Journal of Bioinformatics, Proteomics and Imaging Analysis | Volume 4 | Issue 1 | 2020-12-30

Mutations in structural proteins of SARS-CoV-2 and potential implications for the ongoing outbreak of infection in India

Rimjhim Dasgupta

Abstract

SARS-CoV-2 has spread in India very quickly from its first reported case on 30 January 2020 in Thrissur, Kerala. With the drastic increasing number of positive cases around the world WHO raised the importance in the assessment of the risk of spread and understanding genetic modifications that could have occurred in the SARS-CoV-2. Using available genome sequence in NCBI repository from the samples of different locations in India, we identified the regions (hotspots) of the viral genome with high rates of mutation. We analysed four regions of the genome encoding structural proteins Spike (S), Nucleocapsid (N), envelop (E) and Membrane (M) proteins. Through computational biology approach, we identified multiple substitution mutations in S and N proteins whereas there is only one substitution in E protein and none in M protein. We showed most of these amino acid residues are evolutionary conserved. The changes in the conserved residues may have significant implication on the stability of the proteins and subsequent interaction with other elements, which are essential for virus propagation. This provides a basis for a better understanding of the genetic variation in SARS-CoV-2 circulating in the India, which might provide important clues for identifying potential therapeutic targets, development of efficient vaccines, antiviral drugs and diagnostic assays for controlling COVID-19.

Keywords: Coronavirus, COVID-19, SARS-CoV2, Sequence alignment, Mutation, Infection

Full text

The pandemic Corona Virus Disease 2019 (COVID-19) caused by the RNA coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), has spread over 200 countries and infected millions of people worldwide. The first case in India was reported on Jan 30 in Thrissur, Kerala and two other cases were reported by Feb 3, 2020. All of them were the students studying at a university in Wuhan, China and came home (Kerala) for vacation. As the number of the positive cases increasing drastically the World Health Organization (WHO) raised the importance of understanding genetic modification that could have occurred in the SARS-CoV-2 and whether the variations in genome sequences is impacting person to person transmission and mortality rate. Searching for mutations and their evolutionary conservation while the virus continues to spread within the country can offer opportunities for a better understanding of virus evolution, biopathology, and transmission. Having this motivation, considering Wuhan based genome NC_045512.2 as reference, we report on multiple missense mutations on structural proteins Spike (S), nucleocapsid (N) and envelope (E) in many Indian isolates and absent in the Chinese ones. Methods Wuhan isolate, SARS-CoV-2 sequence NC_045512.2 (length 29903 nt) was used as a reference sequence and for sequence comparisons. For our study, we considered the sequences that were available in GISAID and NCBI. We first compared 20 available sequences from Wuhan. They showed 100% sequence similarities. In the present report we have focussed on sequence alignments, we have used NCBI BLAST, and CLUSTAL OMEGA.~150 genome sequences (sourced from Global Initiative on Sharing All Influenza, GISAID, https://www.gisaid.org/and NCBI) from different regions of India were considered for this study. Results We have taken attempt to conduct genomic comparison by considering ~150 SARS-Cov2 sequences from patient samples of different locations in India. Since COVID19 started from Wuhan, China, we started our analyses from sequences from Wuhan. We first compared 20 sequences of patient samples from Wuhan. It showed 100% sequence similarities (data not shown). We considered NC_045512.2 as reference for our analysis. Our analysis revealed that several sites are possessing non-synonymous substitutions and associated amino acid changes. We analysed four regions of the genome encoding structural proteins, those are Spike (S), Nucleocapsid (N), envelop (E) and Membrane (M) proteins. Multiple non-synonymous substitutions were found in S and N proteins. The list of mutations detected in this set of sequences from ~150 samples are provided in Table 1 and table 2. Table 1: The list of mutations in S (Spike) protein is placed in tabular form. Sequence ID is the accession number followed by sample location (patient sample location), mutated residue (single letter code for amino acid has been used) and region (region in protein sequence) Sequence ID Sample location Mutation Region QIA98583.1 Kerala A930V S2 domain QHS34546.1 Kerala Y145Stop, R407I, D614G, Junction of S1 & S2 QJQ39968.1 Bangalore D614G Junction of S1 & S2 QJQ39980.1 Bangalore D614G Junction of S1 & S2 QJQ39992.1 Bangalore D614G Junction of S1 & S2 QJF77858.1 Hyderabad D614G Junction of S1 & S2 QJF77882.1 Hyderabad D614G Junction of S1 & S2 QKQ30054.1 Ahmedabad D614G Junction of S1 & S2 QKQ30066.1 Ahmedabad D614G Junction of S1 & S2 QKQ30090.1 Ahmedabad D614G Junction of S1 & S2 QKQ30102.1 Ahmedabad D614G Junction of S1 & S2 QKQ30114.1 Ahmedabad D614G Junction of S1 & S2 QKQ30150.1 Ahmedabad D614G Junction of S1 & S2 QKQ30246.1 Ahmedabad D614G Junction of S1 & S2 QKQ30258.1 Ahmedabad D614G Junction of S1 & S2 QKQ30270.1 Ahmedabad D614G Junction of S1 & S2 QKQ63388.1 Ahmedabad D614G Junction of S1 & S2 QJQ28345.1 Ahmedabad D614G Junction of S1 & S2 QJQ28357.1 Ahmedabad D614G Junction of S1 & S2 QJQ28369.1 Ahmedabad D614G Junction of S1 & S2 QJQ28381.1 Ahmedabad D614G Junction of S1 & S2 QJQ28405.1 Ahmedabad D614G Junction of S1 & S2 QKY60165.1 Bharuch D614G Junction of S1 & S2 QJW00363.1 Dahegam D614G Junction of S1 & S2 QJW00375.1 Dahegam D614G Junction of S1 & S2 QJW00387.1 Dahegam D614G Junction of S1 & S2 QKV25909.1 Ahmedabad D614G Junction of S1 & S2 QKV27563.1 Dhanera D614G Junction of S1 & S2 QKV25945.1 Vadodara D614G Junction of S1 & S2 QKV25957.1 Vadodara D614G Junction of S1 & S2 QKV25981.1 Vadodara D614G Junction of S1 & S2 QKV26029.1 Vadodara D614G Junction of S1 & S2 QKV26041.1 Vadodara D614G Junction of S1 & S2 QKV26065.1 Vadodara D614G Junction of S1 & S2 QKV26089.1 Vadodara D614G Junction of S1 & S2 QKG91214.1 Himatnagar D614G Junction of S1 & S2 QKI28661.1 Himatnagar D614G Junction of S1 & S2 QJS39639.1 Hyderabad D614G Junction of S1 & S2 QJY40421.1 Jamnagar D614G Junction of S1 & S2 QJY40445.1 Jamnagar D614G Junction of S1 & S2 QJY40505.1 Junagadh D614G Junction of S1 & S2 QKQ29946.1 Kadi D614G Junction of S1 & S2 QKQ29958.1 Kadi D614G Junction of S1 & S2 QKQ30042.1 Kalol D614G Junction of S1 & S2 QKQ30174.1 Kalol D614G Junction of S1 & S2 QKQ30198.1 Kalol D614G Junction of S1 & S2 QKH78810.1 Khedbrahma D614G Junction of S1 & S2 QKI28637.1 Khedbrahma D614G Junction of S1 & S2 QJY40385.1 Kodinar D614G Junction of S1 & S2 QKY64792.1 Mandvi D614G Junction of S1 & S2 QJW00447.1 Mansa D614G Junction of S1 & S2 QJQ28429.1 Mansa D614G Junction of S1 & S2 QJW39904.1 Modasa D614G Junction of S1 & S2 QKY74628.1 Palanpur D614G Junction of S1 & S2 QKY59941.1 Palanpur D614G Junction of S1 & S2 QJW39916.1 Prantij D614G Junction of S1 & S2 QJT43680.1 Prantij D614G Junction of S1 & S2 QKJ68485.1 Rajkot D614G Junction of S1 & S2 QKY60049.1 Surat D614G Junction of S1 & S2 QKY60085.1 Surat D614G Junction of S1 & S2 QJY40529.1 Una D614G Junction of S1 & S2 QJY40577.1 Una D614G Junction of S1 & S2 QJY51288.1 Vadodara D614G Junction of S1 & S2 QJY51348.1 Vadodara D614G Junction of S1 & S2 QJY51384.1 Vadodara D614G Junction of S1 & S2 QKY65277.1 Bhuj D614G Junction of S1 & S2 EPI_ISL_430465 Kolkata D614G Junction of S1 & S2 EPI_ISL_430467 Kolkata D614G Junction of S1 & S2 EPI_ISL_430468 Kolkata D614G, G1124V Junction of S1 & S2, S2 EPI_ISL_430464 Kolkata D614G, G1124V Junction of S1 & S2, S2 QKQ30078.1 Ahmedabad E583D, D614G RBD, Junction of S1 & S2 QKQ30222.1 Ahmedabad E583D, D614G RBD, Junction of S1 & S2 QJW00327.1 Himatnagar E583D, D614G RBD, Junction of S1 & S2 QJQ27854.1 Bangalore K187X, D614G NTD, Junction of S1 & S2 QKV25969.1 Vadodara L54F, D614G NTD, Junction of S1 & S2 QKJ84943.1 Himatnagar L54F, D614G NTD, Junction of S1 & S2 QKJ84979.1 Himatnagar L54F, D614G NTD, Junction of S1 & S2 QKY64614.1 Kheda L54F, D614G NTD, Junction of S1 & S2 QKY64640.1 Kheda L54F, D614G NTD, Junction of S1 & S2 QKY74640.1 Palanpur L54F, D614G NTD, Junction of S1 & S2 QKY60213.1 Savli L54F, D614G NTD, Junction of S1 & S2 QKY60225.1 Savli L54F, D614G NTD, Junction of S1 & S2 QKY60025.1 Surat L54F, D614G NTD, Junction of S1 & S2 QKY60073.1 Surat L54F, D614G NTD, Junction of S1 & S2 QKY60201.1 Vadodara L54F, D614G NTD, Junction of S1 & S2 QKY60237.1 Vadodara L54F, D614G NTD, Junction of S1 & S2 QKY60264.1 Vadodara L54F, D614G NTD, Junction of S1 & S2 QKI28601.1 Jamnagar L5F, T572I, D614G NTD, RBD, Junction of S1 & S2 QJY40469.1 Jamnagar L5F, S162I, D614G NTD, RBD, Junction of S1 & S2 QJY40409.1 Una Q677H S2 domain QJW69139.1 Modasa R78M, D614G, Q784X, V785X, K786X, Q787X, I788X, Y789X, NTD, S2 domain QJW00315.1 Modasa R78M, D614G NTD, Junction of S1 & S2 QJW39892.1 Modasa R78M, D614G NTD, Junction of S1 & S2 QJW39928.1 Modasa R78M, D614G NTD, Junction of S1 & S2 QJT43704.1 Modasa R78M, D614G NTD, Junction of S1 & S2 QJT43716.1 Modasa R78M, D614G NTD, Junction of S1 & S2 QJY77055.1 Jamnagar D614G, K811X, P812X, S813X, K814X, R815X S2 domain QKQ30126.1 Ahmedabad T572I, D614G, RBD, Junction of S1 & S2 QKQ30186.1 Ahmedabad T572I, D614G, RBD, Junction of S1 & S2 QKV27587.1 Nadiad W152L, D614G NTD, Junction of S1 & S2 QJF77846.1 Hyderabad Y28H NTD Table 2: The list of mutationsin N (Nucleocapsid) protein is placed in tabular form. Sequence ID is the accession number followed by sample location (patient sample location), mutated residue (single letter code for amino acid has been used) and region (region in protein sequence) Sequence ID Sample location Mutation Region QKI28681.1 Khedbrahma P6T NTD QJU70565.1 Hyderabad P13L NTD QJQ27850.1 Bangalore P13L NTD QJQ27874.1 Bangalore P13L NTD QJQ27886.1 Bangalore P13L NTD QJF77878.1 Hyderabad P13L NTD QJF11844.1 Bangalore P13L NTD QJF11856.1 Bangalore P13L NTD QJF11868.1 Bangalore P13L NTD QJR84533.1 Ahmedabad P13L NTD QJY40405.1 Botad P13L NTD QJS39659.1 Hyderabad P13L NTD QJT43688.1 Prantij P13L NTD QJQ28437.1 Mansa P13R NTD QJY77063.1 Jamnagar P13L NTD QJY40417.1 Una P13L, A146S NTD, RNA binding domain QKQ30158.1 Ahmedabad D22Y, S194L NTD, Linker peptide QKY60093.1 Surat D22N, L139F, S194L NTD, RNA binding domain, Linker peptide QJQ28377.1 Ahmedabad S33I NTD QJQ28389.1 Ahmedabad S33I NTD QKV26073.1 Vadodara A134V RNA binding domain QJF77854.1 Hyderabad P344S dimerization domain (DD) QJW69147.1 Modasa D399X, L400X, D401X, D402X, F403X, S404X, K405X, Q406X, L407X, C terminal end QKY60069.1 Surat S194L, S202N Linker peptide QJY40429.1 Jamnagar S194L, D348Y Linker peptide, dimerization domain (DD) QKI28669.1 Himatnagar S194L Linker peptide QKY74648.1 Palanpur S194L, R203G, G204R Linker peptide QJY40585.1 Una R203K, G204R Linker peptide QJY40537.1 Una R203K, G204R Linker peptide QKY74636.1 Palanpur R203K, G204R Linker peptide QKY64800.1 Mandvi R203K, G204R Linker peptide QJF11880.1 Bangalore R203K, G204R Linker peptide QJF11832.1 Bangalore R203K, G204R Linker peptide QJF11820.1 Bangalore R203K, G204R Linker peptide QJH92187.1 Pune R203K, G204R Linker peptide EPI_ISL_430468 Kolkata R203K, G204R Linker peptide EPI_ISL_430464 Kolkata R203K, G204R Linker peptide EPI_ISL_430465 Kolkata R203K, G204R Linker peptide Total 25 different missense mutations were found in S protein. Among them 8 are in N terminal domain (NTD) of S1 subunit, 3 (R407I, T572I, E583D) are in receptor binding domain (RBD), 1 is in C terminal of S1 subunit and 13 is in S2 domain. We found L54F, R78M, D614G mutations in 9%, 4% and 90% of sequences respectively. L54F and R78M mutations were detected only in some patients in Gujarat. We noticed Y145Stop, R407I mutations in one sequence from Kerala (QHS34546.1). S162I substitution (polar to nonpolar) was observed in one sequence (QJY40469.1). 2% of the sequences have T572I substitutions. All these also have D614G substitutions. We conducted multiple sequence alignment of S proteins to analyse the evolutionary conservation of amino acid residues for many of these mutations (Figure 1). Our sequence alignment result shows that Leucine at position 5 is evolutionary conversed through bat, pangolin, SARS-Cov, SARS-CoV2, Leucine at 54F is conserved in RatG13, pangolin, MERS-CoV, SARS-CoV and SARS-CoV2, Arginine in position 78 is conserved in bat and SARS-CoV2, Tyrosine at 146 is conserved in bat, pangolin and SARS-CoV2, Arginine at position 408 is conserved in bat, pangolin, SARS-Cov, SARS-CoV2, Threonine at 572 is conserved in bat, pangolin and SARS-CoV2, Aspartic acid at position 614 is conversed in bat, pangolin, SARS-Cov, SARS-CoV2, Glycine at 1124 is conversed in bat, pangolin, SARS-Cov, SARS-CoV2. Figure 1: Alignment of S proteins (MERS-CoV accession # AFS88936.1; SARS-CoV accession # AAP30030.1; Pangolin coronavirus accession # QIA48614.1; Bat coronavirus RaTG13accession # QHR63300.2; SARS-CoV2 REFSEQ: accession NC_045512.2accession # YP_009724390.1; SARS-CoV2accession # QHS34546.1 of Kerala patient sample; SARS-CoV2 accession # QKY60264.1 of Vadodara patient sample; SARS-CoV2 accession # EPI_ISL_430464 Kolkata patient sample; SARS-CoV2 accession # QJW00315.1 Modasapatient sample; SARS-CoV2 accession # QKQ30126.1 Ahmedabadpatient sample; SARS-CoV2 QKI28601.1 Bayad patient sample); Mutated conserved amino acid positions are highlighted in green and mutated amino acids are in pinkhighlight; The numbers below the alignments denote the residue position ofcorresponding to SARS-CoV-2 S protein Total 23 different missense mutations were found in nucleocapsid (N) protein. One sample from Gujarat (QKI28681.1) has P6T mutation, nonpolar to polar amino acid. P13L mutation was found in 10% sequences. D22Y and D22N were found in 2 sequences and both also have additional mutations. S33I mutation was found in 2 sequences and these do not have any additional mutation in N protein. 4% sequences have S194L (polar to non-polar) mutation and among them 6% also has S202N (both are polar but Ser is polar neutral whereas Arg is hydrophilic) mutation. Two mutations, R203K and G204R were found in ~6% of ~150 sequences. Sample locations are Gujarat, Bangalore, Pune, Delhi and Kolkata. We also conducted multiple sequence alignment of N proteins to analyse the evolutionary conservation of amino acid residues for many of these mutations. Our result shows that Proline at position 6, 13 are conversed in bat, pangolin, SARS-Cov, SARS-CoV2; Aspartic acid at position 22 is conversed in bat, pangolin, MERS-CoV, SARS-Cov, SARS-CoV2; serine at position 194, Glycine at position 204 are conserved in are conversed in bat, pangolin, MERS-CoV, SARS-Cov, SARS-CoV2; Arginine is conversed in bat, pangolin, SARS-Cov, SARS-CoV2 (Figure 2). Figure 2: Alignment of N proteins (MERS-CoV accession # AFS88943.1; SARS-CoV accession # AAP30037.1; Pangolin coronavirus accession # QIG55953.1; Bat coronavirus RaTG13 accession # QHR63308.1; SARS-CoV2 REFSEQ: accession NC_045512.2 accession # YP_009724397.2; SARS-CoV2 accession # QKY74648.1 of Palanpur patient sample; SARS-CoV2 accession # QJY40417.1 of Unapatient sample; SARS-CoV2 accession # QKY60093.1of Suratpatient sample; SARS-CoV2 accession # QKY60069.1 of Surat patient sample; SARS-CoV2 accession # QKI28681.1 of Khedbrahmapatient sample; Mutated conserved amino acid positions are highlighted in green and mutated amino acids are in pink highlight; The numbers below the alignments denote the residue position of corresponding to SARS-CoV-2 N protein SARS- CoV2 envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus life cycle, such as assembly, budding, envelope formation, and pathogenesis. One substitution (V62F) was found at the C terminal end in one sample from Gujarat (Figure 3). Figure 3: Alignment of C terminal end of E protein shows substitution mutation V62F (SARS-CoV2 accession # QKV26079.1 of Nadiad patient sample with Reference sequence NC_045512.2 accession # YP_009724392.1); Mutated residue in pink highlight; The numbers above the alignment denote the residue position of corresponding to SARS-CoV-2 E protein The important point to note here that most of the patient samples where mutations were identified either have international travel history or direct contact to the person travelled. Discussion Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is being intensively studied, particularly its evolution, in the increasingly available sequences with classical phylogenetic tree representation. We report here certain protein mutations which could have potential structural and/or functional implication. Mutations in Spike protein and potential implication Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface [1] . S comprises two functional subunits, responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). For many Corona viruses, S is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the prefusion conformation. This region is reported to be the most potent and indispensable for viral attachment and entry into host system [2] . The miss-sense mutations in S protein those we report are mostly single point mutations with few double and triple mutations. All these mutations could be classified as stabilizing and destabilizing based on the free-energy changes. We found L5F, L54F substitutions in 1.5% and 9% sequences considered in this study. Both amino acids are non-polar, but phenylalanine has a benzoic ring in the side chain which may stiffen the secondary structure by means of aromatic-aromatic, hydrophobic or stacking interactions. Earlier report in Non- Structural Protein 6 (NSP6) by amino acid change stability (ACS) analysis showed that this (leucine to phenylalanine) leads to a lower stability of the protein structure [3] . We noticed R78M mutation in 4% of the sequences studied. Arginine (R) is basic, hydrophilic whereas methionine (M) is nonpolar hydrophobic. Other mutations in N terminal domain (NTD) includes Y145Stop, W152L, S162I, K187X. Previous study [2] (Walls et al., 2020) showed a high thermal factor for the NTD (through the cryo-EM structure of the S protein). As these mutations are found at NTD, this observation puts an open question, whether the virus adopts viability through mutations, stabilizing the flexible NTD. The receptor-binding domain or RBD of the Spike protein of SARS-CoV-2 lies between amino acids 330 and 583. We found R407I, T572I, E583D mutations in RBD. The high-temperature factor of RBD was reported earlier [2] which is associated with its dynamic nature leading to the conformational switch between close and open states. All three substitutions we report here, lie in the RBD, which could be corelated with the alteration in receptor binding affinity. Arginine (R407I) is a positively charged amino acid and Isoleucine is a hydrophobic amino acid. While positively charged amino acid could be more exposed, hydrophobic amino acids secure themselves away from the outer aqueous environment. For T572I, Threonine is polar and Isoleucineis a hydrophobic amino acid. Recent report suggests that S protein binds to ACE2 (angiotensin converting enzyme 2) through Leu455, Phe486, Gln493, Asn501, and Tyr505 [4] . This substitution mutation of neighbouring residues may impact host receptor interaction by altering the protein conformation. All together these may potentially impact on the binding affinity to host receptor. D614G substitution was observed in ~90% sequences studied in our analysis. It occurs either as a single mutation or coupled with other mutations (L5F/S162I/D614G, L54F/D614G, R78M/D614G, W152L/D614G, E583D/D614G, Y145Stop/R407I/D614G). Laha et al (2020) [5] showed that the change from Aspartic acid to Glycine alters the electro-static potential of the surface of the protein. This change creates a favourable environment in a hydrophobic pocket of the S protein. Another report (Flores and Cardozo 2020) [6] revealed that the amino acid at position 614 occurs at an internal protein interface of the viral spike, and the presence of G at this position destabilises a specific conformation, within which the key host receptor binding site is more accessible. It makes G614 is a more pathogenic. In this study we have seen that L5F, L54F, R78M mutations are individually coupled with D614G. Earlier (Benvenuto et al. 2020) [3] . ACS analysis showed that leucine to phenylalanine leads to a lower stability of the protein structure. There is possibility that D614G coupled with Leucine to Phenylalanine mutation brings more favourable environment to enhance accessibility to host receptor and makes the virus more infectious. Another substitution Q677H was observed in one sequence of patient sample from Una. This position lies at the junction of S1 and S2 subunit in the vicinity of furin cleavage site, which plays an important role in virus entry. Glutamine is polar whereas Histidine is a basic hydrophilic amino acid. It was reported earlier that glutamine to histidine in H1N1 had a severe impact in virus entry replication and cross infectivity to other species [7] . Whether both these mutations (D614G, Q677H) have resulted in the evolution of a more stable and transmissible viral subtype needs investigation. Mutation A930V was found in the sequence (QIA98583.1) from Kerala sample which falls in the S2 subunit. Considering the nature of valine being destabilizing causing distortion [5] , this mutation might have implications in viral membrane fusion. The residue alanine at position 930 stabilizes the protein due to its hydrophobic nature. On substitution with Valine in the same position, it can potentially change the affinity of the molecule toward its receptor. Secondly this substitution was observed only in QIA98583.1 (patient from Kerala who was studying in Wuhan). Interestingly this sequence doesn t have D614G mutation. We have not found D614G or any other mutations (we reported here) in any Chinese sequences studied in this study whereas many of these including D614G are reported in sequences from other countries [6] . Probably this is the indication that the virus is getting mutated after multiple passages. Our multiple sequence alignment shows that the amino acid residues which had undergone mutations, are evolutionary conserved (Figure 1). This implies the significance of these residues in respective positions. Any alteration could impact the structural and functional conformation of the protein which makes it more infectious. Although the clinical significance of the observed mutations is not readily available, our findings in Indian patients lay the ground work for India to understand the impact of SARS-CoV2 mutations on disease severity, host immune response, vaccine development and serological response. Mutations in Nucelocapsid (N) protein and potential structural and functional implication The nucleocapsid protein is an important structural protein for the coronaviruses. It is highly abundant in the viruses. Its function involves entering the host cell, binding to the RNA, and forming the ribonucleoprotein core. It consists of RNA binding domain (RBD; residues 44-180) in the N-terminal region (N) of the protein, linker peptide (residues 181-246), the dimerization domain (DD; residues 247-364) in the C-terminal region[8]. Three disordered regions were reported on 1) N terminal (residues 1-43), 2) linker peptide, and 3) C terminal end (residues 365-419). We found P6T, P13L, D22Y, D22N, S33I in first disordered region, A134V, L139F, A146S in RBD, S194L, S202N, R203G, G204R in linker (second disordered region), P344S, D348Y in dimerization domain and D399X, L400X, D401X, D402X, F403X, S404X, K405X, Q406X, L407X in 3rd disordered region. R203K/G and G204R mutations were observed in ~6% sequences. Sometimes it is coupled with S194L. Triple base mutations of 2881-2883 GGG/AAC was detected in coding region that results in two consecutive amino acid changes, R203K and G204R. The sites of these mutations are located in the SR-rich intrinsically disordered region [9] . Moreover, this region surrounds with GSK3 phosphorylation SRGTS (amino acid position 202-206) and CDK phosphorylation SPAR (amino acid position 206-209) motifs [10] . When Ser202 is phosphorylated it incorporates a large negative group tethered to the sidechain of Ser, as seen in many other substrates of kinases. Arg203 is a part of GSK3 phosphorylation motif and its sidechain could potentially contribute to charge neutralization at phosphorylated-Ser202. Earlier structural study [8] (Zeng et al. 2020) reports that multiple disordered regions facilitate the N protein to transiently bind to different partners and maintain a correct conformation. Since we report multiples mutations in these disordered regions, possibly it impacts the binding of different partners to attain the correct conformation. Our protein alignment study shows that most of the positions in which point mutations occurred are conserved in bat, pangolin, SARS-Cov and MERS-CoV (Figure 2). It implies the structural and functional significance of these residues for protein stability and interaction with binding partners. Mutation in envelope (E) protein and its potential implication The envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus life cycle, such as assembly, budding, envelope formation, and pathogenesis. Recent studies have reported its structural motifs and topology. The report also showed its function as an ion-channelling viroporin, and it interacts with both other coronaviruses (MERS-CoV, SARS-CoV) proteins and host cell proteins (Schoeman and Fielding 2019). The SARS-CoV E protein consists of three domains, i.e. the amino (N)-terminal domain (residues ~1-8), the transmembrane domain (TMD, residues 9 -38)), and the carboxy (C)-terminal domain (residues 39- 75) [11] . We found one substitution (V62F) at the C-terminus in one sequence of patient sample from Gujarat. Earlier study showed that V25F hampers the oligomerisation of SARS-CoV E, to some extent. It was also reported that SARS-CoV E protein contain a binding motif known as the postsynaptic density protein 95 (PSD95)/Drosophila disc large tumour suppressor (Dlg1)/zonula occludens-1 protein (zo-1) (PDZ)-binding motif (PBM), located in the last four amino acids of the C terminus [11] . The PBM motif can bind to the C-terminus of target proteins such as the cellular adapter proteins involved in host-cell processes important for viral infection. We identified the similar motif DLLV (residues: 72-75) at the C-terminus in all the sequences of SARS-COV2 analysed in this study (Figure 3). V62F can potentially interfere the interaction with target proteins thereby altering the host-cell processes required for viral infection. This analysis does not find any mutation in Membrane (M) protein in the sequences from ~150 patient samples of different regions in India. This implies that M protein is comparably stable but needs further investigation by increasing sample size and covering more locations. Conclusion All together our findings provide leads which might benefit outbreak tracking, development of therapeutic and prophylactic strategies against the infection. These mutations in the conserved amino acids, likely developed during virus spread, could affect virus host receptor interaction, stability, intracellular survival. This report provides important insights for functional validation to understand the molecular basis of differential disease severity. 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