Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics

Abstract

Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

Keywords: BA.1; BA.2; COVID-19; Omicron; SARS-CoV-2; fusogenicity; growth capacity; immune resistance; pathogenicity.

Fig 1

Evolution of BA.1 and BA.2 lineages. (A and B) The ML trees of 624 SARS-CoV-2 genomes (A) and S gene region (B). Each branch color corresponds to the SARS-CoV-2 PANGO lineage shown on the right. The scale (substitutions per nucleotide site) is shown at the bottom. The trees with mutations and bootstrap values of the trees are shown in Fig. S1. (C) A heatmap showing frequency of mutation occurred in proteins and 3′UTR of SARS-CoV-2 in BA.1 and BA.2 lineages compared to those in B.1.1 lineage.

Fig 2

Impact of L371F substitution on the functions of Omicron S. (A–D) S-based fusion assay. Cell surface expression of BA.1-based derivatives (A) and BA.2-based derivatives (C) is shown. (B and D) S-based fusion assay in Calu-3 cells. The recorded fusion activity (arbitrary units) is shown. The dashed green lines in panels (B) and (D) are the results of BA.1 S and BA.2 S, respectively. The red number in each panel indicates the fold difference between BA.1 (top) or BA.2 (bottom) and the derivative tested at 24 h post coculture. (E and F) Pseudovirus assay and western blotting. Top, HOS-ACE2-TMPRSS2 cells were infected with pseudoviruses bearing each S protein. The amount of input virus was normalized based on the amount of HIV-1 p24 capsid protein. The percent infectivity compared to that of the virus pseudotyped with BA.1 S (E) or BA.2 S (F) is, respectively, shown. The direct comparison between BA.1 S and BA.2 is shown in Fig. S2. The dashed horizontal lines in the left and right panels indicate the values of BA.1 and BA.2, respectively. Bottom, western blot. Representative blots of S-expressing cells (labeled with “Cell”) and supernatants (labeled with “Virus”) are shown. ACTB and HIV-1 p24 were used for the internal controls of “Cell” and “‘Virus.” kDa, kilodalton. In Fig. 2A through F, the three mutations in the NTD, NL211-212I, V213G, and ins214EPE (shown in Fig. 1C) are combined to either IVREPE211-216NLGR (for BA.1 S) or NLGR211-214IVREPE (for BA.2 S). (G) The binding affinity of the RBD of SARS-CoV-2 S protein to ACE2 by yeast surface display. The K _D value indicating the binding affinity of the RBD of the SARS-CoV-2 S protein to soluble ACE2 when expressed in yeast is shown. (H) DSF assay. Representative results (left) and the summarized data of the inflection temperatures of the RBD proteins by BA.1, BA.1 L371F, or BA.2 (right) are shown. (I) Neutralization assay using Sotrovimab. NT₅₀, 50% neutralization titer. The dashed green lines are the results of BA.1. Assays were performed in triplicate. The presented data are expressed as the average ± SD. In panels (A), (C), (E), (F), (G), and (H, right), each dot indicates the result of an individual replicate. Statistically significant differences (*P < 0.05) versus parental BA.1 [panels (A), (E), (G, left), and (H, right)] or parental BA.2 [(C), (F),and (G, right)] were determined by two-sided Student’s t-tests. In panels (B) and (D), statistically significant differences versus parental BA.1 (B) or parental BA.2 (D) across timepoints were determined by multiple regression. The FWERs calculated using the Holm method are indicated with parentheses in the figures.

Fig 3

Impact of S:L371F substitution on the virological features of Omicron. (A) Scheme for the chimeric recombinant SARS-CoV-2 used in this study. The SARS-CoV-2 genome and its genes are shown. The template was SARS-CoV-2 strain WK-521 (PANGO lineage A, GISAID ID: EPI_ISL_408667) (22), and ORF7a gene was swapped with the GFP gene. Two recombinant viruses, rBA.1 S-GFP and rBA.2 S-GFP, were used in our previous study (3). (B and C) Viral growth assay. rBA.1 S-GFP, rBA.2 S-GFP, and rBA.1 S:L371F-GFP were inoculated into Vero cells [B; multiplicity of infection (m.o.i.) = 0.1] and VeroE6/TMPRSS2 cells (C; m.o.i. = 0.01). The copy numbers of viral RNA in the culture supernatant were routinely quantified by RT–qPCR. (D) Fluorescence microscopy. The GFP area was measured in infected VeroE6/TMPRSS2 cells (m.o.i. = 0.01) at 48 h.p.i. Representative panels are shown in the left panel. Scale bars, 400 μm. Middle and right, the summarized results of GFP-positive area (middle) and GFP intensity (right). To measure the GFP-positive area, 1,000 cells per virus were counted. (E) Plaque assay. Representative panels (left) and a summary of the recorded plaque diameters (20 plaques per virus) (right) are shown. (F and G) Viral growth in an airway-on-a-chip system. rBA.1 S-GFP, rBA.2 S-GFP, and rBA.1 S:L371F-GFP were inoculated into an airway-on-a-chip system, and the copy numbers of viral RNA in the top (F, top) and bottom (F, bottom) channels of an airway-on-a-chip were routinely quantified by RT–qPCR. (G) The percentage of viral RNA load in the bottom channel per top channel during 6 d.p.i. (i.e., percentage of invaded virus from the top channel to the bottom channel) is shown. (H and I) Animal experiment. Syrian hamsters (n = 6 per group) were intranasally inoculated with rBA.1 S-GFP, rBA.2 S-GFP, and rBA.1 S:L371F-GFP (10,000 TCID₅₀ in 100 µL per animal). Hamsters of the same age were intranasally inoculated with 100 µL of saline (uninfected). (H) Body weight change of infected hamsters (n = 6 per infection group). (I) Viral RNA loads in the lung hilum of infected hamsters at 5 d.p.i. (n = 4 per infection group). Assays were performed in triplicate [(F) and (G)] or quadruplicate [(B) and (C)]. The presented data are expressed as the average ± SEM. In panels (E) and (G), each dot indicates the result of an individual replicate. In panel (I), each dot indicates the result of an individual hamster. In panels (B), (C), and (F), the dashed green lines are the results of rBA.1 S-GFP. Statistically significant differences (*P < 0.05) versus rBA.1 S-GFP were determined by two-sided Mann-Whitney U test [(D), (E), and (I)] or two-sided Student’s t-test (G). In panels (B), (C), (F), and (H), statistically significant differences versus rBA.1 S:L371F-GFP across timepoints were determined by multiple regression. The FWERs calculated using the Holm method are indicated in the figures.

Fig 4

Modulation of viral growth and pathogenicity by the mutations downstream of S gene. (A) Scheme for the chimeric recombinant SARS-CoV-2 used in this study. The SARS-CoV-2 genome and its genes are shown. The template was SARS-CoV-2 strain WK-521 (PANGO lineage A, GISAID ID: EPI_ISL_408667) (22). A recombinant virus bearing S:D614G mutation (rB.1.1) was used in our previous study (3). (B and C) Animal experiment. Syrian hamsters (n = 4 per group) were intranasally inoculated with rB.1.1, rBA.2up, and rBA.2down (10,000 TCID₅₀ in 100 µL per animal). Hamsters of the same age were intranasally inoculated with 100 µL of saline (uninfected). (B) Body weight change of infected hamsters. (C) Viral RNA loads in the oral swabs of infected hamsters at 1, 3, and 5 d.p.i. (D–I) Viral growth assay. rB.1.1 (black), rBA.2down (red), rBA.2up (blue), or the rB.1.1 derivatives bearing the mutation indicated in the figure were inoculated into Vero cells (D, F, and H; m.o.i. = 0.1) and VeroE6/TMPRSS2 cells (E, G, and I; m.o.i. = 0.01). The copy numbers of viral RNA in the culture supernatant were routinely quantified by RT–qPCR. The presented data are expressed as the average ± SEM. In panels (D–I), assays were performed in quadruplicate, and the dashed black and red lines are the results of rB.1.1 and rBA.2 bottom, respectively. Statistically significant differences versus rB.1.1 across timepoints were determined by multiple regression. The FWERs calculated using the Holm method are indicated in the figures.