Coronavirus Genomes and Unique Mutations in Structural and Non-Structural Proteins in Pakistani SARS-CoV-2 Delta Variants during the Fourth Wave of the Pandemic

Abstract

Genomic epidemiology of SARS-CoV-2 is imperative to explore the transmission, evolution, and also pathogenicity of viruses. The emergence of SARS-CoV-2 variants of concern posed a severe threat to the global public health efforts. To assess the potential consequence of these emerging variants on public health, continuous molecular epidemiology is of vital importance. The current study has been designed to investigate the major SARS-CoV-2 variants and emerging mutations in virus structural and non-structural proteins (NSP) during the fourth wave in September 2021 from the Punjab province of Pakistan. Twenty SARS-CoV-2 positive samples have been collected from major cities were subjected to next-generation sequencing. Among the 20 whole genomes (GenBank Accession SRR16294858-SRR16294877), 2 samples failed to be completely sequenced. These genome sequences harbored 207 non-synonymous mutations, among which 19 were unique to GISAID. The genome sequences were detected: Delta 21I, 21J variants (B.1.617.2). Mutation's spike_F157del, spike_P681R, spike_T478K, spike_T19R, spike_L452R, spike_D614G, spike_G142D, spike_E156G, and spike_R158del have been detected in all samples where K1086Q, E554K, and C1250W were unique in spike protein. These genomic sequences also harbored 129 non-synonymous mutations in NSP. The most common were NSP3_P1469S (N = 17), NSP3_A488S (N = 17), NSP3_P1228L (N = 17), NSP4_V167L (N = 17), NSP4_T492I (N = 17), NSP6_T77A (N = 17), NSP14_A394V (N = 17), NSP12_G671S (N = 18), and NSP13_P77L (N = 18). The mutation, F313Y in NSP12, detected in the current study, was found in a single isolate from Belgium. Numerous other unique mutations have been detected in the virus papain-like protease (NSP3), main protease (NSP5), and RNA-dependent RNA polymerase (NSP12). The most common non-synonymous mutations in the spike protein were subjected to stability analysis, exhibiting a stabilizing effect on structures. The presence of Delta variants may affect therapeutic efforts and vaccine efficacy. Continuous genomic epidemiology of SARS-CoV-2 in Pakistan may be useful for better management of SARS-CoV-2 infections.

Keywords: NSP; Pakistan; SARS-CoV-2; genome; mutations; variants.

Figure 1

Domain organization of structural proteins. ORF: open reading frame, E: envelope, M: membrane, N: nucleocapsid, SP: signal peptide, NTD: N-terminal domain, RBD: receptor-binding domain, RBM: receptor-binding motif, FP: fusion peptide, HR: heptapeptide repeat sequence, TM: transmembrane, CP: cytoplasmic domain, SR: serine rich, CTD: C-terminal domain.

Figure 2

Location of most common mutations in S protein. Yellow indicates the receptor-binding motif, present in the RBD region (raspberry color). The majority of these mutations were found in the loop regions.

Figure 3

Mutation effect on S protein structure stability and flexibility. Mutants G142D, T478K, E156G, and L452R in S protein, exhibited stabilizing effect. The blue region shows rigidification of structure behind mutations and red shows gain in flexibility.

Figure 4

Mutation effect on S protein stability and flexibility. Mutants P681R and T19R in S protein, exhibited a stabilizing effect. The blue region shows rigidification of structure behind mutations and red shows gain in flexibility.

Figure 5

SARS-CoV-2 genomic epidemiology from Pakistan. The graph shows an estimate of divergence over the time period within genomes. (A) Current study isolates (labelled Pakistan Delta isolates). (B) Position of current SARS-CoV-2 isolates in radial tree (UOL) was built using Nextstrain 28 (Nextclade (nextstrain.org). Sub-clades have been color coded in the tree, characterized by some specific mutations in structural proteins (https://covariants.org/variants) (accessed on 28 September 2021).

Figure 6

Structure of NSP12 and NSp3 (PLpro). (A) SARS-CoV-2 RdRp (PDB ID: 6M71) contains an N-terminal β-hairpin (residues 31–50). NiRAN (residues 50–249), interface domain (residues 251–365). The NiRAN domain is comprised of three helices and five β-strands, associated with RdRp domain (residues 366–920). (B) Complex of NSP12, NSP7, and NSP8. (C) Organization of NSP3 protein. (D) Structure of PLpro with mutations labeled NSP3_P822L (P77L), NSP3_L862F (L117F), and H920Y (H175y). PLpro has a small N-terminal ubiquitin-like (Ubl) domain and catalytic domain (thumb–palm–fingers).