Coronaviral RNA-methyltransferases: function, structure and inhibition

Abstract

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.

Figure 1.

Comparison of human and coronaviral mRNA cap formation. RNGTT - RNA guanylyltransferase and 5′-phosphatase, RNMT, RNA guanine-7 methyltransferase; CMTR1, Cap methyltransferase 1; CMTR2, Cap methyltransferase 2.

Figure 2.

Recognition of ‘pre-cap’ and cap-0 structures by IFIT1 and IFIT5. (A, C) Comparison of homologous overall structures of IFIT5 (gold) and IFIT1(light blue). Both of these proteins have a deep RNA binding pocket (B, D). The pocket of IFIT1 cannot accommodate an RNA molecule bearing a cap-1 or cap-2 that have methylated 2′-OH ribose groups (clash space depicted as cyan spheres), while IFIT5 can only accommodate 5′-ppp-RNA.

Figure 3.

Coronaviral 2′-O-MTase - the nsp16 subunit. (A) Primary sequence alignment highlighting the most important residues. Residues forming the SAM and m⁷GpppA binding sites are marked by asterisks and circles, respectively. See Supplementary Figure S1 for full sequence alignment. (B) Structures of SAM, SAH, and sinefungin. Amino acid moiety is shown in green, sugar is in red, and the base is in blue. (C) Cartoon representation of nsp16 from SARS-Cov-2, the structure resembles a sandwich with a β-sheet in the middle (magenta) and the slices of bread made of α-helices (cyan). (D) Superposition of the known structures of coronaviral nsp16s revealed a very high conservation of the SAM binding and of the active site of this MTase. The catalytic tetrad (Lys46, Asp130, Lys170 and Glu203) is highlighted by a circle. (PDB IDs: MERS-CoV:5YNB, SARS-CoV:2XYR, SARS-CoV-2:6YZ1, OC43-CoV:7NH7). (E) Interactions of sinefungin with the active site of the enzyme (SARS-CoV-2). The amino acids involved in the interaction with sinefungin are shown as sticks, the water molecules are shown as red spheres, and selected hydrogen bonds are depicted as dashed lines. The left side of the panel shows to view along sinefungin, whilst the right side is rotated by ∼90° and down by ∼30°.

Figure 4.

RNA binding towards the coronaviral 2′-O-MTase. The crystal structure of nsp10/nsp16 from coronavirus SARS-CoV-2 with cap-1 as the product along with SAH (PDB ID: 7L6R). Nsp16 is in cyan and nsp10 in orange, m⁷G is locked in the tunnel of a long RNA binding pocket spanning across the nsp10/nsp16 heterodimer.

Figure 5.

Crystallographic snapshots of methylation and conformational changes during cap-1 synthesis by coronaviral 2′-O-MTase. (A, B) Opened and closed conformation of Tyr30 and Lys137 of nsp16. (C) Cap-1 locked in the RNA binding pocket by the action of Tyr30 and Lys137 whilst Pro134 and Lys135 are reorganized to accommodate the first nucleoside of RNA. The methyl group of SAM (yellow sphere) is ready to be transferred on the 2′-O position of the ribose ring. (D) Methylated ribose forms a complete cap-1 RNA. (E, F) Detailed view of the interaction and the degree of movements between residues Tyr30 and Lys137 in nsp16. Paired residues from individual crystal structures are color matched. Both RNA bound structure (PDB IDs: 6WKS and 7L6R) display identical orientation of these two residues whilst locking m7G. RNA is in white sticks for clarity.

Figure 6.

Mechanism of the 2′-O methylation reaction. (A) view on the active site with SAM, the catalytic tetrad (Lys46, Asp130, Lys170 and Glu203) and the substrate RNA molecule (cap-0), (B) identical view on the product cap-1, active site and the side product SAH. (C, D) detailed view on catalytic site and the ribose where a methyl group (yellow sphere) is transferred. (E, F) Model of the catalytic mechanism based on the crystal structures (PDB IDs:6WKS and 7L6R).

Figure 7.

Structural alignment of MTases from ZIKV and SARS-CoV-2. (A) overall structures of ZIKV (yellow) and SARS-CoV-2 (cyan) MTases (B) conserved residues of the catalytic tetrad from both enzymes in the vicinity of sinefungin.

Figure 8.

Structure of the coronaviral nsp10/nsp14 complex. (A) Schematic representation of the domain structure of the SARS-CoV nsp14/nsp10 protein complex. (B) The overall fold of the SARS-CoV nsp14/nsp10 protein complex and detailed views of the ExoN active site and the SAH- and GpppA-binding sites (based on PDB ID: 5C8S). (C) Sequence alignment of the active sites of selected coronaviral nsp14 proteins. Residues forming the ExoN domain active center and the SAH- and GpppA- binding sites are marked by triangles, asterisks, and circles, respectively. See SI Fig. 2 for full sequence alignment.

Figure 9.

Assembly of the SARS-CoV-2 co-transcriptional capping complex. Elongation replication-transcription complex (E-RTC) composed of nsp7, nsp8, nsp12, and nsp13 is shown as a grey surface. The NiRAN subdomain of nsp12 is colored black in cartoon representation. The template and primer RNA strands bound to E-RTC are shown as green and red sticks, respectively. The single-stranded RNA binding protein nsp9 is shown as a green surface. The nsp10/nsp14 complex is depicted as in Figure 8, i.e. nsp10 is shown as an orange surface, while nsp14 is shown in cartoon. The figure was generated using the structures with PDB ID: 7EGQ (17), 7CYQ (12) and 5C8S (32).

Figure 10.

Inhibitors of coronaviral MTases and their IC₅₀ values and selectivity based on enzymatic assays. (A) SAM analogues with modified amino acid moiety (1–4). (B) Non-specific SAM analogues with more lipophilic substituents 5 and 6. (C) Adenine dinucleoside inhibitor 7. (D) SAH analogues 8 and 9 with modified nucleobase. (E) The most active compounds (10–12) discovered by HTRF (homogeneous time resolved fluorescence) assay. (F) Three compounds (13–15) with antiviral effect on SARS-CoV-2. (G) Tizoxanide 29 and nitazoxanide 30. Inhibitors are divided into colored boxes according to their structural motive: yellow – SAM derivatives, green – structure mimicking the transit state, purple – random structures received by HTS. Color-coding of SAM analogues structures is: green – amino acid moiety, red – sugar part and blue – nucleobase. BCDIN3D – bicoid interacting three domain containing RNA MTase, DOT1L – disruptor of telomeric silencing-1 like histone lysine MTase, GNMT – glycine N-MTase, hRNMT – human RNA N7-MTase, RNMT-RAM – complex of RNMT and RNMT-activating miniprotein.