Novel Coronavirus Polymerase and Nucleotidyl-Transferase Structures: Potential to Target New Outbreaks.

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.

To date (May 11, 2020), more than 3.9 million worldwide cases of infection and 274 000 deaths have been attributed to the novel coronavirus, SARS-CoV-2, since its emergence in December 2019. This new viral disease has spread to more than 210 countries with an increasing number of people still being infected. Furthermore, human-to-human transmission[1,2] of SARS-CoV-2 has been confirmed and virus survival on hard surfaces for longer time periods has been reported.[3]

Coronaviruses (CoVs), a type of RNA virus, are enveloped viruses with a single-strand, positive-sense RNA genome of approximately 26–32 kilobases in size. Known examples include severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV).[4] The latest reports show that the closest relatives to SARS-CoV-2 are the bat SARS-related coronaviruses found in Chinese horseshoe bats as determined by phylogenetic analysis and next-generation sequencing.[2] The SARS-CoV-2 genome shares 88% sequence identity with two bat-derived SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21), approximately 79% with SARS-CoV, and 50% with MERS-CoV.[2] Homology modeling revealed that SARS-CoV-2 has a receptor-binding domain structure similar to that of SARS-CoV.[2] The RNA-dependent RNA polymerase (RdRp) of SARS-CoV is essential for viral replication and is a potential target for anti-SARS drugs.[5] Crystal structures of RdRps from different RNA viruses have revealed key aspects in the structural biology of RdRps and confirmed the hypothesis that RdRps share a common architecture and mechanism of polymerase catalysis.[6,7] No mammalian cells have been shown to encode any RdRp or its equivalent; therefore, inhibition of RdRp is not anticipated to result in undesirable side effects during therapy.[8] Recently reported attempts to treat SARS-CoV-2 infections by targeting RdRp using an antiviral drug currently under clinical assay, Remdesivir, support the necessity for our structural study on the virus RdRp.[9]

The sequences of SARS-CoV-2 nonstructural protein12 (nsp12, 932 amino acids [a.a.s]), RdRp protein (part of nsp12, 535 a.a.s), and Spike protein (1273 a.a.s) were aligned with 5 other strains of human coronaviruses (Table and Supplementary Table 1). Sequence alignments and comparisons indicate that SARS-CoV-2 RdRp shares a high sequence identity with other coronavirus RdRps (60.9%–98.1%). However, SARS-CoV-2 Spike has a significantly lower sequence identity with other coronavirus Spikes (27.4%–77.4%). Moreover, both protein sequences of SARS-CoV-2 have a higher identity with SARS-CoV compared with other CoVs. This higher sequence conservation among RdRps in the coronavirus family compared with Spike protein supports the argument of finding an inhibitor of RdRp in combating the novel outbreaks.[10] In addition, a sequence comparison between SARS-CoV and SARS-CoV-2 has revealed 96.4% identity between nsp12s, with RdRps showing a slightly higher identity of 98.1%.

Table 1

Percentage Identity Matrix of Different Coronavirus RdRps and Spikes

A. Percentage Identity Matrix of Different Coronavirus RdRps
	HCoV-NL63	HCoV-229E	HCoV-OC43	MERS-CoV	SARS-CoV-2	SARS-CoV
HCoV-NL63		83.74	60.19	63.36	60.93	61.31
HCoV-229E	83.74		60.56	62.43	61.31	61.68
HCoV-OC43	60.19	60.56		73.08	71.96	71.59
MERS-CoV	63.36	62.43	73.08		75.51	75.89
SARS-CoV-2	60.93	61.31	71.96	75.51		98.13
SARS-CoV	61.31	61.68	71.59	75.89	98.13

Structural studies of RdRp and NiRAN were performed using Modeler v9.23. Because of the lack of a suitable template, only a.a.s from 117–895 of nsp12 were modeled. During revision of this Letter, the crystal structure of this RdRp in complex with cofactors was made available in the PDB (ID: 6M71).[11] An r.m.s.d. of 0.5 Å between the crystal structure and our model indicates the quality of the structure we modeled. The SARS-CoV-2 nsp12 (Figure B) and SARS-CoV nsp12 structures (Figure C) showed high similarity. The nsp12 protein has been reported to have an N-terminal nidovirus RdRp-associated nucleotidyl-transferase (NiRAN) (a.a.s 1–250), and a C-terminal RdRp (a.a.s 398–932) connected by an interface domain (Figure A).[7,12] NiRAN is essential for replication of SARS-CoV and other nidoviruses and can be involved in nucleic acid ligation, mRNA capping, and protein-primed RNA synthesis.[13] However, the mechanism of its nucleotidyl-transferase activity is still not completely clear. We found that the NiRAN sequence of SARS-CoV-2 showed 93.2% identity with SARS-CoV. A total of 17 substituted a.a.s appeared in SARS-CoV-2. However, the well-conserved a.a.s, Lys73, Arg116, Thr123, Asp126, Asp218, and Phe219, were unchanged.[7] Meanwhile, RdRp showed a very high similarity of 98.1% with only 10 substitutions. RdRp comprises a finger domain, a palm domain, and a thumb domain. The finger domain revealed two important conserved motifs named motif G and motif F. The palm domain forms the catalytic core of the polymerase and has four conserved motifs, A to D, and part of motif E. Considering the structure of the modeled nsp12 protein, the NiRAN domain may engage in a functional interaction with the palm domain as these two domains are in close spatial proximity. In comparison with SARS-CoV, three substitutions were apparent in the polymerization zone: Tyr766Phe in motif C, one Ala772Ser, and one Ala784Ser in motif D (Figure A,B). The hydrogen bonds between Tyr766Phe and Ala772Ser with other a.a.s are similar, whereas Ala784Ser provides an additional hydrogen bond (3.2 Å to Tyr787) in SARS-CoV-2 when compared with SARS CoV. The thumb domain is essential for nucleic acid binding. SARS-CoV-2 showed 100% a.a. sequence identity with SARS-CoV. However, superimposition analysis of SARS-CoV-2 and SARS-CoV RdRp suggests two structural modifications in the finger and thumb domains (Figure C). These changes are located at the start of the finger domain (a.a.s 405–448, colored orange and cyan, respectively) and thumb domain (a.a.s 835–895, colored pink and cyan, respectively). Because of the high a.a. sequence identity, the difference appears to be minimal.

Figure 1

Structures of SARS-CoV-2 and SARS-CoV nsp12 showing high similarity. (A) Diagram of SARSCoV- 2 nsp12 with conserved motifs in each domain. (B) Modeled SARS-CoV-2 nsp12 (a.a.s 117–895) and (C) SARS-CoV nsp12 (PDB: 6NUR, a.a.s 117–895, 906–919) structure. NiRAN is colored purple, and the interface domain is colored green. Three RdRp domains finger, palm, and thumb are colored yellow, blue, and red, respectively.

Figure 2

Differences between SARS-CoV-2 and SARS-CoV RdRp. Three a.a. substitutions in motif C and motif D in the palm domain of (A) SARS-CoV-2 and (B) SARS-CoV. Hydrogen bonds connected to each atom are shown in cyan and red. (C) Superimposition of SARS-CoV-2 and SARS-CoV RdRp demonstrated two regions of dissimilarity at the start of the finger domain (a.a.s 405–448, orange and cyan, respectively) and thumb domain (a.a. 835–895, pink and cyan, respectively). (D) Superimposition of the nsp7 structure of SARS-CoV-2 (red) and SARS-CoV (blue). The only substitution Arg70Lys is in cyan and yellow, respectively. (E) Superimposition of the nsp8 structure of SARSCoV-2 (red) and SARS-CoV (blue).

Two cofactors for RdRp nsp7 and nsp8 have also been modeled (Figure D,E). Both nsp7 and nsp8 play essential roles in the formation and activity of RNA synthesis machinery.[14] Although the function of nsp7 and nsp8 in RNA synthesis is not completely elucidated, they may act as processivity factors on RdRp by extending primed RNA templates and boosting the activity of RdRp.[15,16] In SARS-CoV, nsp7 and nsp8 may form a super complex assembled from eight copies of nsp8 that are held tightly together by eight copies of nsp7.[12,15] Nsp7 in SARS-CoV-2 and SARS-CoV have 83 a.a.s with only one substitution Arg70Lys. Superimposition analysis showed structural changes at the terminal region, possibly imposed by Arg70Lys substitution (Figure D). The nsp8 sequences showed 97.5% similarity with only 5 substitutions and formation of the well-known “golf-club” structure (Figure E).[15] Superimposition analysis found no significant change to their structure.

Knowledge of the nsp12 structure permitted us to test drug–RdRp interactions. Docking analyses of three drugs that target RdRps including Galidesivir,[17] Favipiravir,[18,19] and Penciclovir[19] were performed using the program CCDC GOLD. Galidesivir (originally developed for hepatitis C), Penciclovir (used for the treatment of various herpesvirus infections), and Favipiravir (used to treat influenza in Japan) are reported to have clinical significance in treating COVID patients. ATP was included as a positive control. The triphosphate of all the molecules docked near the conserved catalytic center Asp760 and Asp761 in motif C[7,12] (Figure and Supporting Information). Analysis of docking poses revealed a significant number of protein–ligand interactions including hydrogen bond and hydrophobic interactions (Table and Supporting Information). Many of the amino acids interacting with ATP were observed to establish contacts with the drug molecules. Galidesivir established four hydrophobic and six hydrogen bond interactions with nsp12. Favipiravir established seven hydrophobic and five hydrogen bond interactions. Penciclovir established five hydrophobic and seven hydrogen bond interactions. It was unsurprising that most of these a.a.s are located in the RdRp motifs with the exceptions of Asp452 and Thr455, indicating that these drugs could significantly affect the function of RdRp. Of importance is that all three drugs showed a hydrophobic interaction or hydrogen bond interaction with the catalytic center Asp760 or Asp761. Arg553 was previously reported to be important in rNTP binding and positioning of template overhang.[20−23] Multiple contacts made by the three drug molecules with these amino acids indicate that they may interfere with rNTP binding. Asp623, an important amino acid in sugar recognition of rNTP,[20−22] was also shown to make contacts with all three drug molecules. Galidesivir and Penciclovir made contacts with Asn691, which help in sugar selection.[20,21] The drug molecules may all interfere with metal ion chelation as they have been shown to interact with Asp618, Asp760, and Asp761, which are important in metal ion chelation.[20−24]

Figure 3

Docked complexes of Galidesivir (A), Favipiravir (B), and Penciclovir (C) with RdRp and their active form 2D structures. RdRp is represented as a surface, and ligands are represented as sticks. The a.a.s interacting with RdRp are colored in cyan. NiRAN is colored purple, and the interface domain is colored green. Three RdRp domains—finger, palm, and thumb—are colored yellow, blue, and red, respectively. The a.a.s that have a hydrogen bond or hydrophobic interaction with the drugs are shown by sticks and labeled.

Table 2

Scores Obtained for Docking of Three Clinically Used RdRp Inhibitors

PLP score	name	hydrophobic interactions with RdRp	hydrogen bond interactions with RdRp
56.45	ATP	Lys621, Arg624, Cys813	Asp452, Ala554, Arg553, Tyr619, Cys622, Asp623, Asp760, Asp761, Ser814
62.09	Galidesivir	Thr455, Arg553, Lys621, Arg624,	Asp452, Ala554, Asp623, Asn691, Ser759, Asp760
63.78	Favipiravir	Arg553, Arg555, Pro620, Lys621, Asp623, Asp760, Asp761	Asp452, Ala554, Asp618, Tyr619, Cys622
49.46	Penciclovir	Arg553, Arg555, Pro620, Cys622, Arg624	Thr556, Tyr619, Lys621, Asp623, Asn691, Ser759, Asp760

In conclusion, docking analysis demonstrated that the clinically used antiviral drugs Galidesivir, Favipiravir, and Penciclovir can bind to the catalytic center of SARS CoV-2 RdRp, establishing various hydrogen bond and hydrophobic interactions. Understanding the binding mechanism of the drug molecules may assist in optimizing these drug molecules to efficiently target the RdRp of SARS-CoV-2. Moreover, the remarkably high structural conservation among RdRps of coronaviruses strongly suggests their potential as therapeutic targets by implementing improvements to available inhibitors of key viral enzymes when an unprecedented rapid response is required.