Analysis of variation in SARS-CoV-2 regarding Spike mutations D614G & N501Y and polymorphism of in vitro isolates.

Differentially Conserved Positions (DCPs) are found between two related groups of protein sequences at selective loci that diverge between the two groups that often signify evolutionarily valuable mutations that affect structure and/or function. This study aims to capture the evolution of DCPs of SARS-CoV-2 regarding two historic S protein mutations; 1. (S)D614G - the most widespread mutation found in all variants and 2 (S)N501Y - a marker of both the Alpha/Beta variants; the intention is to discover new concurrent mutations, exclude "assumed" concurrent mutations and define the subsequent structure function changes for these two flagship mutations using publicly available EM and crystal structures.

Using GISAID data collected up till December 2020, (S)D614G was found to have two mildly conserved DCPs - (S)L18F and (S)A222V. (S)N501Y was separately examined for DCPs which recovered (S)A570D, (S)P681H, (S)T716I, (S)S982A and (S)D1118H. All DCPs were found in S protein and structurally examined from PDB structures in both the "open" (ACE-2 receptive state) and "closed" (non-receptive state). Three S mutations were calculated to specifically destabilise the "closed" form of S(A570D, D614G, and S982A). Of note, inter-protomer salt bridges appear severed for (S)D614 to (S)K853/K854 specifically in the "closed" form, indicating protomer destabilisation.

Additionally, sequencing data from a pair of infected CaCo-2 cultures were analysed for RNA mutations to detect in vitro mutations and identify areas of the SARS-CoV-2 genome that are prone to mutagenesislimited at the cellular level, i.e., without the pressures of the humoral immune system or host transmission. As well as silent mutations, cultivation resulted in two B.1.1.7-associated features - (ORF8)Q27STOP and (S)A570D, deleterious zones in NSP1 and NSP12 (pos. 508-522nt and 14,408-14,414) and several nonsynonymous mutations within NSP3, NSP6, NSP13, NSP15, S, N, ORF3a andORF9c.