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...

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

The gene coding for the protein methyl transferase HemK (ECBD_2409, 834 bp, 277 aa) was amplified from the genomic DNA of E. coli BL21 DE3 by colony PCR using the primers 5'-GTCCGAGCAGGACATATGGAATATCAA-3' and 5'-GCAGTGTAG AAAAACCTCGAGTTGATAAT-3', digested with NdeI and XhoI (New England Biolabs) and ligated into a pET-24a Vector (Novagen). The gene sequence encoding for the N-terminal domain of E. coli HemK (residues 1-73, HemK NTD hereafter) was amplified by PCR from a vector encoding full-length HemK (residues 1-277), using primers containing recognition sequences for NdeI and XhoI restriction enzymes for subsequent cloning into expression vector pET24-a (Novagen). The purified PCR fragment was digested with NdeI and XhoI restriction enzymes for 1 h and ligated into the expression vector pET-24a linearized with the same restriction enzymes. The Leu-Glu sequence (encoded by the XhoI restriction used for cloning) was subsequently removed by inverse deletion PCR. Mutants of wild type NTDHemK were made by PCR using a commercial site-directed mutagenesis kit (Stratagene, Carlsbad, CA) according to the manufacturer's instructions. We note that HemK from E. coli BL21 DE3 contains Lys at position 34, in contrast to HemK from E. coli K12, which contains Arg at position 34 and the structure of which is available. In the following, the sequence ofHemK from E. coli BL21 DE3 is denoted as wild type (wt). Endogenous tryptophan residues at position 6 and 78 were replaced with phenylalanine in all constructs using the quick-change method. For PET experiments tryptophan residues were introduced at position 6, 38, and 49 using site-directed mutagenesis. All constructs were verified by sequencing. Expression and purification of HemK NTDThe plasmid encoding HemK NTD (or its mutants) was transformed into competent E. coli Fluorescence emission spectra and equilibrium chemical unfoldingFluorescence emission spectra were recorded at 25 °C with a Horiba model Fluorolog 3 spectrofluorimeter connected to a circulating water bath. Measurements were performed in a quartz cuvette of 1-cm path length at a protein concentration of 2 µM in buffer E.Fluorescence was excited at 280 nm. Fluorescence emission spectra were collected between 310 and 420 nm in 1 nm increments and were corrected for background (buffer without protein). Slid widths of 1 and 10 nm were used for excitation and emission, respectively. Samples were incubated for 2 hours before measurements were taken. For each collected background-corrected fluorescence emission spectrum, the normalized global emission