Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline

Abstract

<jats:title>Abstract</jats:title><jats:p>In order to explore nonsynonymous mutations and deletions in the spike (S) protein of SARS-CoV-2, we comprehensively analyzed 35,750 complete S protein gene sequences from across six continents and five climate zones around the world, as documented in the GISAID database as of June 24<jats:sup>th</jats:sup>, 2020. Through a custom Python-based pipeline for analyzing mutations, we identified 27,801 (77.77 % of spike sequences) mutated strains compared to Wuhan-Hu-1 strain. 84.40% of these strains had only single amino-acid (aa) substitution mutations, but an outlier strain from Bosnia and Herzegovina (EPI_ISL_463893) was found to possess six aa substitutions. The D614G variant of the major G clade was found to be predominant across circulating strains in all climates. We also identified 988 unique aa substitution mutations distributed across 660 positions within the spike protein, with eleven sites showing high variability – these sites had four types of aa variations at each position. Besides, 17 in-frame deletions at four major regions (three in N-terminal domain and one just downstream of the RBD) may have possible impact on attenuation. Moreover, the mutational frequency differed significantly (p= 0.003, Kruskal–Wallis test) among the SARS-CoV-2 strains worldwide. This study presents a fast and accurate pipeline for identifying nonsynonymous mutations and deletions from large dataset for any particular protein coding sequence and presents this S protein data as representative analysis. By using separate multi-sequence alignment with MAFFT, removing ambiguous sequences and in-frame stop codons, and utilizing pairwise alignment, this method can derive nonsynonymus mutations (Reference:Position:Strain). We believe this will aid in the surveillance of any proteins encoded by SARS-CoV-2, and will prove to be crucial in tracking the ever-increasing variation of many other divergent RNA viruses in the future.</jats:p>

Evolutionary dynamics of SARS‐CoV‐2 nucleocapsid protein and its consequences

“…Among the deletions, nucleotide deletion positioned 578–633 (aa: 193–211) was found in multiple strains of multiple countries including Turkey, Bosnia, Australia, Malaysia, Germany, Scotland, England, Wales, and United States (Table 6 and Supporting Information Data 1). However, previously we explored 12 deletion‐sites of ranged nucleotides in polyprotein ( n = 9; NSP1:6, NSP2:1, NSP8:1, NSP15:1), ORF10 ( n = 1) and 3′‐UTR ( n = 2) and four major deletion‐sites in S protein while no deletions were found among strains globally 5,20 …”

Evolutionary Insights into the Envelope Protein of SARS-CoV-2

“…The Study SARS-CoV-2, the etiologic agent of COVID-19 disease has impacted the entire world, and created a public health emergency since December 2019 1,2 . The inherently higher mutations in the genome of SARS-CoV-2 have already produced many descendants from the original Wuhan strain, thereby escaping the host immune responses [3][4][5][6] . The genome of SARS-CoV-2 virus encodes for four major structural proteins such as the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the small envelope (E) protein, all of which are required to complete a successful infectious event/replication cycle of virus including entry, assembly, packaging and release of new virus particles within the human cells [8][9][10][11] .…”

“…The ongoing rapid transmission, and global spread of COVID-19 have raised intriguing questions whether the evolution and adaptation of SARS-CoV-2 is driven by synonymous mutations, deletions and/or replacements 5,12,13 . Although, the mutational spectra of different structural proteins (S, M, and N) of SARS-CoV-2 has been reported by several research groups 5,6,[13][14][15] over a short period of time, however, available literature on the nucleotide and aa-level mutations of E protein is till limited.…”

“…To comprehensively analyze the mutational spectra of E protein of SARS-CoV-2 as a continuous part of the coronavirus genomic mutational research 5,6,15,16 , we retrieved 83,607 complete or near-complete genome sequences of SARS-CoV-2 (human host) from the global initiative on sharing all influenza data (GISAID) (https://www.gisaid.org/) belonging to 159 countries or territories till 20 August 2020 (Supplementary Data 1). We obtained 81,818 cleaned sequences (97.86%) after removing the low quality sequences.…”

“…Multiple sequence alignment was performed in MAFFT by using Wuhan strain as a reference (NCBI accession no. NC_045512) 17 , and nonsynonymous mutations were retrieved using the previously reported methods 5,6,15 .…”