We use cookies to enhance your experience. By continuing to browse this site you agree to our use of cookies. More info.
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.
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.
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.
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.
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.
Posted in: Medical Science News | Medical Research News | Disease/Infection News
Tags: Adenine, Coronavirus, Coronavirus Disease COVID-19, covid-19, Hepatitis, Hepatitis C, Magnesium, Mass Spectrometry, Microscopy, Molecule, Nucleotide, Nucleotides, Polymerase, Protein, Remdesivir, Research, Respiratory, Ribonucleic Acid, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spectrometry, Structural Protein, Syndrome, Transcription, Virus
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.
Please use one of the following formats to cite this article in your essay, paper or report:
Kunkalikar, Bhavana. (2022, May 24). Study explores the substrate selectivity of the SARS-CoV-2 replicase. News-Medical. Retrieved on May 28, 2022 from https://www.news-medical.net/news/20220524/Study-explores-the-substrate-selectivity-of-the-SARS-CoV-2-replicase.aspx.
Kunkalikar, Bhavana. "Study explores the substrate selectivity of the SARS-CoV-2 replicase". News-Medical. 28 May 2022. <https://www.news-medical.net/news/20220524/Study-explores-the-substrate-selectivity-of-the-SARS-CoV-2-replicase.aspx>.
Kunkalikar, Bhavana. "Study explores the substrate selectivity of the SARS-CoV-2 replicase". News-Medical. https://www.news-medical.net/news/20220524/Study-explores-the-substrate-selectivity-of-the-SARS-CoV-2-replicase.aspx. (accessed May 28, 2022).
Kunkalikar, Bhavana. 2022. Study explores the substrate selectivity of the SARS-CoV-2 replicase. News-Medical, viewed 28 May 2022, https://www.news-medical.net/news/20220524/Study-explores-the-substrate-selectivity-of-the-SARS-CoV-2-replicase.aspx.
Irene Walsh and Professor Claire Smith
In this interview, News Medical speak to Irene Walsh, the director of Product, Design & Content, and 3D4Medical at Elsevier, and Professor Claire Smith, Head of Anatomy at Brighton and Sussex Medical School, about the real-world applications of the Complete Anatomy female model, the most advanced full female anatomy model in the world.
In this interview, we talk to Takayuki Baba about ongoing research using artificial intelligence to achieve earlier diagnosis of pancreatic cancer.
In this interview, we speak to Matt Walz, CEO of Trialbee, about the importance of improving patient recruitment and retention within clinical trials.
News-Medical.Net provides this medical information service in accordance with these terms and conditions. Please note that medical information found on this website is designed to support, not to replace the relationship between patient and physician/doctor and the medical advice they may provide.
This site complies with the HONcode standard for trustworthy health information: verify here.
News-Medical.net - An AZoNetwork Site
Owned and operated by AZoNetwork, © 2000-2022