Coronavirus Replication Machinery: the RNA-dependent RNA Polymerase

In a previous blog post, we highlighted some excellent recent work that has elucidated the molecular mechanism of host-cell invasion by the novel 2019 coronavirus (2019-nCoV). Researchers provided us with three-dimensional structures of the viral spike protein using cryoEM. In this blog post, we will continue this theme and pick up on the replication machinery of 2019-nCoV, which allows the virus to rapidly copy itself and spread quickly.

Replication in hand

Unlike living cells that use double stranded DNA to encode the molecular building blocks of life, 2019-nCoV belongs to a class of viruses that use double stranded RNA. This RNA genome is replicated by an RNA dependent RNA polymerase (RdRp), which in 2019-nCoV is encoded by the non-structural protein (nsp) 12 that forms a complex with two further chaperone proteins nsp7 and nsp8 in a 1:1:2 ratio (Figure 1). A cryoEM structure of the highly-similar SARS-CoV RdRP (96% identical sequence: PDB: 6NUR) (Kirchdoerfer and Ward, 2019), and several structures of the 2019-nCoV RdRP complex itself have already been solved (Figure 1) (Gao et al., 2020).

The polymerase domain has a typical viral polymerase fold, which is often compared to the shape of a hand that grasps the RNA double strand. The active site is located between ‘finger’ and ‘thumb’, and incoming template is held in the ‘palm’ (Figure 1). The polymerase also has an N-terminal extension in comparison to some other viral RdRps, which form nidovirus RdRp-associated nucleotidyltransferase (NiRNA) and interface domains. A unique feature that can now be observed is the role of the two chaperone subunits nsp7 and particularly nsp8. A number of positively charged residues along the extended helices of the nsp8 subunits interact with the RNA. Thus, nsp8 appears to assist in stabilising the growing RNA double strand as it exits the RdRp active site.

Figure 1 – Structure of 2019-nCoV RdRp (nsp7/nsp8/nsp12) complex – Panels a and b show the overall cryo-EM structure of the 2019-nCoV RdRp from the front and rotate 90˚ to the side, respectively. The NiRNA and interface domains are coloured red and purple, respectively. There are two copies of the accessory protein nsp8 (green) and one copy of nsp7 (blue). The polymerase domain is coloured in gold. A double stranded RNA molecule extending from the active site is coloured in orange and blue. Molecular coordinates from the PDB structure 6YTT were used to create this figure.

2019-nCoV RdRp as a therapeutic target

RNA polymerases employed by viruses are very different from those found in human. Thus, inhibiting the activity of 2019-nCoV RdRp provides a potential target for therapeutic intervention with small molecules. Nucleoside analogues are one such class of molecule that can target the RdRp, and the product remdesivir appears to be particularly promising. Remdesivir has been designed as an effective treatment against a number of different viral pathogens such as Ebola, Marburg, MERS, SARS and includes 2019-nCoV. Remdesivir has recently progressed through phase III clinical trials, assessing the drug’s effectiveness in treating COVID-19. Remdesivir is enough like a canonical RNA nucleotide (e.g. adenosine) that it is recognised and incorporated into the growing RNA strand. One of the recent cryoEM structures allows us to visualise where it binds to 2019-nCoV RdRp and suggests a potential mechanism of action (Figure 2). It has been postulated that a collision between the remdesivir nitrile group and residue S⁸⁶¹ of the RdRp prevents RNA strand elongation and therefore inhibits further RNA replication.

Figure 2 – Remdesivir bound to the active site of the 2019-nCoV RdRp. a) Comparison between hydrolysed remdesivir, and the ribonucleic acid adenosine monophosphate, which share a similar chemical substructure. b) Close-up view of the RdRP active site, located between the ‘finger’ and ‘thumb’ of the polymerase domain. Incoming template RNA strand (blue), the primer RNA (red) and nucleotide analogue remdesivir incorporated into the growing chain (green) are displayed. Molecular coordinates from the PDB structure 7BV2 used to create this figure.

It should be noted that remdesivir is not yet licensed or approved anywhere globally and has not yet been demonstrated to be safe or effective for the treatment of COVID-19. However, the initial indications show that remdesivir or a similar nucleotide analogue drug may provide an effective treatment to those suffering from COVID-19, another weapon we can use to fight the disease.

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