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Year : 2021  |  Volume : 64  |  Issue : 4  |  Page : 771-775
Identification of recurrent mutations in exonuclease (nsp14); a potential drug target in SARS-CoV-2


1 Department of Zoology, P.C. Vigyan College, Chapra, Bihar, India
2 Department of Microbiology, Patna Women's College, Patna University, Patna, Bihar, India
3 Department of Botany, Patna University, Patna, Bihar, India

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Date of Submission12-Sep-2020
Date of Decision24-Mar-2021
Date of Acceptance03-Jun-2021
Date of Web Publication20-Oct-2021
 

   Abstract 


Context: The rapid outbreak of SARS-CoV-2 has become a significant global health concern, highlighting the dire need for antiviral therapeutic agents. RNA-dependent RNA polymerase (RdRp) of coronavirus plays crucial roles in RNA synthesis, and hence remains the druggable target for the treatment of this disease. The most potent broad-spectrum inhibitors of viral RdRp are members of nucleoside analogs (NAs). However, SARS-CoV-2 proved to be a challenging one for the novel NA drug designing strategy because coronavirus possesses an exonuclease (ExoN) domain that is capable of excising NAs, thus showing resistance to existing antiviral drugs. Aim: The objective of our study was to compare the SARS-CoV-2 exonuclease (nsp14) protein sequence of Wuhan-type virus with those of Indian SARS-Cov-2 isolates and to study the effect of multiple mutations on the secondary structure alterations of proteins. Subjects and Methods: Multiple-sequence alignment of exonuclease amino-acid sequences followed by phylogenetic analysis and prediction of its secondary structure of the protein was performed. Results: Altogether, seven mutations were detected in the nsp14 of Indian SARS-CoV-2 isolates. Subsequently, prediction of their secondary structures revealed that mutations altered the structural stability of exonuclease proteins. Conclusions: Present findings, therefore, further suggest that evolvability of SARS-CoV-2 is primarily associated with the onset of multiple novel mutations that rapidly spread at several new locations of the viral genome and also provides important insight to develop specific control strategies to fight against COVID-19 infections.

Keywords: Exonuclease, Nucleoside analogs, RNA-dependent RNA polymerases, SARS-CoV-2, Wuhan

How to cite this article:
Jha DK, Yashvardhini N, Kumar A. Identification of recurrent mutations in exonuclease (nsp14); a potential drug target in SARS-CoV-2. Indian J Pathol Microbiol 2021;64:771-5

How to cite this URL:
Jha DK, Yashvardhini N, Kumar A. Identification of recurrent mutations in exonuclease (nsp14); a potential drug target in SARS-CoV-2. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Nov 27];64:771-5. Available from: https://www.ijpmonline.org/text.asp?2021/64/4/771/328516





   Introduction Top


The emergence of a new SARS-CoV-2 (severe acute respiratory syndrome coronavirus) virus in December 2019, originated from the seafood market in Wuhan, China, is the causative agent of COVID-19 that possesses significant global health threats. This virus is closely related to approximately 88% to two bat-derived coronaviruses such as bat-SL-CoVZC45 and bat-SL-CoVZXC21. Coronaviruses are positive-strand, enveloped, RNA viruses belonging to family coronaviridae and order Nidovirales, having 30 Kbp of genome size.[1] Its outbreak has been reported from 216 countries and in all 5 major continents. This virus is infecting more than millions of people daily but unfortunately, no antiviral drug or vaccine has yet been developed against this contagious disease. The World Health Organization has declared public health emergency on March 11, 2020. As of today August 27, 2020, WHO has reported 23,980,044 confirmed positive cases of COVID-19, including 820,763 causalities.

Generally, RNA viruses show a dramatically high rate of mutation, substantially higher than that of their host. Due to this high rate of mutability shown by SARS-CoV-2 over a short period, it appears that viruses acquire adaptation as well genomic variability which enables them to modulate virulence properties and simultaneously evade the host immunity. SARS-CoV-2 consists of structural (spike S, envelope E, membrane M, and nucleocapsid N) as well as nonstructural proteins like RdRp, helicase, exonuclease (ExoN), and others that help these viruses in assembling, and releasing novel copies in the human cells.[2],[3],[4] Previous studies have shown that major activity of exonuclease (nsp14) includes proofreading function as well as ExoN activity. This also increases the fidelity rate of RNA synthesis by adding correct ribonucleotides to the growing RNA chain and its inactivation (SARS-CoV-2 ExoN) leads to a 21 fold decrease in the fidelity rate of viral replication when compared to wild SARS-CoV.[5],[6]

In the present research work, we have identified altogether seven mutations in the SARS-CoV-2 ExoN sequences, isolated from India by comparing it with the Wuhan-type isolate (as a reference sequence). Our observations suggest that mutational analysis of SARS-CoV-2 ExoN might be considered as a new approach for developing antiviral therapeutics to curb COVID-19 pandemic.


   Subjects and Methods Top


Sequence retrieval

SARS-CoV-2 exonuclease protein sequences (528 amino-acid length) were downloaded from the NCBI virus database which was submitted in the month of June from India. Also, the sequence of Wuhan-type SARS-CoV-2 exonuclease was downloaded to be used as a reference in this study (Accession number YP_009724389).[7]

Multiple sequence alignment and phylogenetic analysis

The exonuclease protein sequences downloaded were aligned using CLUSTAL Omega online platform which performs HMM profiling.[8] This alignment file was visualized in Jalview and the variations in the exonuclease sequences were recorded. To study the phylogeny of these isolates a neighbor-joining phylogenetic tree was prepared using MEGAX software with default parameters.[9]

Secondary structure prediction

The secondary structure prediction of exonuclease protein was done using CFSSP (Chou and Fasman secondary structure prediction) online platform.[10] This program predicts the formation or loss of alpha helix, beta-sheet and turns in the protein structure of mutant as compared to wild type.


   Results Top


Identification of exonuclease mutants in SARS-CoV-2

The protein sequences of SARS-CoV-2 exonuclease from Indian isolates (June 2020) were aligned with those of Wuhan-type isolate and the variations were detected as shown in [Figure 1]. A total of 7 mutations were identified, out of them, one occurred at the same site namely, A2S, M73I, P141S, S256R, K350N, and V462L. The isolates with their accession number, site of mutation, wild type, and mutated sequence are shown in [Table 1]. Therefore, only six isolates and their exonuclease were used for further study.
Figure 1: Multiple sequence alignment of exonuclease protein of Indian SARS-CoV-2 with that of Wuhan SARS-CoV-2. The mutated regions are marked with red boxes

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Table 1: The details of exonuclease (nsp14) sequences found mutated from the Wuhan SARS-CoV-2 sequence. Details include the accession number of the isolate position of mutation and the wild type and mutated amino acid sequence

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Phylogenetic analysis

To gain information on the phylogeny of the wild type and mutated isolates, a neighbor-joining phylogenetic tree was prepared using MEGAX. This phylogenetic analysis showed that the exonuclease variants from India and Wuhan formed different clusters, revealing the multivariant nature of SARS-CoV-2 [Figure 2].
Figure 2: Phylogenetic tree of Indian SARS-CoV-2 and Wuhan SARS-CoV-2 isolates with reference to exonuclease protein

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Alteration in secondary structure of exonuclease protein upon mutation

The effect of mutation on the secondary structure of nsp14 was studied using CFSSP to analyze the alterations in loss or formation of beta-sheet, alpha helix, and turns. Only two mutations in the exonuclease showed a change in secondary structure such as A2S and S256R, whereas the other four did not show any alterations [Figure 3]. At position 2, where an alanine is substituted by serine resulted in the formation of turn at point 1 and 3 and also causes loss of helix structure at position 1, 2, 3 and 4. Alpha helix formation is favored by alanine amino acid and hence upon replacement resulted in the loss of helix, whereas serine amino acid forms turn rather than the helix. The point mutation at 256, where serine is replaced by arginine causes the formation of sheet secondary structure at positions 255, 256, 257 258, and 259. Arginine is a polar amino acid with a guanidium ring which has a large side chain forms sheet rather than other secondary structures. Therefore, the mutations in the exonuclease of SARS-CoV-2 lead to its secondary structure alterations and hence assist in its multiplicity and host evasion.
Figure 3: Secondary structure prediction of exonuclease protein. Effect of mutation at different sites on the secondary structure of exonuclease protein, (a-f) represent six mutations that occurred in Indian isolates. The first secondary structure in each (a-f) represents the Wuhan-type sequence, whereas the second represents the mutated one. The mutation location and respective secondary structures are marked with boxes

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   Discussion Top


In this study, we have compared the SARS-CoV-2 sequence of Wuhan virus (reference genome) with that of Indian SARS-Cov-2 isolates, only those mutations which occurred in the exonuclease (nsp14) region were identified and used for further study. Usually, high rate of mutations facilitates viral evolution as well as genome variability and thus enabling viruses to evade the immunity of the host and is accompanied by drug resistance properties. SARS-CoV-2 emerged from Wuhan, China, further began to spread rapidly all over the world. Further, various factors are directly linked with the transmissibility of SARS-CoV-2 infections, such as huge population density, health care system as well as changing environmental and climatic conditions.[11]

In addition, mutations in the SARS-CoV-2 ExoN impairs the fidelity of viral replication because CoV ExoN interacts with highly processive RdRp (RNA dependent RNA polymerase, nsp12) protein and its associated cofactors like nsp7 and nsp8 to accomplish proofreading activity.[12],[13] Viruses having mutated RdRp most commonly showing resistance towards antiviral therapeutic drugs such as remdesivir which is a drug of choice among various repurposing drugs for SARS-CoV-2 treatment nowadays.[14] Present findings therefore further suggest that evolvability of SARS-CoV-2 is primarily associated with the onset of multiple novel mutations that rapidly spread at several new locations of the viral genome and also provides important insight to develop specific control strategies to fight against COVID-19 infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Li Y, Yang X, Wang N, Wang H, Xin B, Yang X, et al. The divergence between SARS-CoV-2 and RaTG13 might be overestimated due to the extensive RNA modification. Future Virol 2020;15. Published Online. https://doi.org/10.2217/fvl-2020-0066  Back to cited text no. 2
    
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Ogando NS, Ferron F, Decroly E, Canard B, Posthuma CC, Snijder EJ. The curious case of the nidovirus exoribonuclease: Its role in RNA synthesis and replication fidelity. Front Microbiol 2019;10:1813.  Back to cited text no. 5
    
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Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, et al. Infdelity of SARSCoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS Pathog 2010;6:e1000896.  Back to cited text no. 6
    
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Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature 2020;579:265-9.  Back to cited text no. 7
    
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Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nuc Acids Res 2019:47:W636-41.  Back to cited text no. 8
    
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Kumar S, Glen S, Michael L, Christina K, Koichiro T. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Bio Evol 2018;35:1547-9.  Back to cited text no. 9
    
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Kumar TA. CFSSP: Chou and Fasman secondary structure prediction server. Wide Spect 2013;1:15-9.  Back to cited text no. 10
    
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Morgenstern B, Michaelis M, Baer PC, Doerr HW, Cinatl J Jr. Ribavirin and interferon-β synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines. Biochem Biophys Res Commun 2005;326:905-8.  Back to cited text no. 11
    
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Ferron F, Subissi L, Theresa A, Morais SD, Le NTT, Sevajol M, et al. Structural and molecular basis of mismatch correction and ribovirin excision from Coronavirus RNA. Proc Natl Acad Sci USA 2018;115:E162-71.  Back to cited text no. 12
    
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Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, et al. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci USA 2014;111:E3900-9.  Back to cited text no. 13
    
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Dufy S. Why are RNA virus mutation rates so damn high? PLoS Biol 2018;16 (8):e3000003.  Back to cited text no. 14
    

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Correspondence Address:
Niti Yashvardhini
Department of Microbiology, Patna Women's College, Patna University, Patna - 800 001, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.328516

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