Structural basis for substrate selection by the SARS-CoV-2 replicase

Abstract

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP3,4. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5’ RNA cap6.

Effects of natural RNA modifications on the activity of SARS‐CoV‐2 RNA‐dependent RNA polymerase

“…At the same time, SARS‐CoV‐2 RdRp cannot extend DNA primers and is also strongly selective for NTP substrates. Structural analysis suggested that active‐site residues of nsp12 (in particular, N691) recognize the 2'‐OH group of the NTP, thereby enabling preferential incorporation of NTPs over dNTPs [ 12 , 40 ], but it remains to be established whether there are additional mechanisms of dNTP exclusion.…”

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19

“…For orthosteric ligands that bind to an active site the relevance is clear, but for allosteric ligands the effect cannot be predicted from a simple free energy of binding [82]. The available data argue that SARS-CoV-2 RdRp [23,24,42] and ExoN [83] are subject to allosteric control. Furthermore, while virtual screening is necessary to evaluate millions of compounds as potential novel antivirals, its value for drug repurposing now appears questionable.…”

“…Small molecules could interfere with NiRAN activity by binding to nsp9, AS2, or an allosteric site. A natural product inhibitor that binds to nsp9 has been recently identified [40], and structures with ligands bound to AS2 [12,18,35,41,42] could guide design of NiRAN inhibitors. An in silico docking study identified several putative NiRAN ligands among FDA-approved protease and kinase inhibitors as well as antibiotics [30].…”

“…While base-pairing is essential for NA accommodation in AS1, no base-specific contacts have been observed in several structures of NiRAN-bound nucleotides [18,35], and natural NTPs appear to bind to AS2 similarly during nsp9 NMPylation [20]. However, preferential binding of GTP is required for RNA capping [22], and recent reports show that NAs may bind to AS2 in strikingly different poses [12,42]: in some transcription complexes, GTP [42] and its analog AT-527 diphosphate [12] bind to AS2 in a flipped orientation (Figure 2A) with the base located in a guanine-specific pocket that is absent in the apo-NIRAN structure [42]. These observations suggest that AS2 has considerable plasticity, which may be a shared feature of AMPylases: E. coli Doc, a toxin from the Fic family of AMPylases, transfers the ATP γ-phosphate (rather than AMP) to a Thr residue of the translation elongation factor EF-Tu, blocking binding of aminoacylated tRNAs [47].…”

“…The bad news is that exceptionally dynamic proteins, such as SARS-CoV-2 RdRp, present challenges for the very axioms of in silico studies. Obviously, both AS1 and AS2 could be used to interrogate potential ligands, but neither active site is fully formed in the absence of a substrate: e.g., AS1 lacks the catalytic Mg 2+ ion and AS2 can bind substrates in different orientations [12,35,42]. Thus, the choice of a receptor structure is not obvious even when targeting either one of SARS-CoV-2 nsp12 active sites, and many other potential targets exist in the multi-subunit transcription-replication complex.…”