The new coronavirus (SARS-CoV-2) is a new member of the genus β-coronavirus and is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) and several bat coronaviruses. Compared with SARS-CoV and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV-2 spreads extremely fast.
RNA-dependent RNA polymerase (RdRp, also known as nsp12) is a core component of coronavirus replication and transcription. It catalyzes the synthesis of viral RNA and plays an important role in the replication and transcription cycle of SARS-CoV-2. nsp12 is the main target of antiviral inhibitors such as Remdesivir. On April 10, 2020, Rao Zihe, an academician of the Chinese Academy of Sciences, and Lou Zhiyong of Tsinghua University, Wang Quan of Shanghai University of Science and Technology, and others analyzed the structure of the cryo-electron microscope of the complex of full-length SARS-CoV-2 nsp12 and cofactors nsp7 and nsp8 The resolution is 0.29nm. In addition, the study found that in addition to the viral polymerase core conserved domain, nsp12 also has a β-hairpin domain at the N-terminus, which may provide a research direction for revealing the biological function of nsp12. This study reveals the internal mechanism of the combination of redoxivir and nsp12 through a comparative analysis model, which provides a research basis for designing new antiviral drugs for nsp12.
After incubating full-length nsp12 (residue S1-Q932) with nsp7 (residue S1-Q83) and nsp8 (residue A1-Q198), the researchers separated and purified the complex by column chromatography. After preliminary screening, the researchers obtained a three-dimensional reconstruction of the nsp12 and nsp7-nsp8 complex with a resolution of 0.29nm, which provided structural information for the analysis of the complete structure of SARS-CoV-2. In addition to the nsp12-nsp7-nsp8 complex, the researchers also observed single-particle classes corresponding to the nsp12-nsp8 dimer, as well as individual nsp12 monomers, but these did not give a reconstructed structure of atomic resolution. The structure of nsp12 contains a “right-handed” RdRp domain (S367-F920) and a unique N-terminal extension domain (D60-R249). The two are connected through the interface domain (A250-R369). Among them, the RdRp domain includes the finger subdomains (residues S367-A581 and K621-G679), palm subdomains (residues T582-P620 and T680-Q815), and thumb substructures Domain (residue H816-E920).
The active site of the nsp12 domain is formed by the conserved polymerase motifs A-G in the palm domain and is arranged like other RNA polymerases. Motif A rich in residues (611-TPHLMGWDYPKCDRAM-626) contains the classic divalent cation-binding residue D618. Motif B is located in the region of 680-710 amino acids (TSSGDATTAYANSVFNICQAVTANVNALLST). Motif C (753-FSMMILSDDAVVCFN-767) contains catalytic residues (759-SDD-761) in sequence between 25 beta chains. These catalytic residues are also conserved in most viral polymerases. For example, the first residue in HCV ns5b (317-GDD-319) and PV 3Dpol (327-GDD-329) is serine or glycine. Motif D is located in the amino acid region of 775-796 (LVASIKNFKSVLYYQNNVFMSE). Motif E (810-HEFCSQHTMLV-820) forms two tight loops. Motif F (912-KKNQHGGLRE-921) forms an ordered loop (β loop) connecting two antiparallel β chains. Motif G is located in the 500-518 amino acid region (KSAGFPFNKWGKARLYYDS).
During the assembly of nsp12 and RNA, the RNA template, primer entry channel, nucleoside triphosphate (NTP) entry channel, and nascent strand exit channel are all positively charged and converge in the central cavity of nsp12. In the central cavity, the nsp12 motif mediates RNA templates to guide RNA synthesis. The NTP entry channel is formed by a set of hydrophilic residues, including some residues in motif F. The RNA template is expected to enter the active site composed of motifs A and C through the grooves sandwiched by motifs F and G. The motif E and the “thumb-like” subdomain support the primer chain. The product-template hybrid leaves the active site through the RNA exit channel on the front of the polymerase.
Sofosbuvir (Sofosbuvir) is a prodrug targeting chronic hepatitis C (HCV) ns5b polymerase and has been approved for the treatment of HCV infection. It mainly works by binding to the catalytic site of HCV ns5b polymerase. In view of the fact that both redoxil and sofosbuvir are nucleotide analogues and the structure of the catalytic site between nsp12 and HCV ns5b polymerase is conserved, the researchers reconstructed redox based on the combination of sofosbuvir and HCV ns5b polymerase A model of binding of civir diphosphate to nsp12. The researchers found that ridxivir retains the complete ribose group, which can use the hydrogen bonding structure like a natural substrate. In addition, T680 in nsp12 may also form a hydrogen bond with the 2′-hydroxyl group of redoxivir and the introduced natural NTP. At the same time, the hydrophobic side chain of V557 in motif F may stack and stabilize with the uridine base of the +1 position RNA template, and base pair with the incoming ppp-remdesivir triphosphate.
Embedding model of redcive in SARS-CoV-2 nsp12
The rapid spread of SARS-CoV-2 around the world shows the necessity of developing vaccines and therapeutic agents. As an ideal target in new therapies, nsp12 is mainly based on the inhibitory effect of redoxivir on viral replication. Based on the structural similarity of the nucleoside analogues, the mechanism of inhibition of redoxivir and nsp12 discussed in this study may also be applicable to other such drugs or drug candidates, such as pirpiravir proved effective in clinical trials Favipiravir). In addition, the research also provides a reference for supporting the development of anti-SARS-CoV-2 mixed drugs.