Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

Fig. 1. Fe-S cluster incorporation into nsp12 occurs through its interactions with components of the Fe-S biogenesis machinery.

(A) Representative Coomassie blue staining of pull-down assays performed with purified proteins. Purified nsp12-FLAG (0.25 μg) or the variants wherein either or both LYR motifs were replaced by alanines (VYR-AAA, LYR-AAA, and VYR/LYR-AAA, respectively) were combined with 0.25 μg of HSC20, as indicated. Immunoprecipitations (IPs) were performed with anti-FLAG antibody to immunocapture nsp12 proteins. The presence of HSC20 (i.e., HSCB) in the eluates after IPs of nsp12 proteins was analyzed by SDS–polyacrylamide gel electrophoresis and Coomassie staining. Aliquots corresponding to 20% of the inputs were run on the gel for comparison (n = 5 biological replicates). (B) Eluates after IPs of nsp12 WT or variants recombinantly expressed in Vero E6 cells, as indicated, were probed with antibodies against FLAG to verify the efficiency of IP and against components of the Fe-S cluster (HSC20, HSPA9, and NFS1) and of the cytoplasmic Fe-S (CIA) assembly machinery (CIAO1, MMS19, and FAM96B) (n = 6). (C) Mass spectrometry identification of affinity purified interacting partners of nsp12 that are components of the Fe-S cluster biogenesis pathway (see data S1 for a complete list). The protein ratios were calculated as reported in the methods (n = 6). The maximum allowed fold change value was set to 100. In the instances (marked with a superscript P) in which the interacting partner was detected in the nsp12-only samples and not in the negative controls, the nsp12/control ratios were set to 100 and reported without P values. (D) Levels of radiolabeled iron (⁵⁵Fe) incorporated into nsp12 WT or the variants in control cells transfected with nontargeting siRNAs (NT siRNAs) and in cells transfected with siRNAs directed against the main scaffold protein ISCU (si-ISCU). Levels of iron stochastically associated with the beads in lysates from cells transfected with the backbone plasmid (empty-vector, p3XFLAG-CMV-14) are also reported (accounting for 587 ± 292.62 cpm/mg of cytosolic proteins) and were not subtracted from measurements of radiolabeled iron incorporated into nsp12 WT or variants in the chart (n = 4). Significance was determined by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Mean ± 95% confidence interval (CI). ***P < 0.001. (E) Representative Coomassie staining showing levels of nsp12 WT or variants in control and ISCU-depleted cells that were quantified in (D) for their iron content. Immunoblots to ISCU, showing the efficiency of its silencing (knock down), and to α-tubulin (TUB), used as a loading control, are also shown.

Fig. 2. Evidence for ligation of two Fe-S metal cofactors by nsp12.

(A) UV-vis spectra of nsp12 WT or variants of the cysteine residues in the two metal ligating centers. (B) Representative Coomassie blue staining of purified nsp12 WT or variants analyzed in (A). (C) Mössbauer spectra of nsp12 WT and variants exhibited the parameters typical of [Fe₄S₄] clusters. For each of the two nsp12 Cys-to-Ser variants, ~95% of iron was still associated with a quadrupole doublet that matched parameters of WT nsp12. (D) RNA polymerase activity of anoxically purified RdRp ([Fe-S]-RdRp at 1 μM) and aerobically purified RdRp reconstituted with zinc and containing two zinc ions per protomer (Zn-RdRp at 1 μM) (n = 4). (E) Conserved zinc-binding motifs in SARS-CoV-2 nsp12 [Protein Data Bank (PDB) ID 7BTF] (8) rendered in the ribbon representation. H295-C301-C306-C310 ligate zinc at the interface between the NiRAN and the catalytic domain, whereas the C487-H642-C645-C646 residues ligate zinc in the catalytic domain. (F) Levels of radiolabeled iron (⁵⁵Fe) incorporated into nsp12 WT or variants, as indicated. ⁵⁵Fe content of nsp12 treated with the chelator EDTA is also reported to provide a control for the complete loss of ⁵⁵Fe in the protein (n = 4). Significance was determined by two-way ANOVA and Sidak’s multiple comparisons test. Mean ± 95% CI. ***P < 0.001.

Fig. 3. Fe-S cluster sites in nsp12 are important for activity and interactions with nsp13.

(A) RNA polymerase activity of anoxically purified RdRp (all lanes except Zn-nsp12) (RdRp at 1 μM) and of aerobically purified and Zn-reconstituted RdRp containing two zinc ions per protomer (three technical replicates are shown; n = 4). (B) Schematic of the complex required for coronaviral replication (10), in which the two Fe-S clusters and their coordination spheres are highlighted. ExoN, exoribonuclease; SSB, single-stranded DNA-binding protein; 2′-O MTase, 2′-O methyltransferase. (C) Co-IP of nsp12 WT or variants recombinantly expressed in Vero E6 cells cotransfected with helicase nsp13 and accessory factors nsp7 and nsp8 (Strep II tagged) probed with antibodies against FLAG, Strep II, or nsp13 (three technical replicates are shown; n = 4).

Fig. 4. The stable nitroxide TEMPOL potently inhibited the RdRp by causing disassembly of its Fe-S clusters and blocked viral replication in cell culture models of SARS-CoV-2 infection.

(A) UV-vis spectra of nsp12 anoxically purified from Expi293F control cells and from cells treated with TEMPOL. (B) UV-vis spectra of purified nsp12 and of purified nsp12 incubated with TEMPOL (1:2 ratio nsp12:TEMPOL) for 10 min. (C) RNA polymerase activity of the RdRp complexes anoxically purified from control and TEMPOL-treated (T) Expi293F cells. (D) Representative Coomassie staining of the RdRp complexes analyzed for activity in (C). (E) RNA polymerase assay of the RdRp complexes (at 1 μM) anoxically purified from control or DEA/NO- or TEMPOL-treated Vero E6 cells, as indicated (n = 4). (F) Titer of infectious virus produced at 48 hours measured by TCID₅₀ (median tissue culture infectious dose) assay in Vero E6 cells infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.1 or 0.01 (n = 3).