A non-spike nucleocapsid R204P mutation in SARS-CoV-2 Omicron XEC enhances inflammation and pathogenicity

Abstract

The global circulation of SARS-CoV-2 in human populations has driven the emergence of Omicron subvariants, which have become highly diversified through recombination. In late 2024, SARS-CoV-2 Omicron XEC variant emerged from the recombination of two JN.1 progeny, KS.1.1 and KP.3.3, and became predominant worldwide. Here, we investigate virological features of the XEC variant. Epidemic dynamics modeling suggests that spike substitutions in XEC mainly contribute to its increased viral fitness. Additionally, four licensed antivirals are effective against XEC. Although the fusogenicity of XEC spike is comparable to that of the JN.1 spike, the intrinsic pathogenicity of XEC in male hamsters is significantly higher than that of JN.1. Notably, we find that the nucleocapsid R204P mutation of XEC enhances inflammation through NF-κB activation. Recent studies suggest that the evolutionary potential of spike protein is reaching its limit. Indeed, our findings highlight the critical role of non-spike mutations in the future evolution of SARS-CoV-2.

Fig. 1. Estimation of effects of mutations on viral fitness.

A Estimated effect of each mutation on relative effective reproductive number (R_e) estimated by a hierarchical Bayesian model. A group of highly co-occurred mutations (e.g., those acquired on the stem branch of the BA.2.86 or JN.1 lineage) was treated as mutation clusters. The red and blue dots indicate the substitutions with significant positive and negative effects, respectively. Mutations characteristic to XEC are highlighted in pink. B, C Estimated effect of each mutation on relative R_e. B shows the top 25 mutations with the highest estimated effects, while C presents the 5 mutations with the lowest estimated effects. The posterior mean value (dot) and 95% credible interval (line) are shown.

Fig. 2. Virological features of the SARS-CoV-2 XEC.

A S-based fusion assay in Calu-3 cells. The dashed green line indicates the result of JN.1. The red number in each panel indicates the fold difference between JN.1 and the derivative tested at 24 h post coculture. Assays were performed in quadruplicate. Statistically significant differences versus JN.1 across time points were determined by multiple regression. B Effect of antiviral drugs against XEC. Antiviral effects of the four drugs (EIDD-1931, nirmatrelvir [also known as PF-07321332], remdesivir, and ensitrelvir) in human iPSC-derived lung organoids. The assay of each antiviral drug was performed in triplicate, and the 50% effective concentration (EC₅₀) was calculated. The viral RNA amount without treatment with antiviral drugs was set as 100%. C JN.1 and XEC were inoculated into VeroE6/TMPRSS2 cells (MOI = 0.01). The 50% tissue culture infectious dose (TCID₅₀) of the culture supernatant were routinely quantified. (n = 3 independent experiments). D, E Syrian hamsters were intranasally inoculated with JN.1 and XEC. Six hamsters of the same age were intranasally inoculated with saline (uninfected). D Six hamsters per group were quantified viral RNA load in oral swab by RT-qPCR. E Six hamsters per group were used to routinely measure the body weight. Uninfected (saline) hamster data is also shown. The familywise error rates (FWERs) calculated using the Holm method are indicated in the figures. h.p.i: hours post-infection; d.p.i: days post-infection. F H&E staining of the lungs at 2 d.p.i. of infected hamsters. Representative figures and uninfected lung alveolar space are shown. The presented data are expressed as the average ± SD (A, B) or SEM (C–E). Scale bars, 250 µm.

Fig. 3. The impact of nucleocapsid R204P mutation on replication and pathogenicity.

A Frequency of N protein’s mutations in XEC and other lineages of interest. Only mutations with a frequency >0.5 in at least one representative lineage are shown. B The efficiency of VLP assembly was measured for different N-protein species indicated and quantified in relative luminescence units. C Growth kinetics of rJN.1, rXEC/N:P204R, and rXEC were inoculated into VeroE6/TMPRSS2 cells (MOI = 0.01). The 50% tissue culture infectious dose (TCID₅₀) of the culture supernatant were routinely quantified. (n = 3 independent experiments). D Recombinant viruses were inoculated into an airway-on-a-chip system. The copy numbers of viral RNA in the top and bottom channels of an airway-on-a-chip were routinely quantified by RT-qPCR (left). The percentage of viral RNA load in the bottom channel per top channel at 6 d.p.i. (i.e., % invaded virus from the top channel to the bottom channel) is shown (right). (n = 3 independent experiments). E The percentage of viral RNA load in the bottom channel per top channel at 6 d.p.i. (i.e., % invaded virus from the top channel to the bottom channel) is shown. F–H Syrian hamsters were intranasally inoculated with the recombinant viruses. Six hamsters per group were used to routinely measure the respective parameters. F Body weight of infected hamsters (n  =  6 per infection group). Uninfected hamster data is also shown. G Viral RNA loads in the oral swab (n  =  6 per infection group) at 2 and 5 d.p.i. H Viral RNA loads in the lung hilum (left) and lung periphery (right) of infected hamsters (n  =  4 per infection group) at 2 and 5 d.p.i. The FWERs calculated using the Holm method are indicated in the figures. The presented data are expressed as the average ± SEM (B–H). Statistical significance was determined using Tukey’s multiple comparison test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001).

Fig. 4. Nucleocapsid R204P mutation enhanced inflammation by NF-κB activation.

A IHC of the viral N protein in the lungs at 2 d.p.i. (left) and 5 d.p.i. (right) of infected hamsters. Representative figures (N-positive cells are shown in brown). Images are from comparable lung lobes, not identical microscopic fields. Uninfected hamster data is also shown. B H&E staining of the lungs at 2 d.p.i. (left) and 5 d.p.i. (right) of infected hamsters. Representative figures and uninfected lung alveolar space are shown. C The structure of N (JN.1 N and XEC N) was predicted by Alphafold3. D HEK293/ACE2/TMPRSS2 cells were transfected with NF-κB reporter vector. At 24 h after transfection, cells were infected with rJN.1, rXEC/N:P204R, and rXEC for 1 h. Luciferase activity was measured at 12 and 24 h.p.i. Horizontal lines in figures represent the average value of the negative control group. (n = 3 independent experiments). E mRNA of the lung tissues obtained at 2 d.p.i. was used to measure expression levels of inflammatory genes (Il-1β, Il-6, Il-8, and Ccl2) with normalization using the housekeeping gene Rpl18. (n  =  4 per infection group). The presented data are expressed as the average ± SEM (D, E). Statistical significance was determined using Tukey’s multiple comparison test (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). Scale bars, 250 µm.