In a recent study posted to the bioRxiv* preprint server, researchers explored factors affecting substrate selection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicase.

To date, there have been over 5.26 million confirmed cases of coronavirus disease 2019 (COVID-19), including 6.27 million deaths reported globally. This has necessitated extensive research to understand the SARS-CoV-2 lifecycle and develop therapeutic methods to prevent it.

About the study

In the present study, researchers used cryoelectron microscopy to investigate the replication-transcription complex (RTC) bound to each nucleotide triphosphate (NTP) found in SARS-CoV-2.

The team assessed various chemical approaches to block the pre-incorporation complexes of the SARS-CoV-2 RTC after substrate binding. The methods investigated included the usage of nucleotide diphosphate (NDP) substrates, α-β non-hydrolyzable nucleotide analogs, alternative metal co-factors including Ca²⁺, and 3’-deoxy p-ribonucleic acid (RNA) scaffolds. The team demonstrated the effectiveness of 3’-deoxy RNA scaffolds by employing native mass spectrometry (nMS) to assess the primer extension by one base or the generation of a ternary complex. Furthermore, cryo-EM samples of RTCs were prepared for each naturally occurring nucleotide, ATP, GTP, CTP, UTP, or RDV-TP. 

Results

The study results showed that NDP substrates efficiently trapped the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) recognition complex. However, the RTC of SARS-CoV-2 showed sufficient incorporation of guanosine diphosphate (GDP), adenine DP (ADP), and to a minor extent, antivirals remdesivir diphosphate (RDV-DP). This indicated the efficiency of SARS-CoV-2 in NDP incorporation as well as the relative promiscuity of the active site at RdRp. The team also noted that RTC synthetic activity was reduced after the introduction of a 3’-deoxynucleotide in the product-RNA (p-RNA).

The researchers observed that in the case of control scaffolds comprising 3’-OH, extension by only one nucleotide monophosphate (NMP) occurred in the almost complete p-RNA. Notably, in the absence of 3’-OH in the p-RNAs, no incorporation was found in the next nucleotide, which could be ATP, GTP, or RDV-TP. This indicated that the present strategy efficiently trapped pre-incorporation complexes. 

Preparation of the RTC cryo samples showed that two of the resulting structures, the RTCs with GTP or CTP had a nominal resolution of more than 3 Å with a local resolution of almost 2.2 Å allowing near-atomic level resolution. According to the existence of magnesium chloride (Mg²⁺) in the cryo buffer and the octahedral coordination geometries, the team assigned the density features of CTP and GTP as Mg²⁺_A and Mg²⁺_B. The density peaks for Mg²⁺_A in the GTP and the CTP structures were weaker than those found in Mg²⁺_B while the density features for Mg²⁺_A were not found in the ATP and RDV-TP structures.

Interestingly, the closure of the active site found around the incoming ATP was a consistent feature across all the structures. The team noted that the closure of the motif stabilized substrate binding by: (1) facilitating the backbone carbonyl of Y619 in coordinating Mg²⁺_B and the catalytic residue D618 in coordinating both Mg²⁺_A and Mg²⁺_B; (2) enabling the generation of a hydrogen-bonding (H-bonding) network to improve the detection of the substrate ribose 3’-OH; (3) allowing weak bonding interactions between the motif residues and β- and γ-phosphates; and (4) breaking the polar in the RTC to allow repositioning for metal coordination.

The team noted that the interactions of the non-structural protein (nsp) 12 residues and the natural NT substrates were comparable except for the conserved residues. Moreover, the SARS-CoV-2 RdRp motif closure structure was similar to the substrate-bound RdRp complexes found in the HCV and the poliovirus. Furthermore, the residue structures were conserved across major CoV clades as well as in SARS-CoV-2 sequences found in infected patients. This indicated that the CoV RdRp active site is nearly immutable.

Assessment of structural selectivity showed that the RDV-TP and the ATP nucleotides were both base-paired; however, the 1’-cyano moiety found on the RDV-TP ribose formed the key difference between the two nucleotides. This cyano moiety created a network of polar interactions across the residues. Moreover, a stable water molecule was found in the absence of the cyano group, which suggested that RDV-TP had a higher affinity than ATP.      

Conclusion

Overall, the study findings highlighted the higher selectivity of RDV as enabled by the presence of the cyano moiety while mutations affecting the hydrophilicity of the RdRp active site increased RDV resistance. The researchers believe that the NTP recognition can be employed to design nucleotide analogs to develop therapeutic approaches that target the SARS-CoV-2 RdRp.      

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Bhavana Kunkalikar

Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.