Structure of replicating SARS-CoV-2 polymerase

Abstract

The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes 1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses 3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19) 4. Coronaviruses are positive-strand RNA viruses that pose a major health risk 1 : SARS-CoV-2 has caused a pandemic of the disease known as COVID-19 5,6. Coronaviruses use an RdRp complex for the replication of their genome and for the transcription of their genes 2,3. This RdRp complex is the target of nucleoside analogue inhibitors-in particular, remdesivir 7,8. Remdesivir inhibits the RdRp of multiple coronaviruses 9,10 , and shows antiviral activity in cell culture and animal models 11. Remdesivir is currently being tested in the clinic in many countries 12 and has recently been approved for emergency treatment of patients with COVID-19 in the United States 4. The RdRp of SARS-CoV-2 is composed of a catalytic subunit known as nsp12 13 as well as two accessory subunits, nsp8 and nsp7 3,14. The structure of this RdRp has recently been reported 15 ; it is highly similar to the RdRp of SARS-CoV 16 , a zoonotic coronavirus that spread into the human population in 2002 1. The nsp12 subunit contains an N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain, an interface domain and a C-terminal RdRp domain 15,16. The RdRp domain resembles a right hand, comprising the fingers, palm and thumb subdomains 15,16 that are found in all single-subunit polymerases. Subunits nsp7 and nsp8 bind to the thumb, and an additional copy of nsp8 binds to the fingers domain 15,16. Structural information is also available for nsp8-nsp7 complexes 17,18. To obtain the structure of the SARS-CoV-2 RdRp in its active form, we prepared recombinant nsp12, nsp8 and nsp7 (Fig. 1a, Methods). When added to a minimal RNA hairpin substrate (Fig. 1b), the purified proteins gave rise to RNA-dependent RNA extension activity, which depended on nsp8 and nsp7 (Fig. 1c). We assembled and purified a stable RdRp-RNA complex with the use of a self-annealing RNA, and collected single-particle cryo-electron microscopy (cryo-EM) data (Ex...

RNA-Dependent RNA Polymerase From SARS-CoV-2. Mechanism Of Reaction And Inhibition By Remdesivir

“…1 E). Overall the active site emerging from EM structures 12,14,39,40 and atomistic simulation strongly suggest that despite the reduced evolutionary refinement, SARS-CoV-2 RdRp has all the structural requirements to be an efficient RNA polymerase, both in terms of catalysis and substrate specificity. The RNA-pol reaction mechanism.…”

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

“…In addition, protein/RNA condensates can recruit additional proteins to promote reactions. To investigate if the RNA-dependent RNA polymerase (RdRp; Figure 5A) concentrates within N SARS-CoV-2 /RNA droplets, we recombinantly prepared the non-structural protein (nsp) 12 of SARS-CoV-2, together with the accessory sub-units nsp7 and nsp8, which are required for transcription (Hillen et al, 2020). First we used nsp12, in order to investigate if the catalytic component of RdRp is recruited to N SARS-CoV-2 /polyU droplets.…”

“…Next, nsp12, nsp7 and nsp8 were reconstituted in a 1:1:2 stoichiometry together with a RNA template-product duplex, which carried fluorescein at the 5' end (Hillen et al, 2020). The RdRp/RNA-complex was added to preformed N SARS-CoV-2 /polyU droplets into which the RdRp/RNA-complex was recruited ( Figure 5C).…”

“…SARS-CoV-2 nsp12 was expressed in insect cells and purified as described previously (Hillen et al, 2020). SARS-CoV-2 nsp7 and nsp8 were expressed in E.coli and purified as described previously (Hillen et al, 2020).…”

“…SARS-CoV-2 nsp12 was expressed in insect cells and purified as described previously (Hillen et al, 2020). SARS-CoV-2 nsp7 and nsp8 were expressed in E.coli and purified as described previously (Hillen et al, 2020). An RNA scaffold for RdRp/RNA complex formation was annealed by mixing equimolar amounts of two RNA strands (5'-rUrUrUrUrCrArUrGrCrArUrCrGrCrGrUrArG rGrCrUrCrArUrArCrCrGrUrArUrUrGrArGrA -3'; 56-FAM/rUrCrUrCrArArUrArCrGrGrUrArUrGrArGrC CrUrArCrGrCrG-3') (IDT Technologies) in annealing buffer (10 mM Na-HEPES pH 7.4, 50 mM NaCl) and…”