Identification and molecular characterization of mutations in nucleocapsid phosphoprotein of SARS-CoV-2

Abstract

SARS-CoV-2 genome encodes four structural proteins that include the spike glycoprotein, membrane protein, envelope protein and nucleocapsid phosphoprotein (N-protein). The N-protein interacts with viral genomic RNA and helps in packaging. As SARS-CoV-2 spread to almost all countries worldwide within 2-3 months, it also acquired mutations in its RNA genome. Therefore, this study was conducted with an aim to identify the variations present in N-protein of SARS-CoV-2. Here, we analysed 4,163 reported sequence of N-protein from United States of America (USA) and compared them with the first reported sequence from Wuhan, China. Our study identified 107 mutations that reside all over the N-protein. Further, we show the high rate of mutations in intrinsically disordered regions (IDRs) of N-protein. Our study show 45% residues of IDR2 harbour mutations. The RNA-binding domain (RBD) and dimerization domain of N-protein also have mutations at key residues. We further measured the effect of these mutations on N-protein stability and dynamicity and our data reveals that multiple mutations can cause considerable alterations. Altogether, our data strongly suggests that N-protein is one of the mutational hotspot proteins of SARS-CoV-2 that is changing rapidly and these mutations can potentially interferes with various aspects of N-protein functions including its interaction with RNA, oligomerization and signalling events.

Keywords: COVID-19; Infectious disease; Mutations; Nucleocapsid Phosphoprotein (N protein); SARS-CoV-2; USA.

Figure 1. The schematic structure of Nucleocapsid Phosphoprotein (N protein) of SARS-CoV-2.

(A) The N-protein is comprised of 419 residues. The RNA-binding domain, dimerization domain, intrinsically disordered regions including IRD1, IRD2 and IRD3 are labelled. (B and C) Cartoon representation of crystal structure of the RNA-binding domain (RCSB protein ID-6VYO) and dimerization domain (RCSB protein ID-6WJI) of N-protein. (B) Demonstrates the RBD while (C) represents dimerization domain of N-protein. The identified mutated amino acids (single letter code) along with its respective position in polypeptide sequence are shown. The structural representations were made using UCSF chimera software tool.

Figure 2. The weblogo diagram showing the conservation status of polypeptide sequence of N-protein.

The sequence weblogo was generated by multiple sequence alignment of 4,164 N-protein sequences. The overall height of the stack indicates the sequence conservation at that position.

Figure 3. Visual representation of Δ vibrational entropy energy between wild-type and mutant N protein.

The amino acids residues are colored according to the vibrational entropy change as a consequence of mutation of N-protein. Blue (A–C) represents a rigidification of the structure and red (D–F) represents, a gain in flexibility. (A–C) The top three mutants that show rigidification in structure upon mutation. (D–F) The top three mutants that show gain in flexibility upon mutation. Each panel also shows the mutation and the location of the residues.

Figure 4. Analysis of interatomic interactions.

Visual representation of interatomic interactions contributed by T271I and I292T of N-protein. Both of these mutants showed maximum positive and negative ΔΔG among mutants present in RBD and dimerization domain of N-protein. (A & B) Threonine to isoleucine substitution at 271st position; (C & D) isoleucine to threonine substitution at 292nd position. Wild-type and mutant residues are represented in light-greencolor. The interactions made by wild type and mutant residues are highlighted in each panel. The polar interactions are depicted in red dotted line, hydrophobic interaction in green and weak hydrogen bonds in orange.