp13 (PDBs: 6XEZ 3 , 7CXM 14 , 7CXN 14 , 7CYQ 15 ). In all these, the Nsp8 CTD appears well structured, adopting the same globular fold which interacts with Nsp7.…”

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

“…Structures of the core SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex (consisting of the enzyme nsp12 and the accessory subunits nsp7 and nsp8) have revealed the structural basis for RNA replication (Chen et al, 2020; Hillen et al, 2020; Wang et al, 2020b; Yan et al, 2020a; Yan et al, 2020b; Yin et al, 2020). However, the mechanism of action of remdesivir, an adenosine analog that was recently approved by the FDA for COVID-19 treatment, remains to be established (Beigel et al, 2020; Wang et al, 2020a).…”