Variations in Orf3a protein of SARS-CoV-2 alter its structure and function

Abstract

Severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread worldwide and acquired multiple mutations in its genome. Orf3a, an accessory protein encoded by the genome of SARS-CoV-2, plays a significant role in viral infection and pathogenesis. In the present in-silico study, 15,928 sequences of Orf3a reported worldwide were compared to identify variations in this protein. Our analysis revealed the occurrence of mutations at 173 residues of Orf3a protein. Subsequently, protein modelling was performed that revealed twelve mutations which can considerably affect the stability of Orf3a. Among the 12 mutations, three mutations (Y160H, D210Y and S171L) also lead to alterations in secondary structure and protein disorder parameters of the Orf3a protein. Further, we used predictive tools to identify five promising epitopes of B-cells, which resides in the mutated regions of Orf3a. Altogether, our study sheds light on the variations occurring in Orf3a that might contribute to alteration in protein structure and function.

Keywords: B-cell epitopes; Mutations; Orf3a; Protein disorder; Protein stability; SARS-CoV-2.

Fig. 1

A) The cartoon structure of Orf3a. The red color represents helical structure while yellow color represents beta sheets. B) The various properties of residues of Orf3a. The data were obtained from Innovagen's peptide calculator (https://pepcalc.com/). The colour coded display of amino acid classification and peptide hydropathy plot are shown. C) The sequence of SARS-CoV-2 Orf3a protein. The mutated amino acids are shown in red color. The number denotes the position of residues in Orf3a. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Analysis of the secondary structure and intrinsic disorder predisposition of the unique mutations of SARS-CoV2 Orf3a in comparison with the reference Orf3a protein (YP_009724391) from China, Wuhan. (A, C and E) Secondary structure predictions, the amino acid sequences near the mutation site were uploaded on CFSSP web tool that predict secondary structure. Each panel (A, C and E) shows the secondary structure of the wild type and mutated input sequences. The panel (i) represents the wild type or Wuhan sequence while panel (ii) represents the mutated Indian sequence. The mutation site is highlighted in the rectangular box. (B,D and F) protein disorder prediction, the analysis was conducted using PONDR-VSL2 algorithm. A disorder threshold is depicted at a score of 0.5). Residues/regions with the disorder scores >0.5 are considered as disordered.

Fig. 3

Prediction of B-cell epitopes. A) On the graphs, the Y-axis depicts for each residue the correspondent BepiPred score (averaged in the specified window); while the X-axis depicts the residue positions in the sequence. The larger score for the residues might be interpreted as that the residue might have a higher probability to be part of epitope (those residues are colored in yellow on the graphs). B) The top five peptides of Orf3a that showed the highest score. The sequences of all five peptides are shown. The number in parentheses represents the location of the peptide in the primary sequence of Orf3a. The red font shows the location of mutant residues of Orf3a. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)