MINIREVIEW: SARS-CoV-2 RNA-dependent RNA polymerase: structure and target for pharmacological intervention

Author of summary: Livia Tepshi, Giulia Poggi; Reviewer: Valeria Montis

Resolving SARS-CoV-2 protein structures is essential to develop appropriate and specific pharmacological intervention to fight the current CoViD-19 pandemic. The RNA-dependent RNA polymerase (RdRp, also called Nsp12) is the central component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug Remdesivir.  Notably, the resolved structure of RdRp can also serve as a model to design new active compounds targeting Nsp12.

CoViD-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to SARS-CoV and MERS-CoV, SARS-CoV-2 exhibits faster human-to-human transmission [1]. Nsp12 (non-structural protein) of SARS-CoV-2 was shown to be fundamental in its replication and transcription cycle, acting as the catalytic subunit of an RNA-dependent RNA polymerase (RdRp) with the help of Nsp7 and Nsp8 as cofactors, (Fig.1). RdRps indeed are multi-domain proteins able to catalyze RNA-template dependent formation of phosphodiester bonds between ribonucleotides. Nsp12 is therefore considered as the primary target for nucleotide analogue antiviral inhibitors such as Remdesivir, a potential treatment against CoViD-19 [2].

Figure 1, adapted from Huang et al. 2020
Figure 1, adapted from Huang et al. 2020

SARS-CoV-2 RdRp structure

Via cryo-electron microscopy, Gao et al. and Hillen et al. have recently resolved (2.9-Å resolution) the structures of inactive and active RdRp, respectively [3, 4]. The main structural information is summarised below:

Subunit Abbreviation Type Structural information
Non-structural protein 12 Nsp12 catalytic N-terminal norovirus RdRP-associated nucleotidyltransferase (NiRAN)
      Interface domain connects N-terminal domain and C-terminal domain
      C-terminal RdRp domain that resembles a right hand: finger subdomains (residues L366-A581 and K621-G679); palm subdomain (residues T582-P620 and T680-Q815); thumb subdomain (residues H816-E920);
      Active site is localised in the palm domain. Motif A (611-TPHLMGWDYPKCDRAM-626) within the palm domain contains the classic divalent-cation-binding residue D618, which is conserved in most viral polymerases including HCV ns5b (residue D220) and poliovirus (PV) 3Dpol (residue D233). Motif C contain the catalytic residues
      Binds the first turn of RNA: motif C interacts with the RNA 3′-end motif F and G position the RNA template
      Nucleoside triphosphate (NTP)-binding site is conserved (comparison with Norovirus RdRp)
Non-structural protein 8 Nsp8 accessory The two copies bind to the fingers and thumb subdomains of Nsp12
      The two Nsp8 copies interact differently with Nsp7 and Nsp12
      Quite flexible and adaptive to the incoming RNA
      13 additional amino acid residues at the N-terminal, probably to prevent premature dissociation of the polymerase during replication. Mutation in K58 to alanine in this region is lethal for the virus.
      N-terminal regions flank the  RNA duplex that exits the Nsp12
      Fundamental for RNA extension activity
      Extended protein region when bound to RNA
Non-structural protein 7 Nsp7 accessory binds to the thumb of Nsp12
      Fundamental for RNA extension activity
      Nsp7/Nsp8 pair shows a conserved structure similar to the SARS-CoV nsp7/nsp8 pair

Pharmacological interventions against RdRp

Currently, one of the most studied inhibitors of RdRp in the context of CoViD-19 treatment is Remdesivir. Remdesivir (GS-5734, Gilead), initially developed to prevent Ebola Virus (EBOV) replication [6], is a nucleoside analogue prodrug reported to inhibit SARS-CoV-2 proliferation. By resembling an RNA building block ATP (adenosine triphosphate), Remdesivir can be incorporated by RdRp and stop the processing of viral RNA. Importantly, Remdesivir can effectively distinguish the viral RNA polymerase from the human mitochondrial RNA polymerase (panel from Owen et al.) [6]. The detailed mechanism of action of Remdesivir has still to be confirmed. In insect cells expressing SARS-CoV and SARS-CoV-2 RdRp complexes, the triphosphate form of Remdesivir (RDV-TP) appears to act as a non-obligate chain terminator; it is incorporated into the RdRP complex and stops RNA synthesis after few further endogenous ribonucleotide incorporation [7]. These additional nucleotides could be fundamental to protect from the proofreading and exonuclease activity of Nsp14 [7]. Yin et al. [8] provided further structural data in support of the proposed mechanism of action of Remdesivir. Via Cryo-EM, they resolved the structure of the Nsp12/Nsp7/Nsp8 bound to the template-primer RNA and to the triphosphate form of Remdesivir with a 2.5 Å resolution. They showed an extensive interaction between the template-primer RNA and Nsp12; furthermore, they confirm that Nsp7 and Nsp8 are not involved in the interaction with the RNA even if these two proteins may be required for RNA binding by RdRp complex. Importantly, they detected one single Remdesivir mono-phosphate (RMP) at the 3’ end of the RNA strand, followed by additional ribonucleotides, indicating  that Remdesivir is likely to act as a non-obligated RNA terminator [8]. Notably, the RNA binding residues and the catalytic active site residues are highly conserved, which makes them a potential target for a broad-spectrum antiviral treatment.

Besides Remdesivir, other nucleoside analogue drugs have shown inhibitory activity on SARS-CoV-2 replication in vitro. These include Favipiravir, Ribavirin, Galidesivir, and EIDD-2801. Notably, EIDD- 2801 has been shown to be 3-10 times more potent than Remdesivir in blocking SARS-CoV-2 replication, therefore it could be an efficient alternative to Remdesivir that needs further testing.

Molecular docking methods and virtual screening have also proposed other potential candidates to target RdRp pocket and potentially inhibit its activity, including herbal compounds from Chinese medicine, like theaflavin [9]. Yet, further studies are required to evaluate and validate the efficacy of these alternative compounds.

In conclusion, resolving the structure of the RdRp subunits has provided fundamental knowledge on new potential pharmacological targets to treat SARS-CoV-2 infection. In addition to targeting other promising candidates, such as the main protease (Mpro), pharmacologically addressing highly conserved domains in RdRp could contribute to the development of anti-coronavirus cocktail treatments that potentially can be used as broad-spectrum antivirals.

Bibliography:

[1] Chan J.F.W. et al., A familial cluster of pneumonia associated with the 2019 Novel Coronavirus indicating person-to-person transmission: a study of a family cluster, Lancet, 24 January 2020.

[2] Wang M. et al., Remdesivir and Chloroquine effectively inhibit the recently emerged novel Coronavirus (2019-nCoV) in vitro, Cell Res., March 2020.

[3] Gao Y. et al., Structure of the RNA-dependent RNA polymerase from COVID-19 virus, lScience, 10 April 2020.

[4] Hillen H.S. et al., Structure of replicating SARS-CoV-2 polymerase, lNature, 21 May 2020.

[5] Kirchdoerfer R. et al.,  Structure of the SARS-CoV Nsp12 Polymerase Bound to Nsp7 and Nsp8 co-factors, Nature Communication, 28 May 2019.

[6] Huang J. et al., Pharmacological Therapeutics Targeting RNA-Dependent RNA Polymerase, Proteinase and Spike Protein: From Mechanistic Studies to Clinical Trials for COVID-19, Journal of Clinical Medicine, 15 April 2020.

[7] Gordon C.J. et al., Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency, lJBC, 13 April 2020.

[8] Yin W. et al., Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir, lScience, 01 May 2020.

[9] Lung J. et al., The potential chemical structure of anti‐SARS‐CoV‐2 RNA‐dependent RNA polymerase,lJournal of Medical Virology, 13 March 2020.

[10] Scudellari M., The sprint to solve coronavirus protein structures — and disarm them with drugs, Nature, 15 May 2020.

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