Dissecting Naturally Arising Amino Acid Substitutions at Position L452 of SARS-CoV-2 Spike

Abstract

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.

Keywords: L452; SARS-CoV-2; coronavirus; fitness; mutational studies; spike; spike protein; substitution.

FIG 1

Recurrent mutations at spike L452 in multiple SARS-CoV-2 lineages. (A) Maximum-likelihood phylogenetic tree based on full-length genome sequences of a total of 25,796 major genotypes of SARS-CoV-2. The sequences were examined for amino acid mutations at spike position 452, and their numbers are shown in red. (B) Global epidemic dynamics of the L452Q-harboring lambda, as well as the L452R-harboring epsilon, kappa, and delta variants spanning from October 2020 to April 2022. (C) Recent emergence of the omicron lineages, including L452R-harboring BA.4, BA.5, and BA.2.11, L452Q-harboring BA2.12.1, and L452M-harboring BA.2.13 and BA.2.9.1 from January to April 2022. All data were retrieved from the GISAID database as of 27 April 2022.

FIG 2

Preference of SNAMs at spike L452. (A) Alignment of spike sequence showing the single base change at nucleotide numbers 22916/22917 that leads to nonsynonymous L452 mutation among circulating SARS-CoV-2 variants. (B) Likelihood for each amino acid substitution from leucine (encoded by CUG) based on a neutral probability estimation model. (C) Likelihood versus actual frequency of L452 amino acid substitutions. Actual global frequency of each amino acid was analyzed using the Table of Mutation Sites tool (https://cov.lanl.gov/content/sequence/TBLS_MUT_SITES/tbls_mut_sites.html; updated on 27 April 2022). Amino acids characterized by a SNAM are highlighted in red.

FIG 3

Biochemical and virological characterization of spike L452 variants. (A) Immunoblots showing total cellular expression and virion incorporation of spike L452 mutants in producer cell and virion (left panel). The spike incorporation level was quantified and normalized to p24 Gag levels and is expressed relative to the WT in three independent replicates (right panel). Statistical significance was determined by one-way ANOVA with multiple-comparison tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Titers (ng/mL) of pseudoviruses collected from the culture supernatant at 48 h posttransfection. (C) Infectivity of reporter lentiviruses pseudotyped by SARS-CoV-2 spike L452-mutants and WT D614G. The indicated titers of pseudoviruses (1, 3, and 5 ng) were exposed to 293T cells expressing ACE2 and TMPRSS2. The amount of pseudoviruses successfully infected into target cells was determined from the luciferase activity, and the relative infectivity is expressed as the percentage normalized to the WT (infection at 5 ng). (D) Infectivity of reporter lentiviruses pseudotyped with SARS-CoV-2 BA.2 spike and its L452 mutants (infection at 5 ng). (E) Fusion formation between the spike-expressing cells and ACE2-TMPRSS2-expressing cells was continuously monitored at intervals of 3, 6, 12, 24, and 48 h. The fusion activity was expressed relative to WT at 3 h after coincubation. (F) Correlation between infectivity of pseudoviruses (infection at 5 ng) and cell-to-cell fusion activity (at 48 h). The statistical significance was determined by using the Pearson’s correlation coefficient test. (G) Structure of SARS-CoV-2 spike trimer with single RBD up-conformation (PDB 7KRR). Each protomer is differently colored. The L452 residues are shown as spheres, while glycans are indicated as sticks. In the RBD with the up-conformation, the L452 residue is exposed (yellow box). In contrast, in the RBD with the down-conformation, the L452 residue is buried at the interface with the NTD of another protomer (orange box). The data shown are means ± the SD of triplicate determinations.

FIG 4

Changes in neutralizing sensitivity by L452 spike variants. (A and B) Neutralizing sensitivity expressed as the pseudovirus neutralizing titer (pNT50) of sera from nine donors receiving two and three doses of the vaccines (Table 1) against a panel of lentiviruses pseudotyped with L452 spike mutants in the context of WT D614G (A) and omicron BA.2 (B), respectively. The relative pNT50 (%) values normalized to their parental pairs are shown. The data shown are means ± the SD of triplicate determinations. Statistical significance was determined by Wilcoxon paired signed rank test (*, P < 0.05; **, P < 0.01).

FIG 5

Changes in TCR recognition by L452 spike variants. (A) Schematic diagram of TCR recognition assay. NFAT-luciferase Jurkat T reporter cells (TCRαβ^–/–) expressing the defined αβ TCR clonotypes were used as effector cells. A549 cells expressing human ACE2 receptor and HLA-A*A24:02 (A549/hACE2/HLA-A*24:02) were loaded with a panel of synthetic peptides or transfected with a panel of genes expressing spike proteins and used as target cells. (B and C) TCR activation level as measured by luciferase activity. A549/hACE2/HLA-A*24:02 target cells loaded with a panel of NF9 and the indicated mutant peptides (B) and transfected by DNAs encoding BA.2 spike and its L452 variants (C) were cocultured with the NFAT-luciferase Jurkat T reporter cells that had been engineered to stably express the indicated NF9-specific TCR clones. The data shown are means ± the SD of triplicate determinations.