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.

Using available cryo-EM and x-ray crystal structures of the nonstructural proteins that are responsible for SARS-CoV-2 viral RNA replication and transcription, we have constructed an atomistic model of how the proteins assemble into a functioning superstructure.  Our principal finding is that the complex is hexameric, centered around nsp15.  The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10) 2 , where nsp14 is seen to undergo a large conformational change between its two domains.  This conformational change facilitates binding of six nsp12/nsp7/(nsp8) 2 polymerase subunits to the complex.  To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed.  Two of the six polymerase subunits are each proposed to carry dimers of nsp13, while two others are proposed to carry monomers.  The polymerase subunits that coordinate nsp13 dimers also bind the nucleocapsid, which positions the 5’-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication.  Analyzing the path of the viral RNA indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14.  The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping (the nsp12 NiRAN and the nsp14 and nsp16 methyltransferases).  The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping and the role of the endonuclease.  It provides a platform to guide biochemical and structural research to address the stoichiometric and spatial configuration of the replication-transcription complex.