Point mutation of COVID-19 proteins: A study on noval corona virus (nCov) correlation with MERS and H1N1 viruses and in silico investigation of nCoV proteins for future applications

2.1Sequence collection

We collected 34 complete genome sequences of nCoV freshly submitted in NCBI database till 18 March 2020. Additionally, we have collected 4 MERS-CoV sequences of China and U.S from the Viral Genome Database and 9 H1N1 influenza virus sequences (submitted from India and China) from Influenza Virus Database using Open Flu Database (Table 1a & b). Next, we collected all types of proteins sequences for the same nCoV from NCBI which was used for comparison with MERS and HIN1 genome.

2.2Multiple sequence alignment using clustal omega

The collected genome sequences were used for multiple sequence alignment (MSA) using online tool, Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) which helped us to collect Jalview format of the aligned sequences [13, 14].

2.3Jalview based analysis of genome and protein sequences

Jalview (https://www.jalview.org/) is a free bioinformatics tool for the analysis of DNA, RNA, and proteins. After performing MSA in clustal omega, we download Jalview format of MSA result and then we use Jalview offline software to visualize the result and export the data in FASTA format [15].

2.4MEGA (Molecular evolutionary genetics analysis) for phylogenetic analysis

The exported FASTA file of the MSA was opened using an offline tool, MEGA-X [https://www.megasoftware.net/] for phylogenetic analysis and construction of a phylogenetic tree was generated using maximum likelihood method [16].

2.5InterPro scan database for domain analysis

The InterPro Scan database has the information to understand the protein families and its functional domain. To understand the functional domain of each protein we used an Indian submitted sequence for nCoV (MT012098). We obtained the domain information from the database of individual proteins of the virus and correlated the mutation result [17].

3.1Phylogenetic analysis of nCoV with MERS-CoV and H1N1

To understand the relationship among the nCoV and other respiratory diseases we collected 34 complete genome sequences of coronavirus of various countries which were submitted till 18 March 2020 in the NCBI database. The evolutionary relationships for these nCoV sequences were analysed with 4 MERS sequences and 9 H1N1 influenza virus sequences. The obtained phylogenetic tree revealed that the nCoV is distantly related to H1N1 (influenza virus) and MERS is closely related (Fig. 1). Our data supports the recent study shown its relation with various SARS viruses including MERS [6, 16].

Fig. 1

Phylogenetic analysis of nCoV, MERS and H1N1 (influenza virus): The 34 complete genome sequences of nCoV were compared with 4 MERS sequences and 9 H1N1 sequences in order to build a phylogenetic tree in the MEGA-X software using the Maximum Likelihood method and Tamura-Nei model [18, 19]. In the analysis the codon positions included were 1st, 2nd, 3rd and the non-coding regions. There was a total of 29945 positions in the final dataset. The default settings were used for the analysis.

3.2Mutation analysis using jalview in nCoV proteins

Viruses are known for changing their coat proteins during their life-cycle since it utilises host expression system. Considering the possibility of changes in nCoV associated proteins during the pandemic we compared all the 34 entries for the mutation occurred in the viral proteins. The individual proteins were studied using MSA in CLUSTAL omega followed by Jalview analysis for mutation search. We compared 10 different proteins present in nCoV: orf1ab polyprotein, surface glycoprotein (spike protein), orf3a protein, envelop protein, membrane glycoprotein, orf6 protein, orf7a protein, orf8 protein, nucleocapsid phospho-protein and orf10 protein. Interestingly, we found mutation in the 6 proteins among various countries’ submitted sequences. However, no mutations were observed among the 34 countries’ sequences for the four viral proteins (membrane glycoprotein, orf6 protein, orf7a protein and orf10 protein) during our analysis (Table 2).

The highest point mutations were observed in orf1ab (35 different positions), orf 8 protein (12 positions) and nucleocapsid phosphor-protein (11 positions) among the sequences used for analysis [20]. Next, we needed to know the protein domains affected by the mutation which directed us to do domain analysis.

3.3Domain analysis of individual proteins of an Indian sequence of SARS-CoV-2

To understand the domains of individual proteins in nCoV we used the coronavirus sequence submitted from India (with the Acc. no.- MT012098). In order to perform the domain analysis of individual protein of the virus, first we collected the amino acid sequence of individual proteins then sequence of the individual protein was uploaded on the InterPro Scan online tool separately and the results were obtained [17, 21]. The obtained results for all 10 proteins were represented in Table 3 along with the gene ontology (GO).

The proteins of SARS-CoV-2 of an Indian sequence in which the domains are present are as follows:

orf1ab polyprotein was the largest protein with 20 domains, surface glycoprotein has two domains but other proteins (orf3a, M-protein, orf7a, orf8 and nucleocapsid phosphor-protein) has one domain each. Interestingly, we did not observe any domains in the analysis of envelope (E) protein, orf6 protein and orf10 protein.

The domain analysis of one submission (MT012098) for SARS-CoV-2 revealed the information about the domains of nCoV proteins. Later the MSA based mutation analysis results were mapped with domain analysis results considering all 34 entries must have similar domain distributions. The mapped results were represented in Table 4.