Identification and Characterization of Novel Mutants of Nsp13 Protein among Indian SARS-CoV-2 Isolates

1. INTRODUCTION

The recent emergence of an outburst of coronavirus disease 2019 (COVID-19) pandemic caused by a novel beta coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1], was first reported in Wuhan, China, in late December 2019 [2-5]. COVID-19 spread rapidly worldwide and was declared a global pandemic on 11^th March, 2020, by World Health Organization (WHO). It has infected hundreds of millions of people and killed more than three million people worldwide. The world is in the midst of the second wave of a pandemic with rapidly evolving new variants of SARS-CoV-2, resulting in a surge of COVID-19 cases worldwide. The highly contagious nature of SARS-CoV-2 has now developed into a severe threat to global public health. The manifestations of SARS-CoV-2 range from the asymptomatic common cold to lethal viral respiratory illness in infected individuals [6].

Coronaviruses are enveloped in a nonfragmented, large single-stranded (positive sense) RNA virus with a GC content ranging from 32% to 43%. They are classified into four genera, namely, alphacoronavirus (α CoV), beta coronavirus (β CoV), gamma coronavirus (γ CoV), and delta coronavirus (δ CoV), respectively [7]. SARS-COV-2 belongs to the β coronavirus 2b lineage with a genome of length 29.9 kb, which encodes 29 proteins [8]. The genomic sequence of the novel SARS-COV-2 exhibits 79.6% similarity to SARS-CoV and about 50% with MERS-CoV (Middle East respiratory syndrome coronavirus), respectively. The phylogenetic analysis has illustrated that SARS-CoV-2 is relatively more similar to SARS-CoV than MERS-CoV. Based on homology modelling studies, SARS-CoV-2 shares 96.2% nucleotide similarity with RaTG13, a bat CoV from Rhinolophus Affinis. The genomic organisation of SARS-CoV-2 includes '5 UTR, ORF1ab, S, E, M, N, and 3'UTR. Its genome comprises ORFs, which encode for four major structural proteins, Spike glycoprotein (S), Envelope (E), Membrane (M), and Nucleocapsid (N) proteins, 16 Nsps (non-structural proteins), and nine other accessory proteins [9-11]. ORF1a/b region is the largest ORF that covers about two-thirds of the whole genome length and encodes known sixteen non-structural proteins (16Nsps). ORFs in the remaining one-third of the genome near the 3' end code for major structural and other accessory proteins. The ribosomal frameshift (-1) between ORF1a and ORF1b produces two polypeptides (pp1a and pp1ab), which are proteolytically cleaved by viral proteases, namely, main protease (M pro) or Chymotrypsin-like protease (3CLpro) and papain-like protease (PL pro) to release 16 Nsps of 7096 amino acids in length. These Nsps are crucially important in viral replication and transcription processes [8]. Nsps are considered more conserved than SARS-CoV-2 structural proteins [12]. Among the 16 known SARS-CoV-2 Nsp proteins, the Nsp13 helicase is a crucial component for viral replication [13] and the most conserved nonstructural protein with 99.8% sequence identity to SARS-CoV Nsp13 [14]. The highest sequence conservation is shared by SARS-CoV-2 Nsp13 within the CoV family, reflecting its significant role in viral viability. Nsp13, an RNA helicase enzyme, also represents a promising therapeutic target for anti-CoV drug development [15-18].

The ongoing rapid transmission and global spread of SARS-CoV-2 have acquired various mutations. The global advancements in the sequencing efforts of SARS-CoV-2 have reported several mutations in its proteins, like spike protein, nucleocapsid, PLpro, and ORF3a, from different populations [19-21]. Emerging variants of SARS-CoV-2 with crucial mutations is a sparkling challenge in the context of therapeutics, drugs, and vaccines developing globally. To date, there are no specific therapeutics and vaccines available to combat SARS-CoV-2. Given this, the present study aimed at identifying and characterizing mutations in SARS-CoV-2 Nsp13 protein that might prove to be useful towards the development of drugs and effective vaccines against this highly mutable coronavirus. It could also help in improving the current diagnostic approaches to viral detection, thereby controlling the transmission of the virus. As a consequence of the indispensable role of SARS-Cov-2 Nsp13 in the replication of the virus, the study aimed at understanding the possible structural and functional implications of Nsp13 mutations among Indian isolates.

2. METHODS

3. RESULTS

3.4. Prediction of B-cell Epitopes

Linear continuous B-cell epitopes of SARS-CoV-2 Nsp13 were predicted by the IEDB web server tool, as shown in Fig. (3A). The BepiPred score of Nsp13 residues depicted a minimum score of -0.161 and a maximum score of -0.651 at a threshold value of 0.482. The data set identified the top seven linear epitopes with at least eight amino acid residues, of which both their sequences and locations are mentioned in Fig. (3B). These epitopes were further analyzed for antigenicity, allergenicity, and toxicity by in-silico based approaches. The predicted data showed that all of the seven peptides are non-toxic based on the Toxin Pred prediction tool. Out of the seven peptides, three peptides were identified as allergens and four as non-allergens based on allergenicity prediction (Fig. 3B). Similarly, two of these predicted linear epitopes exhibited antigenicity and the remaining five peptides were non-antigenic as analyzed by the VaxiJen prediction tool (Fig. 3B). In addition, discontinuous B-cell epitopes of Nsp13 were predicted using Discotope 2.0 server tool. The available 3D structure of Nsp13 in PDB format (PDB ID: 6ZSL) with a chain A of protein was used as input in the Discotope 2.0 server tool (Fig. 3C). The residue with Discotope score equal to or greater than the threshold value (-6.6) was identified as discontinuous epitope residues of Nsp13. The data sets identified 18 B cell epitope residues out of the total residues of Nsp13 protein at a threshold value of -6.6 (Fig. 3D). Histidine and valine at positions 245 and 247, out of 18 predicted residues were significantly positively predicted with high DiscoTope score values, as depicted in Fig. (2D). Overall, this data revealed B-cell epitopes contributed by Nsp13.

4. DISCUSSION

In the present study, we identified 38 mutations in Nsp13 among Indian isolates. The variations in the SARS-CoV-2 protein enable us to better understand its diversity, dynamics, and genetic epidemiology [31], which might provide an opportunity in developing effective and safe therapeutic drugs and vaccines for this highly mutable coronavirus. Our data revealed that the observed mutations at different positions (H164Y, A237T, T214I, C309Y, S236I, P419S, V305E, G54S, H290Y, P53S, A308Y, and A308Y) in the Nsp13 significantly affected the stability and flexibility of Nsp13 protein. In addition, the alterations in the function of Nsp13 were also predicted upon mutational effect by predicted PROVEAN scores. Furthermore, B-cell epitopes contributed by Nsp13 were identified using various predictive immunoinformatic tools. Immunological parameters of Nsp13, such as antigenicity, allergenicity, and toxicity were assessed to predict the potential B-cell epitopes. Predicted in silico epitopes are in consideration for the development of epitope-based peptide vaccine candidates against SARS-CoV-2. The predicted peptide sequences were correlated with the observed mutants. Our predicted data showed that there are seven high-rank linear epitopes as well as 18 discontinuous B-cell epitopes based on immunoinformatic findings. Moreover, it was investigated that, out of the total 38 identified mutations among Indian SARS-CoV-2 Nsp13 protein, four mutant residues at positions 142 (E142), 245 (H245), 247 (V247), and 419 (P419) were localised in the predicted B-cell epitopic region (Fig. 3). These mutant residues may help the SARS-CoV-2 variants in eliciting a distinct immune response from the wild-type SARS-CoV-2. Several previous studies evidently supported the findings with similar observations using the immunoinformatic approach [32-34]. Hence, our study provides some insights into understanding the Nsp13 epitopes, which could regulate host immune responses against SARS-CoV-2. The present in silico study revealed the impact of the identified mutations on the stability and flexibility of Nsp13 protein. Further in vivo studies are necessary to better understand and validate the effect of mutations on the immunogenicity of epitopes of SARS-CoV-2.

CONCLUSION

The emergence of a novel coronavirus, SARS-CoV-2, has become a major global concern with an unprecedented public health crisis. Understanding the genomic variations in SARS-CoV-2 could help in improving the current diagnostic techniques and developing suitable vaccine candidates against SARS-CoV-2 infections. Altogether, the results of the present in silico study showed various mutations in Nsp13 among Indian SARS-CoV-2 and their implications on the stability, flexibility, and function of Nsp13 protein using predictive tools of immunoinformatics. Non- structural proteins exhibited low glycation density compared to the structural proteins of SARS-CoV-2. Therefore, epitopes of Nsp13 could be used as effective and promising targets against SARS-CoV-2.

AUTHORS’ CONTRIBUTION

AK conceived the idea of the study and contributed to study design, data interpretation, and manuscript preparation. DK performed the experiments, analyzed the data, and prepared the manuscript. NK, SK, PKS, SKS, and NRB contributed to the conceptualization of the study and reviewing of the manuscript.

REFERENCES