December07, Unitedkingdom  2021 


Abstract Volume: 3 Issue: 4 ISSN:

Mutations in Nsp10 of SARS-CoV-2 in Pakistani Isolates and their Effect on Protein Stability and Flexibility

Muhammad Sohail.

Corresponding Author: Muhammad Sohail,

Copy Right: © 2021 Muhammad Sohail, this is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.  

Received Date: October 18, 2021

Published date: November 01, 2021



The main public health issue is COVID-19 diseases provoked by SARS-CoV-2 (severe acute respiratory syndrome corona virus 2). The disease begins in China and then proliferated very speedily throughout the entire world. The CoV-2 has an RNA genome of 30kb in size, encoding 16 non-structural proteins (NSP) and 4 structural proteins including envelope, membrane, nucleocapsid, and spike proteins. The goal of this study was to look at the most frequent mutations in one of the most significant NSP10 protein among SARS-CoV-2 Pakistani isolates. We screened the genomic data of SAR-CoV-2 of Pakistani isolates and investigated mutations in NSP10. In the Global Initiative on Sharing All Influenza Data, CoV-surver was used to align the SARS-CoV-2 genome with the reference genome (Pakistan). The impact of mutations on the structure of NSP10 was investigated using the DynaMut server. Among these mutations, 21 increased the stability of NSP10 while 10 were involved in decreasing the protein stability. Similarly, 4 were found, to enhance the flexibility of NSP10 while 27 decreasing the protein flexibility.  NSP10_L92stop is stopped, Deletion is occurred at NSP10_C103del and insertions occurred at these 2 mutations NSP10_ins86stopIIQIL, NSP10_ins93VSM.

The presence of a large number of mutations in NSP10 may be an obstacle to effective drug design. Moreover, inhibitors should be designed based on mutations, present in the local isolates of CoV-2. Further large studies on the whole genome of CoV-2 are needed to explore the variation in all drug target proteins. Geographic specific drugs should be designed against local SARS-COV-2 for better management of CoV-2 infections.

Keywords: MERS-CoV, SARS-CoV, SARS-CoV-2, mutations, NSP10, stability and flexibility.

List Of Abbreviations

NSPS    -          Non-Structural proteins

NSP-10 -          Non-Structural protein10

NSP-14-           Non-Structural protein 14

NSP-16 -            Non-Structural protein 16

RTC    -           Replication Transcription complex

N         -           Nucleocapsid 

E          -           Envelope

M         -           Membrane

MERS- CoV  -Middle East Respiratory Syndrome Virus

WHO  -          World Health Organization

SARS-CoV-2- Sever Acute Respiratory Syndrome –Coronavirus-2

CoV-1 -           Sever Acute Respiratory Syndrome –Coronavirus-1

N7-MTase  -    N7-methyltransferase

RdRp   -           RNA-dependent RNA polymerase,

CoVs   -           Coronaviruses

WT      -         Wild Type

MT      -         Mutant Type

Mutations in Nsp10 of SARS-CoV-2 in Pakistani Isolates and their Effect on Protein Stability and Flexibility


World Health Organization (WHO) alarmed last month 2019 regarding the flare of disease with a hidden etiology, which began in the Chinese province of Wuhan, Hubei (Chan et al., 2020; Dong et al., 2020). After some time, scientists identified that the cause of the illness was the coronaviruses, which relates to SARS. The respiratory infection was because of the SARS-CoV-2 (P. Zhou et al., 2020). This disease covered all the world in four months causing a global emergency. Due to this governments of all countries ordered people to stay at home.  WHO elaborates that a total of 2,074,529 people was infected due to SARS-CoV-2, these infected patients 139, 378 were dead due to this lethal virus. This data was similar to that, which was published by the server of Johns Hopkins University (Dong et al., 2020). Many species like birds and mammals, including pigs’ camels and humans, can be infected by this lethal Coronaviridae family.  This virus can cause diseases like acute and dangerous gastrointestinal infections, organ problems, and illnesses in lower vertebrates. There are three categories of coronaviruses that can cause infection having no symptoms and   the little common cold. Those are hCoVB-OC43 hCoV-229E hCoV-NL63 (Algahtani et al., 2016; Chau et al., 2004; Chen et al., 2011). The other 4 viruses cause acute diseases like pneumonia due to hCoV-HKU1, Middle East Respiratory Syndrome Virus (MERS-CoV) with death caused 37%, SARS-CoV-1 with death caused 10% (P. Zhou et al., 2020), and present-day death due to SARS-CoV-2 at 6% (Dong et al., 2020). The continuity of spreading this virus is still going on, the requirement for potent vaccines and effective medicines is rising. On the other hand, we have less knowledge about the immune reaction to a virus or the aptitude for reinfection (Moon, 2020; Vaninov, 2020; Weiss & Navas-Martin, 2005; L. Zhou et al., 2020). SARS-CoV-2 has a length of 30 kb single-stranded, (+) sense RNA genome (Wu et al. 2020). 16 non-structural proteins (NSPS) are located in the Open Reading Frame 1ab (ORF1ab) which is two-thirds of the whole genome. They are known as NSP 1 to NSP 16 (A. Wu et al., 2020). The other part at 3’prime end of the CoVID-19 genome code the Spike (S), 

Nucleocapsid (N), Membrane (M) and Envelope (E) are 4 structural proteins, these cover 1/3 of whole genome, there are also some extra proteins that vary in the family of CoV (Chen, Liu, et al., 2020).

The RTcomplex is regulated by the main 4 proteins NSP8, NSP7, NSP14, and NSP12, these are originated by polyprotein Orf1ab cleaved by the enzyme turned them into developed proteins. NSP-12 also known as RdRp (RNA-dependent RNA polymerase); it performs function of synthesizing novel RNA STRAND by retrieving message from viral genome acting as a template. For the polymerase unit, the most important co-factors are NSP7 and NSP8. By joining with each other they form (Kirchdoerfer & Ward, 2019; Q. Peng et al., 2020), exonuclease function is exhibit by NSP14, this enzyme removes the mismatch from the RNA production complex, consequently letting the SARS-CoV-2 to sustain its huge genome (Ma et al., 2015; Ogando et al., 2019; Romano et al., 2020; Subissi et al., 2014).

NSP-10 is the main protein which has a major role in genomic replication. Nsp10 is protein made of 140 constituents of amino acid, only present in viruses, and does not exist in eukaryotes and prokaryotes. The most necessary stimulator in SARS is NSP 10 for enhancing the function of nsp14 and NSP 16, and scaffolding protein. Dual functional protein NSP14 has 2 terminals N-terminal and C-terminal 3’-5’end. Both terminals possess 2 different enzymes, an exoribonucleases and N7-methyltransferase (N7-MTase). When NSP 10 binds with ExoN, this enzyme activates but for the activation of N-7MTase this protein (NSP-10) does not seem to be crucial (Chen et al., 2009). For the triggering function of   2’-O-MTase in NSP 16, you need NSP -10 (Bouvet et al., 2010), We came to know that machinery for RNA methylation required NSP-10 MOST IMPORTANT STRIKING PROTEIN (Chen & Guo, 2016).

NSP-10 of SARS-CoV-2 has a hydrophobic surface that is positively charged. This creates a link with the negatively charged region of the hydrophobic area of NSP 16 due to this SAM binding site stabilizes. The NSP-10 contains a central anti-parallel pair of β-strands surrounded on one side by a crossover large loop. The other side is a helical domain with loops that form two zinc fingers. These structures are involved in the non-specific binding of RNA in other coronaviruses (Chen et al., 2011; Matthes et al., 2006). There are 2 zinc ions in NSP 10, the first zinc ion arranged with the C74, C77, H83 and C90, the second zinc ion interacts with the C117, C120, C128 and C130. (Rosas-Lemus et al., 2020). NSP-10 links with the two proteins named NSP14 AND NSP- 16. The physical linking of these complexes promotes the activity. The NSP-10 acts as a cofactor for these two proteins. The Complex with NSP 10/ NSP 14 act as methylation of the nascent mRNA which is known as the capping process which help in altering the host immune system, the other activity of this complex acts as ExoN, this enzyme removes the mismatch that occurred in sequence and acts as a proofreading mechanism (Chen et al., 2009). In the same way at C terminal capping is done   NSP-16, 2′-O methyltransferase (MTase) with help of co-factor NSP-10 (Azad, 2020). Making NSP10 an adominant participant in the fundamental   RNA methylation mechanism (Chen & Guo, 2016).

The mutation occurred when the virus transferred from an old place to a new area. The mutation was recognized in India, in the complex of NSP10- NSP16. Due to this mutation the secondary structure is changed. The mutation occurred at the 20 (A20V) position of the protein. Mutation in wild type occurred where the residue of alanine was changed to the valine residue in the mutated protein. The mutation is situated at the N TERMINUS of the protein. In the same way mutation in NSP 16 was recognized in 5 locations. P7S, L11F, A116S, P236L, N285D. Two of these mutations were recognized at BOTH TERMINUS at N AND C NAMED AS (P75 AND N285D) of NSP-16(Azad, 2020).

The arrangement of proteins relies on the sequence of the amino acid sequence as the amino sequence is altered due to which protein’s structure also changed because the occurrence of a new mutation. Mutation at position 20 causes the arrangement to change secondary structure. Wild type has helical formation between the 17- 20 residue. Due to a change in amino acid residue helix is converted into the beta-sheet. The NSP-16 amino acid residue change produced few alterations in the secondary structure. The four amino acid residues do not cause any alteration but one of these P236L produced structural rearrangement (Azad, 2020).


Materials and Methods



For the retrieval of data, I had used many websites like Google Scholar and PubMed. From those online websites, I had downloaded 20-22articles from a different year, using those articles I had gain mine required data from almost 8- 10 articles which has data about the history of the pandemic, structural proteins, Nonstructural proteins, Function of those proteins, their arrangements, location of protein at the genomic level, how these proteins are interconnected with each other in RTC mechanism, how viruses invade the host’s Immune system form new viruses copies using the self-protein for the replication-transcription due the process of methylation due to this host immune system cannot be recognized that this is an invading particle or no, mutation in nsp10 and effect on structure.


Global Initiative on Sharing Avian Influenza Data (GISAID) (Shu & McCauley, 2017) is a database of the genome sequences of SARS-CoV-2. GISAID shares the data about lethal influenza viruses. For getting data, user would first have to register themselves, so information could be observed.

• Registered ID

• Select for search option

• Searched for Pakistani genomic sequences

For extracting the genome, Pakistani SARS-CoV-2 genomic isolates were downloaded from the GISAID server. Downloaded whole genome sequence in FASTA format. For getting the mutation, using the online CoV- SURVER application. Download the extracted mutation analysis, with their frequency. Extracted the patient history.

Structural Extraction

Protein Data Bank

The Protein Data Bank (PDB ID: 6ZCT) (Berman et al., 2000) is a database of 3D structures of macromolecules and their complexes, The PDB document right now houses ~130,000 sections (May 2017). Protein Data Bank organization supervised PDB (wwPDB;, which incorporates the RCSB Protein Data Bank (RCSB PDB;, the Protein Data Bank Japan (PDBj;, the Protein Data Bank in Europe (PDBe;, and BioMagResBank (BMRB;


DynaMut, a web server that presents the flow component to transformation investigation. This can be accomplished by actualizing and coordination well set up typical mode approaches with our graph-based marks in an agreement indicator for protein solidness changes upon transformation, which we appear optimizes by and large expectation. DynaMut to give a precise analysis about the influence of a mutation on protein stability, and give a complete information about protein motion and flexibility examination and imagining via an easy-to-use web interface (Rodrigues et al., 2018).

Different data has been given; 40 genomic samples belong to Karachi. 1 sample belong to Punjab, 1 sample belong to Sindh, 4 samples belong to Islamabad, 2 belongs to Rawalpindi, 1 sample belong to KPK, 1 sample belong to Gilgit, 7 samples belong with name of Pakistan. 8 Genomes belongs to females and 6 belongs to males’ rest of gender details are not available here.  Mostly clade belongs to GH clade they are 37, 4 clade belongs to O, 3 belong to L and 3 belong to S clade, 9 clades belong to GR, 1 belong to G clade. Age of females are given in table   25, 39, 40, 46, 49, 55 and age of males are 22, 23, 41 and 47. Few genders information is available but age is not mentioned. Mostly both information about gender and age is not present.

For extraction of these mutation, we utilized GISAID server. Total number of the retrieved mutation were 35 from different cities of Pakistan. This data demonstrates that 1 of these mutations is STOP (NSP10_L92stop) and 2 mutations are insertion (NSP10_ins86stopIIQIL, NSP10_ins93VSM). has 0 substitution and has 1 deletion mutation (NSP10_C103del).

Eight mutations occurred on N-terminal (NSP10_M63L, NSP10_D64H, NSP10_Q65R, NSP10_E66K, NSP10_S67P, NSP10_F68I, NSP10_G69W, NSP10_G70I) respectively. Other remaining 27 are occurred on the C-terminal of the protein, NSP10. SO, by these results we came to know that CTD region have excessive mutation than the NTD. Mutation on CTD (NSP10_A71K, NSP10_S72N, NSP10_C73P, NSP10_C74L, NSP10_L75V, NSP10_Y76V, NSP10_C77H, NSP10_C79V, NSP10_H80V, NSP10_I81C, NSP10_D82T, NSP10_H83A, NSP10_P84V, NSP10_N85A, NSP10_P86T, NSP10_ins86stopIIQIL, NSP10_G88D, NSP10_C90V, NSP10_D91T, NSP10_L92stop, NSP10_ins93VSM, NSP10_G94Y, NSP10_P100E NSP10_T101R, NSP10_T102R, NSP10_C103del, NSP10_A104V respectively.

For protein modelling studies table demonstrate the data of mutant and wild type Nsp10.For   this analysis DynaMut webserver was used. The values of difference in vibrational entropy (ΔΔS Vib)’ and ‘difference in free energy (ΔΔG)’ between wild-type and mutant Nsp10. Stabilization of protein is related to the positive ΔΔG value related, and protein destabilization denoted by negative value.

These all mutation belongs destabilizing  of NSP10 protein, NSP10_A104V, NSP10_C73P, NSP10_F68, NSP10_G88D, NSP10_Y76V, NSP10_S67P, NSP10_L75V, NSP10_H83A, NSP10_G94Y, with negative ΔΔG(kcal/mol) values of, -0.616, -0.928, -1.015, 0.282, -1.37, -0.944, -0.944, 1.170, -0.616 respectively. In this data mutation NSP10_L92stop is stopped, Deletion is occurred at this mutation NSP10_C103del and insertions occurred at these 2 mutations NSP10_ins86stopIIQIL, NSP10_ins93VSM. Rest of the mutations are Stabilizing. Flexibility of 4 mutations are increased NSP10_H83A, NSP10_L75V, NSP10_F68I and NSP10_Y76V. Rest of the flexibility of these mutations is decreased.

Our records exposed the visible increase or decrease in free energy in several mutations as represented in table 3. The highest increase in ΔΔG was observed for NSP10_G69W (1.495 kcal/mol) and the highest negative ΔΔG was obtained for NSP10_H83A (1.170 kcal/mol). The flexibility maximum value is (0.893 kcal.mol-1. K-1) for mutation NSP10_H83A and decreasing flexibility have (-1.425 kcal.mol-1. K-1) value for mutation NSP10_G69W.

In these interactions we came to know that the stabilization of protein is effected by mutation occurs at any one of the domain of NSP 10 protein. Where ever mutation occurred stability of protein converted in Destabilizing form which effect function and due this mutation immune system cannot recognize it easily.

In the same way mutation also effect the interaction between the wild type and mutant. Further we will recognize that which interaction is greater or smaller numbers. As we have extracted our figure from DynaMut webserver, which helped us in visualization of the different mutation of NSP10 protein. Now we will do the comparison of the data that which type of mutations have higher numbers of the interaction at the mutant site. Which mutation form maximum interaction and which are equal.

Figure 4.1. Interaction in wild type (WT) and mutant A71K and A104V. WT of A71K exhibited 6 Interactions at mutation site while mutant A71K has also 6. WT A104V of exhibited 15 Interactions at mutation site while mutant A104V has 26. Mutant -type and Wild- residues are dyed in bright-green and are also symbolized as sticks near the neigh boring residues which are engaged in several type of interactions.



Coronaviruses (CoVs)(Stadler et al., 2003) are respirational and enteric pathogens of humans and housetrained animals and also seem to be universal in wildlife, specific in bats and rodents (Corman et al., 2014; Peiris et al., 2004; Smith & Wang, 2013). The ability to cross species obstacles seems to be a widespread CoV feature, and all endemic human CoVs are supposed to spread from animal hosts at some point in the past. CoVs can source of life-threatening zoonotic infections, and the emergence in humans, less than a decade apart, of CoVs causing severe acute respiratory syndrome (SARS) (Snijder et al., 2003) and Middle East respiratory syndrome (MERS) (van Boheemen et al., 2012; Zaki et al., 2012).

The shape of NSP10 SARS-CoV-2 incorporates residues of 19-133 (the residues from 4272-4392 of pp1a). It contains a hydrophobic surface area and a positive charged particles that interconnect with a pocket of hydrophobic molecule and a surface of negative charged particle from NSP16, which facilitates within side of SAM binding stabilization. The shape of NSP 10 of SARS-CoV-2 consists of an important anti-parallel BETA chain pair which is surrounded on one facet with the aid of using a crossover massive loop. The different facet is a helical area with loops that shape the two zinc fingers. These systems are included with inside the non-unique binding of RNA in different coronaviruses (Chen et al., 2011; Matthes et al., 2006). The site 1 of Zn-binding is coordinated with the aid of using the residues C74, C77, H83 and C90. The site 2 of Zn binding is correlated with the aid of using C117, C120, C128, and C130.

The core interaction domain of NSP10 is essential for SARS-CoV replication. The machinery of RNA-synthesizing severe acute respiratory syndrome Coronavirus (SARS-CoV) has 16 nonstructural proteins (NSP1–16) encoded by ORF1a/1b. NSP10 has 148- amino acid, subunit comprises of two zinc fingers and is recognized to cooperate with both NSP14 and NSP16, stimulating their respective 3_-5_ exoribonuclease and 2_-O-methyltransferaseactivities(Bouvet, Lugari, Posthuma, Zevenhoven, Bernard, Betzi, Imbert, Canard, Guillemot, & Lécine, 2014)  Important protein involved in RNA replication is NSP10. Nsp10 exists exclusively in viruses and not in prokaryotes or eukaryotes. In SARS, NSP10 was demonstrated to be essential for the stimulation of nNSP14 and NSP16, acting primarily as a stimulatory and scaffolding protein. Nsp14 exoribonuclease (ExoN) activity and C-terminal N7-methyltransferase (N7-MTase) activity. Nsp10 binds to and stimulates ExoN activity but does not seem to be required for the stimulation of the N7-MTase (Chen et al., 2009). Nsp10 is also required for the stimulation of 2-O-MTase activity in nsp16 (Chen & Guo, 2016), making NSP10 a central player in the essential RNA methylation machinery (Bouvet et al., 2010).

NSP14 reveals the activity of exonuclease by its own, this work observed that bind to NSP10 improved the activity of nsp14 by 35-fold, applying that nsp10 performs a position in keeping the ExoN the active site within side the proper function for the catalysis of substrate (Bouvet et al., 2012; Bouvet, Lugari, Posthuma, Zevenhoven, Bernard, Betzi, Imbert, Canard, Guillemot, Lécine, et al., 2014, p. 7). The nsp10 attract the nsp14's N-terminal Ala1-Arg76 and a beta-hairpin shape accommodate the nsp14's b5 and beta 6 Ala119-Asp145, exposing the important thing player of a 1:1 ratio of nsp10 and nsp14 molecules. (Ferron et al., 2018; Ma et al., 2015) NSP10 interacts with NSP14 within side the identical manner that a hand (nsp14) interconnects with a fist (NSP10).

The variation in Genetics of nsp10 region that coordinates with the nsp14 included in a lower in replication fidelity of viruses, and the association of the complicated proteins suggested that nsp10 offers integrity of structures and balance to the ExoN area of nsp14(Bouvet, Lugari, Posthuma, Zevenhoven, Bernard, Betzi, Imbert, Canard, Guillemot, Lécine, et al., 2014).

The mechanism of 5′-capping of the mRNA in eukaryotes and a few viruses together with coronaviruses (CoVs) are important for keeping the RNA balance and protein translation in the virus. SARS-CoV-2 encodes S-adenosyl-L-methionine (SAM) structured methyltransferase (MTase) enzyme characterised via way of means of NSP16 (2′-O-MTase) for producing the capping structure. The current studies shows the binding mechanism of NSP16 and NSP10 to become aware of the function of nsp10 in activity of MTase.

Thus, for the robust binding of SAM to NSP16 the NSP10 acts as an activator. The hydrophobic interrelations have been predominately diagnosed for the NSP16-NSP10 interconections. Also, the strong bonding of hydrogen among Ala83 (NSP16) and Tyr96 (NSP10), and among Gln87 (NSP16) and Leu45 (NSP10) play essential function for the dimerization of NSP16 and NSP10.

Except two mutations (NSP10-H83A and NSP-10 A104V) all of the mutation reported here are new in my study. With respective to NSP-10 Protein Data Bank (PDB ID: 6ZCT).

There are only 2 mutation named as NSP10-H83A(Donaldson, Sims, et al., 2007) NSP-10 A104V (Countries who reported: England, USA and Australia)(Patro et al., 2020). With respective to NSP-10 Protein Data Bank (PDB ID: 6ZCT).

Eight mutations occurred on N-terminal. Other remaining 27 are occurred on the C-terminal of the protein, NSP10. So, according to result we came to know that N terminal have less mutation than C terminal.

These all mutation belongs destabilizing of NSP10 protein, NSP10_A104V, NSP10_C73P, NSP10_F68, NSP10_G88D, NSP10_Y76V, NSP10_S67P, NSP10_L75V, NSP10_H83A, NSP10_G94Y, with negative ΔΔG(kcal/mol) values of, -0.616, -0.928, -1.015, 0.282, -1.37, -0.944, -0.944, 1.170, -0.616 respectively. In this data mutation NSP10_L92stop is stopped, Deletion is occurred at this mutation NSP10_C103del and insertions occurred at these 2 mutations NSP10_ins86stopIIQIL, NSP10_ins93VSM. Rest of the mutations are Stabilizing. Flexibility of 4 mutations are increased NSP10_H83A, NSP10_L75V, NSP10_F68I and NSP10_Y76V. Rest of the flexibility of these mutations is decreased.

Our records exposed the visible increase or decrease in free energy in several mutations as represented in table 3. The highest increase in ΔΔG was observed for NSP10_G69W (1.495 kcal/mol) and the highest negative ΔΔG was obtained for NSP10_H83A (1.170 kcal/mol). The flexibility maximum value is (0.893 kcal.mol-1. K-1) for mutation NSP10_H83A and decreasing flexibility have (-1.425 kcal.mol-1. K-1) value for mutation NSP10_G69W.



According to my study using GISAID-CoV surver I had retrieved 36 mutation 34 are new variations. The drug designer should be designed the drug according to our mutations that are helpful for human health.


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