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. 2021 Nov 17;75:29–36. doi: 10.1016/j.biologicals.2021.11.002

Emerging genetic diversity of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) alters its B-cell epitopes

Sushant Kumar b, Khushboo Kumari b, Gajendra Kumar Azad b,∗
PMCID: PMC8595351  PMID: 34802866

Abstract

The RNA dependent RNA polymerase (RdRp) plays crucial role in virus life cycle by replicating the viral genome. The SARS-CoV-2 is an RNA virus that rapidly spread worldwide and acquired mutations. This study was carried out to identify mutations in RdRp as the SARS-CoV-2 spread in India. We compared 50217 RdRp sequences reported from India with the first reported RdRp sequence from Wuhan, China to identify 223 mutations acquired among Indian isolates. Our protein modelling study revealed that several mutants can potentially alter stability and flexibility of RdRp. We predicted the potential B cell epitopes contributed by RdRp and identified thirty-six linear continuous and twenty-five discontinuous epitopes. Among 223 RdRp mutants, 44% of them localises in the B cell epitopes region. Altogether, this study highlights the need to identify and characterize the variations in RdRp to understand the impact of these mutations on SARS-CoV-2.

Keywords: COVID-19, SARS-CoV-2, Mutations, B cell epitopes, RNA dependent RNA polymerase (RdRp), India

1. Introduction

SARS-CoV-2 genome encodes 29 protein molecules which are categorised into three groups including structural, non-structural and accessory proteins. SARS-CoV-2 has four structural proteins namely Spike glycoprotein, Membrane protein, Envelope protein and Nucleocapsid Phosphoprotein [1]. It also encodes sixteen non-structural proteins (Nsp1-16) and nine accessory proteins. The 16 non-structural proteins are synthesised as a single polypeptide molecule of 7096 amino acids known as Orf1ab that is subsequently cleaved into 16 separate proteins [2]. The RNA dependent RNA polymerase (RdRp), also known as Nsp12, is a non-structural protein that replicates SARS-CoV-2 RNA genome [1]. It associates with Nsp7 and Nsp8 and exist as a trimeric complex inside the viral envelope structure [3]. By itself, RdRp has a very weak polymerase activity; however, the complex of RdRp with Nsp7 and Nsp8 significantly increases RdRp processivity and template affinity [4].

RdRp of SARS-CoV-2 is 932 residues in length and contains distinct polymerase and nucleotide binding domains with a central connecting domain. Structurally, RdRp is comprised of an N-terminal β-hairpin (residues 31–50) followed by an extended nidovirus RdRp-associated nucleotidyl-transferase domain (NiRAN, residues 115–250) [5]. Following the NiRAN domain is an interface domain (residues 251–365) connected to the RdRp domain (residues 366–920). Further, the domains of RdRp arranges in such a way that it forms a canonical right-handed cup configuration [6], with the finger subdomain (resides 397–581 and residues 621–679) forming a closed circle with the thumb subdomain (residues 819–920) [5].

Bioinformatics has enabled researchers to study large number of epitopes and their properties without the risk of growing pathogens. It has drastically reduced the cost of study and faster output over the conventional methods of vaccine study. Further, the amalgamation of various genome-wide studies with the immunoinformatics has revolutionised the identification of epitopes contributed by a protein or virus and accelerated our understanding of vaccine design and action [7,8]. Several studies show that SARS-CoV-2 RdRp participates in host immune response and thus provides insights into viral pathogenesis [[9], [10], [11], [12]]. RdRp stimulates a considerable amount of immunogenicity due to its lower glycosylation density as compared to other structural proteins and several studies have revealed that RdRp induces both innate and adaptive immune response of host [[9], [10], [11]]. Another study has revealed that RdRp, suppresses host antiviral responses by inhibiting IRF3 nuclear translocation [12]. Furthermore, RdRp is one of the most conserved enzyme across several viral species, such as influenza virus, hepatitis C virus (HCV), ZIKA virus (ZIKV), and coronavirus (CoV), suggesting that its function and mechanism of action might be well conserved [13,14]. As the SARS-CoV-2 spread to new geographical areas, it started to mutate [14]. The mutations acquired by the SARS-CoV-2 are retained as a consequence of natural selection, if the variants are more adaptable. In order to understand the variations occurring in RdRp among Indian geographical area, we analysed 50217 RdRp sequences reported from India to identified 223 mutations. The B cell epitopes contributed by RdRp were predicted in silico and the mutations were also mapped.

2. Material and methods

2.1. Protein sequences retrieval and analysis

The sequences used in this study are available on publicly accessible CoVal database (https://coval.ccpem.ac.uk/). A total of 50217 SARS-CoV-2 sequences reported between Jan 2020 till Sept 2021 from different geographical locations within India were used in this study. The mutations occurring in SARS-CoV-2 were obtained from CoVal webserver. The CoVal Webserver uses sequences from the GISAID repository and updates its information at frequent intervals.

2.2. Identification of RdRp mutants by multiple sequence alignments (MSAs)

In order to identify the variations present in the RdRp sequences among Indian isolates of SARS-CoV-2, the MSAs were conducted by Clustal omega programe [15] as described earlier [16].

2.3. B cell epitope prediction

The prediction of linear continuous B cell epitopes were conducted by IEDB [17]. The IEDB webserver provides training set for evaluation of existing epitope prediction methods and constitute platform for development of novel and better algorithm for prediction. IEDB webserver also provides tools for the prediction of linear B-cell epitopes from protein sequence including amino acid scales and HMMs, DiscoTope, ElliPro, Paratome, and PIGS. The IEDB contains epitopes derived from the peer-reviewed literature, patent applications, direct submission, and other publicly available databases, for example, FIMM, HLA Ligand database, and MHC binding database. The IEDB prediction method known as ‘Bepipred linear epitope prediction method 2.0’ was used in this study. For this prediction the threshold value of 0.500 was used during the evaluation. The prediction of discontinuous B cell epitopes was performed by an online tool ‘DiscoTope 2.0’. For this prediction the threshold value was set at −3.7.

2.4. Protein modelling studies

We performed protein modelling studies by DynaMut programe [18] as described earlier [19]. The DynaMut web server was used for analysing thermodynamic stability of different RdRp mutations observed in this study. The DynaMut webserver introduces dynamics component for mutational analysis to predict difference in free energy (ΔΔG) and vibrational entropy (ΔΔS). This webserver implements Normal mode analysis (NMA) through two different approaches, Bio3D and ENCoM, that provide rapid and simplified access to analyse protein dynamics and stability resulting from vibrational entropy changes [18]. It also enables to assess the effects of missense mutations on protein stability and provide comprehensive suite for protein motion and flexibility analysis and visualization (http://biosig.unimelb.edu.au/dynamut/). For this study, we used recently reported structure of RdRp (PDB ID: 7BV1) [5]. The effect of mutations on protein is shown in terms of difference in free energy (ΔΔG). DynaMut provides difference in vibrational entropy (ΔΔSvib ENCOM) between the wild type and mutant protein. We ran DynaMut webserver to calculate the ΔΔG and ΔΔSvib ENCOM that provides the impact of mutation on protein structure and stability.

3. Results

3.1. RdRp is frequently mutated among Indian isolates of SARS-CoV-2

In order to identify the mutations in RdRp, we used CoVal webserver that compares the first reported sequence of RdRp from Wuhan, China with the sequences reported from India. Till Sept 2021, a total of 50217 sequences has been analysed by CoVal webserver. The data revealed 223 mutations present among the Indian sequences of RdRp as shown in Table 1 . The mutations are also demonstrated on the schematic representation of RdRp as shown in Fig. 1 A. Our result show that the mutations are spreading all over the RdRp polypeptide sequence. The distribution of mutations in different domains of RdRp has been highlighted in Fig. 1A. This data strongly indicates that RdRp is one of the most frequently mutated proteins of SARS-CoV-2 because we observed 223 mutations till Sept 2021. Furthermore, we looked at the time course of the samples used for mutational study by CoVal webserver. This webserver shows the monthly appearance of new mutations from India. Based on the mutational analysis of RdRp by CoVAL webserver, we observe that during initial phase of COVID19 pandemic, the rate of occurrence of new mutations were high but it slowed down as the time progresses (Fig. 1B), suggesting that the virus is attaining mutational stability over time.

Table 1.

The list demonstrates the location and details of mutations of RdRp identified by CoVal webserver. The RdRp sequence reported from Wuhan, China was used as wild type sequence for this analysis. The 50217 sequences of RdRp reported from India (till Sept 2021) were used for identifying mutations and their frequency. The ΔΔG and ΔΔSvib ENCOM values were obtained by protein modelling using DynaMut programe. The positive and negative ΔΔG represents increase and decrease in protein stability upon mutation. Similarly, the positive and negative ΔΔSvib ENCOM represents the increase in flexibility and rigidity upon mutations. The mutation that localises in the unmodeled region of RdRp was not used in the analysis of ΔΔG and ΔΔSvib ENCOM and they were left blank (denotes by -) in table. P stands for Polar, NP stands for Non-Polar.

Serial
Number		 Nsp12 mutations Polarity changes Charge changes Frequency of mutation ΔΔG (kcal/mol) ΔΔSVib ENCoM (kcal.mol-1.K-1)
1 A2V NP to NP Neutral to Neutral 3 – –
2 S6L P to NP Neutral to Neutral 12 – –
3 C12R P to P Neutral to Basic 2 – –
4 A16V NP to NP Neutral to Neutral 8 – –
5 A16E NP to P Neutral to Acidic 2 – –
6 T20A P to NP Neutral to Neutral 11 – –
7 G25D NP to P Neutral to Acidic 4 – –
8 T26I P to NP Neutral to Neutral 13 – –
9 Y32H P to P Neutral to Basic 2 −0.349 0.090
10 D40A P to NP Acidic to Neutral 4 −0.139 0.021
11 V42L NP to NP Neutral to Neutral 2 0.347 −0.342
12 G44V NP to NP Neutral to Neutral 2 −1.011 −0.103
13 A46T NP to P Neutral to Neutral 2 −1.031 −0.174
14 K59 N P to P Basic to Neutral 12 – –
15 D62Y P to P Acidic to Neutral 3 – –
16 N64D P to P Neutral to Acidic 2 – –
17 D67 N P to P Acidic to Neutral 4 – –
18 T85I P to NP Neutral to Neutral 14 −0.707 −0.074
19 K91R P to P Basic to Basic 11 −0.143 −0.066
20 K91E P to P Basic to Acidic 2 −0.187 0.416
21 K91 N P to P Basic to Neutral 2 −0.311 0.295
22 P94L NP to NP Neutral to Neutral 6 0.858 −0.242
23 P94S NP to P Neutral to Neutral 3 −0.035 −0.131
24 A95S NP to P Neutral to Neutral 5 −1.317 −0.172
25 A95V NP to NP Neutral to Neutral 4 −0.669 −0.627
26 A97V NP to NP Neutral to Neutral 438 0.469 −1.020
27 G108V NP to NP Neutral to Neutral 8 – –
28 D109G P to NP Acidic to Neutral 3 – –
29 D109Y P to P Acidic to Neutral 2 – –
30 R118C P to P Basic to Neutral 4 0.005 0.055
31 M124I NP to NP Neutral to Neutral 6 −0.088 0.250
32 V128I NP to NP Neutral to Neutral 5 1.237 −0.268
33 G137S NP to P Neutral to Neutral 2 −1.389 0.029
34 C139S P to P Neutral to Neutral 2 −0.460 −0.227
35 D140Y P to P Acidic to Neutral 6 −0.105 0.035
36 D140G P to NP Acidic to Neutral 3 −0.180 0.035
37 T141I P to NP Neutral to Neutral 11 1.041 −0.231
38 I145V NP to NP Neutral to Neutral 4 −0.190 0.408
39 D153Y P to P Acidic to Neutral 10 0.961 0.049
40 D154G P to NP Acidic to Neutral 2 −0.771 0.111
41 K160 N P to P Basic to Neutral 6 −0.089 −0.029
42 D161Y P to P Acidic to Neutral 2 1.107 −0.621
43 W162C NP to P Neutral to Neutral 3 −1.169 1.004
44 P169S NP to P Neutral to Neutral 3 0.478 −0.232
45 D170G P to NP Acidic to Neutral 3 −0.527 0.156
46 D170Y P to P Acidic to Neutral 3 −0.089 0.154
47 R173S P to P Basic to Neutral 4 −0.412 0.203
48 R173C P to P Basic to Neutral 2 −0.283 0.168
49 R173H P to P Basic (strongly) to Basic (weakly) 2 −0.284 0.010
50 A176T NP to P Neutral to Neutral 2 0.032 −0.721
51 A185V NP to NP Neutral to Neutral 11 −0.179 −0.369
52 A185S NP to P Neutral to Neutral 2 – –
53 A195D NP to P Neutral to Acidic 2 −0.478 −0.397
54 M196I NP to NP Neutral to Neutral 7 −0.554 0.094
55 R197Q P to P Basic to Neutral 3 −0.483 0.096
56 R197L P to NP Basic to Neutral 2 −0.109 0.109
57 I223 M NP to NP Neutral to Neutral 13 −0.051 −0.084
58 T225I P to NP Neutral to Neutral 2 0.090 −0.076
59 P227L NP to NP Neutral to Neutral 245 0.360 −0.332
60 P227T NP to P Neutral to Neutral 2 0.459 −0.375
61 G228S NP to P Neutral to Neutral 18 −1.113 −0.158
62 V231I NP to NP Neutral to Neutral 3 −0.529 0.229
63 I244T NP to P Neutral to Neutral 85 −3.236 0.746
64 T248I P to NP Neutral to Neutral 7 0.452 −0.080
65 R249S P to P Basic to Neutral 7 −0.672 0.320
66 R249G P to NP Basic to Neutral 2 −0.382 0.504
67 R249 M P to NP Basic to Neutral 2 0.825 −0.111
68 A250V NP to NP Neutral to Neutral 5 −0.237 −0.482
69 T252I P to NP Neutral to Neutral 4 0.265 −0.060
70 A253V NP to NP Neutral to Neutral 2 −0.415 −0.586
71 E254D P to P Acidic to Acidic 2 −1.136 0.395
72 H256Y P to P Basic to Neutral 3 −0.411 −0.074
73 V257F NP to NP Neutral to Neutral 5 −0.485 0.162
74 K263 N P to P Basic to Neutral 5 −0.336 0.316
75 P264S NP to P Neutral to Neutral 4 −0.715 0.098
76 D269 N P to P Acidic to Neutral 34 −0.258 0.080
77 L270F NP to NP Neutral to Neutral 3 0.390 −0.209
78 E278D P to P Acidic to Acidic 3 −0.714 0.103
79 R279S P to P Basic to Neutral 3 −2.849 1.063
80 L282I NP to NP Neutral to Neutral 3 −0.287 0.096
81 D284Y P to P Acidic to Neutral 7 0.346 −0.459
82 Q292H P to P Neutral to Basic 2 0.171 0.070
83 T293I P to NP Neutral to Neutral 5 0.264 −0.016
84 V299F NP to NP Neutral to Neutral 4 0.240 −0.143
85 L302F NP to NP Neutral to Neutral 3 −0.363 0.045
86 A311S NP to P Neutral to Neutral 8 −0.229 −0.014
87 V315A NP to NP Neutral to Neutral 3 −1.724 0.711
88 F317L NP to NP Neutral to Neutral 2 0.045 0.379
89 T319I P to NP Neutral to Neutral 35 1.052 −0.244
90 P323L NP to NP Neutral to Neutral 11094 0.530 −0.252
91 P323F NP to NP Neutral to Neutral 24 0.297 −0.199
92 P323V NP to NP Neutral to Neutral 3 0.578 −0.313
93 S325I P to NP Neutral to Neutral 2 1.763 −0.574
94 P328S NP to P Neutral to Neutral 2 −0.425 0.065
95 L329I NP to NP Neutral to Neutral 15 0.081 0.231
96 V330A NP to NP Neutral to Neutral 38 – –
97 V330L NP to NP Neutral to Neutral 5 0.025 −0.103
98 G337C NP to P Neutral to Neutral 2 −1.187 0.219
99 Y346H P to P Neutral to Basic 2 −0.021 0.619
100 V354L NP to NP Neutral to Neutral 21 −0.162 −0.387
101 V354A NP to NP Neutral to Neutral 4 −1.811 0.277
102 Q357H P to P Neutral to Basic 7 1.218 −0.094
103 D358G P to NP Acidic to Neutral 3 −0.096 0.357
104 A379V NP to NP Neutral to Neutral 7 0.624 −0.529
105 A379S NP to P Neutral to Neutral 2 1.586 −0.510
106 M380I NP to NP Neutral to Neutral 6 −0.487 0.551
107 A382V NP to NP Neutral to Neutral 3 0.596 −0.438
108 A383S NP to P Neutral to Neutral 2 −0.131 −0.021
109 T394 M P to NP Neutral to Neutral 3 0.947 −0.648
110 A400S NP to P Neutral to Neutral 52 0.437 −0.127
111 T402I P to NP Neutral to Neutral 6 0.301 0.039
112 V405F NP to NP Neutral to Neutral 3 0.955 −0.639
113 A406S NP to P Neutral to Neutral 7 0.497 −0.246
114 A406V NP to NP Neutral to Neutral 2 −0.148 −0.047
115 D418E P to P Acidic to Acidic 2 0.915 −0.258
116 A423V NP to NP Neutral to Neutral 12 0.732 −0.338
117 S434Y P to P Neutral to Neutral 2 0.277 −0.606
118 V435I NP to NP Neutral to Neutral 4 0.150 −0.391
119 V435F NP to NP Neutral to Neutral 2 −0.063 −1.096
120 A443V NP to NP Neutral to Neutral 4 0.937 −0.250
121 A443S NP to P Neutral to Neutral 2 −0.013 −0.135
122 Q444H P to P Neutral to Basic 5 1.310 −0.346
123 A449V NP to NP Neutral to Neutral 7 0.871 −0.560
124 A449S NP to P Neutral to Neutral 2 −0.265 0.016
125 I450V NP to NP Neutral to Neutral 2 −1.121 0.580
126 Y458H P to P Neutral to Basic 3 −0.169 0.293
127 P461S NP to P Neutral to Neutral 8 −0.475 0.055
128 M463I NP to NP Neutral to Neutral 19 0.483 0.250
129 C464F P to NP Neutral to Neutral 2 1.251 −1.092
130 I466T NP to P Neutral to Neutral 2 −2.428 0.355
131 V473F NP to NP Neutral to Neutral 24 −0.802 −0.941
132 V476A NP to NP Neutral to Neutral 3 −0.477 0.578
133 K478 N P to P Basic to Neutral 60 −1.105 0.533
134 I494V NP to NP Neutral to Neutral 2 −0.044 0.027
135 L514F NP to NP Neutral to Neutral 2 −0.300 −0.066
136 Y521H P to P Neutral to Basic 4 −0.048 0.764
137 A526V NP to NP Neutral to Neutral 4 0.305 −0.109
138 A526S NP to P Neutral to Neutral 3 −0.315 −0.121
139 A529V NP to NP Neutral to Neutral 16 −0.273 −0.068
140 A529T NP to P Neutral to Neutral 3 −0.874 0.002
141 A529S NP to P Neutral to Neutral 2 −0.884 −0.059
142 I536T NP to P Neutral to Neutral 3 – –
143 L544I NP to NP Neutral to Neutral 7 0.224 0.060
144 A544V NP to NP Neutral to Neutral 2 0.325 −0.547
145 I562V NP to NP Neutral to Neutral 2 −0.801 0.215
146 I579V NP to NP Neutral to Neutral 3 −0.573 0.414
147 T586P P to NP Neutral to Neutral 2 −0.213 0.662
148 V588L NP to NP Neutral to Neutral 4 0.599 −0.117
149 T591I P to NP Neutral to Neutral 4 1.218 −0.416
150 M601I NP to NP Neutral to Neutral 18 −0.362 −0.033
151 V605A NP to NP Neutral to Neutral 18 −1.528 0.596
152 H613Y P to P Basic to Neutral 20 0.836 −0.213
153 M629I NP to NP Neutral to Neutral 6 −0.428 0.170
154 L636F NP to NP Neutral to Neutral 4 −0.115 −0.435
155 L636I NP to NP Neutral to Neutral 2 0.569 0.041
156 L638F NP to NP Neutral to Neutral 6 −0.167 −0.297
157 T643I P to NP Neutral to Neutral 10 −0.383 −0.074
158 T644 M P to NP Neutral to Neutral 3 −0.084 −0.037
159 S647I P to NP Neutral to Neutral 29 −0.272 0.166
160 S649L P to NP Neutral to Neutral 2 0.684 −0.422
161 A656S NP to P Neutral to Neutral 20 −1.416 0.063
162 M666I NP to NP Neutral to Neutral 6 −0.229 0.365
163 M668I NP to NP Neutral to Neutral 11 0.104 0.066
164 G671S NP to P Neutral to Neutral 2665 0.786 −0.246
165 G671A NP to NP Neutral to Neutral 2 1.315 −0.906
166 V675I NP to NP Neutral to Neutral 49 0.050 −0.115
167 C697F P to NP Neutral to Neutral 3 −1.055 −0.979
168 A699S NP to P Neutral to Neutral 4 −2.233 −0.185
169 T710 N P to P Neutral to Neutral 2 0.703 −0.185
170 A716T NP to P Neutral to Neutral 2 0.285 −0.035
171 K718 N P to P Basic to Neutral 5 −0.208 0.051
172 H725Y P to P Basic to Neutral 6 0.240 0.166
173 E729K P to P Acidic to Basic 2 0.825 −0.139
174 N734D P to P Neutral to Acidic 4 −0.338 0.227
175 D736 N P to P Acidic to Neutral 3 0.006 −0.049
176 D738Y P to P Acidic to Neutral 5 0.861 −0.137
177 T739I P to NP Neutral to Neutral 4 0.272 0.008
178 E744D P to P Acidic to Acidic 11 −0.566 0.229
179 M756I NP to NP Neutral to Neutral 11 1.019 0.205
180 S768 N P to P Neutral to Neutral 2 −0.152 −0.012
181 G774C NP to P Neutral to Neutral 4 0.107 −0.086
182 L775P NP to NP Neutral to Neutral 2 −0.600 0.754
183 V776L NP to NP Neutral to Neutral 3 −0.017 −0.160
184 S778C P to P Neutral to Neutral 3 0.409 −0.305
185 S778 N P to P Neutral to Neutral 2 0.687 −0.195
186 K780R P to P Basic to Basic 5 1.115 −0.590
187 E796D P to P Acidic to Acidic 4 −0.269 0.244
188 T801I P to NP Neutral to Neutral 5 0.114 −0.047
189 L803I NP to NP Neutral to Neutral 2 0.397 −0.105
190 L805I NP to NP Neutral to Neutral 3 −0.189 0.217
191 T806I P to NP Neutral to Neutral 29 0.151 −0.065
192 P809R NP to P Neutral to Basic 5 −0.615 −0.234
193 M818I NP to NP Neutral to Neutral 2 0.129 0.221
194 Q822H P to P Neutral to Basic 60 0.006 0.514
195 Q822K P to P Neutral to Basic 2 1.801 −0.467
196 G823C NP to P Neutral to Neutral 4 0.442 −0.644
197 G823D NP to P Neutral to Acidic 2 0.467 −0.400
198 D824Y P to P Acidic to Neutral 6 −0.451 0.111
199 V827L NP to NP Neutral to Neutral 3 0.471 −0.340
200 L829F NP to NP Neutral to Neutral 4 −1.408 0.045
201 P834L NP to NP Neutral to Neutral 3 1.266 −0.943
202 V848L NP to NP Neutral to Neutral 12 0.194 −0.201
203 V848I NP to NP Neutral to Neutral 2 −0.732 0.119
204 M855I NP to NP Neutral to Neutral 6 −0.312 0.223
205 I856V NP to NP Neutral to Neutral 2 −0.635 0.307
206 T870I P to NP Neutral to Neutral 10 0.654 −0.440
207 P873S NP to P Neutral to Neutral 6 −0.054 0.023
208 E876D P to P Acidic to Acidic 2 −1.338 1.031
209 A878S NP to P Neutral to Neutral 2 1.053 −0.488
210 D879Y P to P Acidic to Neutral 6 1.218 −0.559
211 V880I NP to NP Neutral to Neutral 34 −0.087 −0.146
212 D893Y P to P Acidic to Neutral 7 0.088 −0.168
213 T896I P to NP Neutral to Neutral 2 – –
214 M899I NP to NP Neutral to Neutral 4 – –
215 V905I NP to NP Neutral to Neutral 38 – –
216 M906I NP to NP Neutral to Neutral 2 – –
217 T908I P to NP Neutral to Neutral 5 – –
218 S913L P to NP Neutral to Neutral 19 −0.116 0.012
219 R914K P to P Basic to Basic 9 −0.240 0.168
220 P918L NP to NP Neutral to Neutral 4 0.199 −0.711
221 E922D P to P Acidic to Acidic 8 −1.106 0.193
222 T929I P to NP Neutral to Neutral 2 0.075 4.551
223 Q932H P to P Neutral to Basic 2 1.077 −4.934
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Fig. 1.

Fig. 1

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The details of the mutation identified in RdRp among Indian SARS-CoV-2 isolates. A) Schematic diagram of the domain architecture of RdRp. Each domain of RdRp is represented by a unique color. The interdomain borders are labeled with residue numbers.The mutations present in RdRp among the Indian isolates of SARS-CoV-2 are demonstrated in the schematics. B) The monthly data of ‘new sequences’ of SARS-CoV-2 and ‘appearance of new mutations’ were obtained from CoVal webserver. These data provided the time course of the SARS-CoV-2 samples reported from India. Based on the mutational analysis of RdRp by CoVal webserver, we observe that during initial phase COVID19 pandemic, the rate occurrence of new mutations were high but it slowed down as the time progresses. The X-axis shows months/year and Y-axis present on the left side of the graph represents number of SARS-CoV-2 samples reported per month while Y –axis on the right side shows the appearance of new mutation in RdRp per month. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.2. Mutations affect RdRp protein dynamic stability and flexibility

We performed protein modelling studies using DynaMut programe to understand, if the mutation observed in RdRp can alter protein structural integrity. Our data revealed that mutations at 89 positions cause stabilisation in protein structure (positive ΔΔG) as shown in Table 1, maximum positive ΔΔG was obtained for Q822K (1.801 kcal/mol). Similarly, the mutations at 111 positions cause destabilisation (negative ΔΔG) in protein structure upon mutation (Table 1), maximum negative ΔΔG was obtained for the mutant I244T (-2.233 kcal/mol).

Subsequently, we measured the changes in vibrational entropy energy (ΔΔSVibENCoM) between the wild type and the mutant. Our data revealed that mutation at 89 positions causes increase in flexibility of mutant protein (positive ΔΔSVibENCoM). The maximum positive ΔΔSVibENCoM was obtained for T929I (4.55 kcal.mol-1.K-1) mutant. Similarly, the mutations at rest of the 111 positions cause rigidification of protein structure (negative ΔΔSVibENCoM) in protein structure upon mutation (Table 1). The maximum negative ΔΔSVibENCoM was obtained for Q932H (-4.93 kcal.mol-1.K-1) mutant. Altogether, our data revealed that the mutation observed in RdRp affects both protein dynamicity and flexibility.

3.3. Identification of B cell epitopes of RdRp

The continuous B-cell epitopes of RdRp were predicted by IEDB webserver tool and the epitopes are shown in Fig. 2 A. The yellow area of the graph corresponds to those regions of the RdRp that can potentially contribute to the B cell epitopes. Our data demonstrated thirty-six epitopes of varying lengths that could potentially act as B cell epitopes (Fig. 2B). Among those peptides, the ‘peptide 18’ is the largest epitope of 44 amino acids (from RdRp residue 482 to 525). Similarly, peptide 5, 19, 30, 31 and 34 are comprised of single amino acid only (Fig. 2B).

Fig. 2.

Fig. 2

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Prediction of B-cell epitopes of RdRp. A) Linear continuous B-cell epitopes contributed by RdRp, the Y-axis of the graph corresponds to BepiPred score, while the X-axis depicts the RdRp residue positions in the sequence. The data was generated by IEDB webserver using ‘Bepipred Linear Epitope Prediction 2.0’ method. The chart is divided into two parts, yellow and green. The RdRp residues present in the yellow area have higher probability to be part of the linear continuous B cell epitope. B) The details of the linear continuous B cell epitopes are listed. The sequence of each peptide along with its start and end point in the RdRp polypeptide sequence is also mentioned. C) Prediction of discontinuous B-cell epitopes of RdRp by DiscoTope 2.0 web tool. The position of each predicted epitope is mentioned along with its propensity and DiscoTope score. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Subsequently, we predicted the B cell epitopes of RdRp based on its three dimensional structure using DiscoTope 2.0 webserver tool [20]. Our analysis revealed twenty-five discontinuous epitopes of RdRp having high score. The locations of these epitopes are listed in Fig. 2C along with its propensity and DiscoTope score. Among discontinuous epitopes, approximately 80% of them (20 out of 25) reside towards the C-terminal end of RdRp (from residue 800 to 932) as shown in Fig. 2C. Altogether, our data revealed B cell epitopes contributed by RdRp.

3.4. RdRp mutants preferentially localises in the B cell epitopes region

Next, we analysed and compared the RdRp mutations that reside in the linear-continuous and discontinuous B cell epitopes. Our data revealed that out of 223 mutants observed in this study, 98 resides in the B cell epitope region of RdRp (Fig. 3 A). These 98 mutants correspond to 44% of the total mutants observed among Indian isolates. The details of all 98 mutants that localises in B cell epitope region are shown in Fig. 3B. Altogether, our data strongly suggest that several RdRp mutations localises in the B cell epitope region.

Fig. 3.

Fig. 3

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Correlation of RdRp mutants and B cell epitopes. A) The graph shows the distribution of RdRp mutants observed among Indian SARS-CoV-2 isolates. Out of 223, 98 mutants localises in B cell epitope region of RdRp. B) Detail of the RdRp mutants that localises in B cell epitope region.

4. Discussion

The coronaviruses belongs to RNA viruses that exhibits high rate of mutations in their genome [21]. As these viruses spread to new locations they keep on acquiring mutations and few of them are naturally selected because of their beneficial effect on the virus. The investigation on the genomic variation acquired by SARS-CoV-2 is indispensable for understanding the epidemiology, pathogenesis; devise preventive measures and treatment strategies against COVID-19. The earlier variation studies on SARS-CoV-2 revealed that RdRp is among the mutational hotspot protein [14]. In the similar directions, this study was conducted with an aim to identify mutations in RdRp from Indian isolates. Our earlier study revealed seven crucial mutations in RdRp of SARS-CoV-2 [22] that can have potential impact on this protein function. The present study identifies and characterises B cell epitope contributed by RdRp and correlate them with the observed mutants. In this study, we analysed 50217 RdRp sequences reported from India till Sept 2021 and identified 223 mutations in RdRp, which indicates that RdRp is one of the mutational hotspot protein of SARS-CoV-2. Furthermore, our data revealed that there are thirty-six high rank linear-continuous B cell epitope as well as twenty-five discontinuous B cell epitopes. Moreover, we also identified that out of 223 mutants identified among Indian isolates, 98 resides (44%) in these B cell epitope region.

We used bioinformatics approach to identify probable epitopes that offer various advantages over conventional approaches. However, despite advantages of immunoinformatics, there are certain limitations. Such as the final selection of epitopes from the probable epitopes identified using bioinformatics is still a challenging task. The RdRp epitopes revealed in this study requires validation using in vivo experiments, which is slow and herculean task. Furthermore, the algorithms used for predicting epitopes are liable to alter if the criteria are changed during the tool selection. Therefore, the algorithms are constantly improved to get better output and more reliable data [7,8]. The variations in RdRp or any other protein of SARS-CoV-2 will possibly tell us how the virus is evolving. Earlier studies with RNA viruses have also shown that these viruses keep on mutating to better adapt and survive in the host [23]. Here, in this study, we have reported RdRp mutations, its correlation with B cell epitopes. However, it warrants future studies to understand the possible effect of these mutations on virus infectivity and life cycle.

Acknowledgements

We would like to acknowledge Patna University, Patna, Bihar (India) for providing infrastructural support for this study. This work has been partly funded by (Science and Engineering Board, Department of Science and Technology, Government of India) a project awarded to GKA (Project number: SRG/2020/000808).

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