Spike And Nsp6 Are Key Determinants Of SARS-CoV-2 Omicron BA.1 Attenuation

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1–12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus-bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

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As of December 2022, the successive waves of the COVID-19 pandemic have been driven by five major SARS-CoV-2 variants, known as variants of concern (VOC): Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2 and AY lineages) and Omicron (BA lineages)13. Omicron is the most recently recognized VOC, and was first documented in South Africa, Botswana, and by a traveler from South Africa in Hong Kong in November 2021 (GISAID ID: EPI_ISL_7605742)14,15. It quickly swept through the world, displacing the previously dominant Delta variant within weeks and accounting for most new SARS-CoV-2 infections by January 2022 (refs. 16–18). At least five lineages of Omicron have so far been identified: BA.1, BA.2, BA.3, BA.4, and BA.5. BA.1 (hereafter referred to as Omicron) exhibits a remarkable escape from infection- and vaccine-induced humoral immunity3,19. Furthermore, it is less virulent than other VOCs in humans and in vivo models of infection4,5,7,11,12,20. Omicron differs from the prototype SARS-CoV-2 isolate, Wuhan-Hu-1, by 59 amino acids; 37 of these changes are in the S protein, raising the possibility that S is at the heart of Omicron's pathogenic and antigenic behavior. 

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S mutations affect Omicron replication in cell culture 

The Omicron S protein contains 30 amino acid substitutions, 6 deletions, and one insertion of 3 amino acids in length, as compared to Wuhan-Hu-1 (Extended Data Fig. 1). Twenty-five of these changes are unique to Omicron relative to other VOCs, although some of them have been reported in wastewater and minor SARS-CoV-2 variants21,22. To test the role of the S protein in Omicron phenotype, we generated a chimeric recombinant virus containing the S gene of Omicron (USA-lh01/2021) and all other genes of an ancestral SARS-CoV-2 (GISAID EPI_ISL_2732373)23 (Extended Data Fig. 2a). This chimeric virus, named Omi-S, was made by using a modified form of circular polymerase extension reaction (CPER)24 (Extended Data Fig. 2b) that yielded highly concentrated virus stocks, containing 0.5 × 106 –5 × 106 plaque-forming units (PFU) per ml, from transfected cells within two days of transfection (Extended Data Fig. 2c,d), obviating the need for further viral amplification.

Fig. 1 | Effect of S on the in vitro growth kinetics of Omicron. a Schematic of viruses. S, spike; N, nucleocapsid. b–e, ACE2/TMPRSS2/Caco-2 cells (b,d) and Vero E6 cells (c,e) were infected at an MOI of 0.01, and the percentage of N-positive cells (n = 6 replicates) (b,c) and the release of infectious particles (n = 3 replicates) (d,e) were determined by flow cytometry and by plaque assay, respectively. f, ACE2/TMPRSS2/Caco-2 cells were infected with virus mixtures at a 1:1 ratio to obtain the final MOI of 0.005 for each virus. The cells were fixed at the indicated times and subjected to flow cytometry. Left, representative dot plot; right, percentage of uninfected, Omi-S/mCherry-infected, Omicron/ mNeoGreen (Omicron/mNG)-infected and doubly infected cells. Singly infected cells were used for compensation. Error bars, mean ± s.d. (n = 3 replicates). g, Plaque sizes. Left, representative images of plaques on ACE2/TMPRSS2/ Caco-2 cells. Right, the diameter of plaques is plotted as the mean ± s.d. of 20 plaques per virus. h, Human iAT2 epithelial cells were infected at an MOI of 2.5 for 48 h or 96 h. The apical side of cells was washed with 1× phosphate-buffered saline (PBS) and the levels of infectious virus particles were measured by plaque assay. Error bars, mean ± s.d. (n = 4 replicates). Experiments were repeated twice, with each experimental repeat containing 3 (b–g) or 4 (h) replicates. P values were calculated by a two-tailed, unpaired t-test with Welch's correction. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; NS, not significant. The gating strategy for flow cytometry is shown in Supplementary Fig. 1.

We first compared the infection efficiency of Omi-S with an ancestral D614G-containing virus (GISAID EPI_ISL_2732373; generated by CPER; hereafter referred to as wild type (WT)) and an Omicron isolate (USA-lh01/2021) in cell culture (Fig. 1a). For this, we infected ACE2- and TMPRSS2-expressing Caco-2 (hereafter, ACE2/TMPRSS2/Caco-2) and Vero E6 cells with Omi-S, WT and Omicron at a multiplicity of infection (MOI) of 0.01, and monitored viral propagation by flow cytometry and plaque formation assay. The WT virus and Omi-S spread rapidly in ACE2/TMPRSS2/Caco-2 cells, yielding 89% and 80% infected cells, respectively, at 24 hours post-infection (HPI) (Fig. 1b). By contrast, Omicron replicated more slowly, leading to 48% infected cells at 24 hpi. A similar pattern was seen in Vero E6 cells, in which 60% and 41% of cells were positive for WT and Omi-S, respectively, at 48 hpi, as opposed to 10% for Omicron (Fig. 1c). The plaque assay showed that although both Omi-S and Omicron produced lower levels of infectious virus particles compared with WT, the viral titer of Omi-S was significantly higher than that of Omicron. In ACE2/TMPRSS2/Caco-2 cells, Omi-S produced 5.1-fold (P = 0.0006) and 5.5-fold (P = 0.0312) more infectious particles than Omicron at 12 hpi and 24 hpi, respectively (Fig. 1d). Similarly, in Vero E6 cells, the infectious virus titers of Omi-S were 17-fold (P = 0.0080) and 11-fold (P = 0.0078) higher than that of Omicron at 24 hpi and 48 hpi, respectively (Fig. 1e). The difference between viruses became less obvious at later time points owing to higher cytotoxicity caused by Omi-S compared to Omicron (Extended Data Fig. 3a). The increased replication efficiency of Omi-S relative to Omicron was preserved when tested at varying MOIs (Extended Data Fig. 3b). We further confirmed the fitness advantage of Omi-S over Omicron by a direct competition assay. For this, we first generated recombinant Omicron (micron), which, in our cell culture assays, mimicked the replication kinetics of natural Omicron (Extended Data Fig. 4). Next, we created mCherry-containing Omi-S and mNeonGreen-containing Omicron, and inoculated ACE2/TMPRSS2/Caco-2 cells with these viruses mixed at a 1:1 ratio. Flow cytometric analysis of infected cells at various times of infection showed that Omi-S/mCherry was clearly superior to Omicron/mNeonGreen in terms of replication (Fig. 1f). Finally, the higher infection efficiency of Omi-S was also reflected in the plaque size; although WT virus produced the largest plaques (around 4.1 mm), the size of Omi-S plaques (around 2.2 mm) was twofold (P < 0.0001) larger than that of Omicron plaques (around 1.1 mm) (Fig. 1g). These results indicate that although mutations in the S protein influence the infection efficiency of Omicron, they do not fully explain the Omicron phenotype.

Several lines of evidence indicated that the S protein incorporated into Omi-S behaved the same way as in natural Omicron. For instance, as described previously20,25, Omicron S was poorly cleaved compared to WT S; whereas 71% of S in WT virions was in the cleaved form, only 45% and 47% was cleaved in Omi-S and Omicron, respectively (Extended Data Fig. 5a). The same pattern of S cleavage was evident in virus-infected cells (WT, 63% cleaved; Omi-S, 33% cleaved; Omicron, 42% cleaved) (Extended Data Fig. 5b). These experiments also revealed that Omicron S was inefficiently incorporated into virus particles compared to WT S (S to nucleocapsid (N) ratio: 3.40 for WT virus, 1.91 for Omi-S and 2.04 for Omicron) (Extended Data Fig. 5a). Similarly, both Omi-S and Omicron produced smaller syncytia compared to the WT virus, an observation that has previously been reported for Omicron20,26 (Extended Data Fig. 5c). Finally, consistent with the published literature25, Omi-S and Omicron showed a preference for cathepsinmediated entry, as reflected by their higher sensitivity to the cathepsin inhibitor E64d (Extended Data Fig. 6). We next compared the replication kinetics of WT, Omi-S and Omicron in lung epithelial cells, which form a major viral replication site in patients with COVID-19 (refs. 27,28). Accordingly, we used human induced pluripotent stem (iPS)-cell-derived lung alveolar type 2 epithelial (iAT2) cells. AT2 cells represent an essential cell population in the distal lung and constitute one of the primary targets of SARS-CoV-2 infection28,29. We infected iAT2 cells, grown as an air–liquid interface (ALI) culture, at an MOI of 2.5 and monitored the secretion of viral progeny on the apical side of cells at 48 hpi and 96 hp. In congruence with the results obtained from cell lines, the WT virus produced the highest levels of infectious virus particles (Fig. 1h). Among Omi-S and Omicron, the former yielded around fivefold (P = 0.0008) higher infectious viral titer at 48 hp. The viral titers for WT and Omi-S decreased at 96 dpi compared to 48 hp owing to the cytopathic effect (CPE) of infection. However, no CPE was seen for Omicron, leading to sustained production of infectious virions. Overall, these results corroborate the conclusion that mutations in S do not fully account for the attenuated replication capacity of Omicron in cell culture. 

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Minimal role of S in Omicron pathogenicity in mice 

To examine whether Omi-S exhibits higher in vivo fitness compared with Omicron, we investigated the infection outcome of Omi-S relative to WT SARS-CoV-2 and Omicron in K18-hACE2 mice. In agreement with the published literature4,5 , intranasal inoculation of mice (aged 12–20 weeks) with Omicron (104 PFU per mouse) caused no significant weight loss, whereas inoculation with WT virus triggered a rapid decrease in body weight, with all mice losing over 20% of their initial body weight by 8 days post-infection (dpi) (Fig. 2a). Notably, 80% of mice infected with Omi-S also lost over 20% of their body weight by 9 dpi (Fig. 2a and Extended Data Fig. 7a). The evaluation of clinical scores (a cumulative measure of weight loss, abnormal respiration, aberrant appearance, reduced responsiveness and altered behaviour) also revealed a similar pattern; whereas Omicron-infected mice exhibited few to no signs of clinical illness, the health of those infected with WT and Omi-S rapidly deteriorated, with the WT virus causing a more severe disease (P = 0.0102) (Fig. 2b and Extended Data Fig. 7b). Because SARS-CoV-2 causes fatal infection in K18-hACE2 mice4 , we compared the survival of mice after viral infection. Agreeing with the results of body-weight loss and clinical score, WT, and Omi-S caused mortality rates of 100% (6/6) and 80% (8/10), respectively. By contrast, all mice infected with Omicron survived (Fig. 2c). These findings, which are consistent with a recent publication30, indicate that the S protein is not the exclusive determinant of Omicron's pathogenicity in K18-hACE2 mice.

Next, we compared the propagation of Omi-S with Omicron and WT SARS-CoV-2 in the lungs and nasal turbinates of K18-hACE2 mice. The mice (12–20 weeks old) were intranasally challenged with 104 PFU (seven mice per virus), and viral titers in mice lungs were measured at 2 and 4 dpi. Consistent with in vitro findings, the infectious virus titer in the lungs of WT-infected mice was higher than that detected in mice infected with the other two viruses (Fig. 2d). Notably, however, Omi-S-infected mice produced 30-fold (P = 0.0286) more infectious virus particles compared with Omicron-infected mice at 2 dpi. The titer decreased at 4 dpi for WT- and Omi-S-infected mice, but it showed an increasing trend for Omicron-infected mice, pointing to the possibility of mild but persistent infection by Omicron in K18-hACE2 mice. All three variants recovered from the lungs of mice maintained the same plaque size phenotype as the original inoculum, indicating that replication in mice had no detectable effect on genotypes of these viruses (data not shown).

To evaluate the viral pathogenicity in lungs and nasal turbinates of K18-hACE2 mice, we performed a histopathological analysis of these tissues at 2 dpi. As previously reported4,31, an extensive near-diffused immunoreactivity of the SARS-CoV-2 N protein was detected in lung alveoli of mice infected with WT virus (Fig. 2e). By contrast, Omi-S and Omicron infection produced localized foci of alveolar staining with fewer foci for Omicron compared with Omi-S. The most marked phenotype was seen in bronchiolar epithelium, in which Omi-S caused pronounced, routinely circumferential infection, with around 10–15% of bronchioles being positive for viral N protein at 2 dpi, whereas only 3–5% of bronchioles were N-positive for Omicron (Fig. 2f). WT virus infected around 1% of bronchioles and in all cases only included a single isolated epithelial cell per bronchiole. Furthermore, bronchiolar infection was associated with epithelial necrosis in Omi-S-infected mice, as determined through serial hematoxylin and eosin (H&E) section analysis, whereas no histological evidence of airway injury was observed in Omicron- or WT-infected mice (Extended Data Fig. 8a,b). The nasal turbinates of mice inoculated with WT and Omi-S viruses both contained abundant SARS-CoV-2-positive cells, which were associated with overt cytopathic effects, whereas Omicron produced rare, sporadic positive cells, with no apparent signs of epithelial injury (Extended Data Fig. 8c). Overall, these findings suggest that replication of Omicron in the mice respiratory tract is substantially attenuated compared to Omi-S, supporting our conclusion that mutations in S are only partially responsible for the attenuated pathogenicity of Omicron. 

Mutations in S and nsp6 define Omicron attenuation

In addition to the S protein, Omicron has amino acid changes in non-structural protein 3 (nsp3), nsp4, nsp5, nsp6, nsp14, envelope (E), membrane (M), and N proteins, when compared with the WT virus (Extended Data Fig. 9a). To identify non-spike proteins that are involved in Omicron attenuation, we generated a large panel of fluorescently labeled chimeric viruses, each containing Omicron S in combination with one non-spike protein of Omicron, with the remaining proteins coming from WT virus (Extended Data Fig. 9b). When we combined Omicron S with Omicron nsp6 (Omi-S/nsp6), we observed a strong decrease in viral replication, with infection kinetics mimicking those of Omicron in cell culture (Fig. 3a–d); no such decrease was seen for other chimeric viruses. Poor replication efficiency of Omi-S/nsp6 was also corroborated by our finding that both Omi-S/nsp6 and Omicron took almost five to six days to recover by CPER, whereas all other variants were recovered in two days (data not shown). Finally, like Omicron, Omi-S/nsp6 was clearly outcompeted by Omi-S in a direct competition assay (Fig. 3e).

Fig. 2 | Role of S in Omicron pathogenicity. a–c, Male and female K18-hACE2 mice (aged 12–20 weeks) were intranasally inoculated with 1 × 104 PFU of WT (n = 6 mice), Omi-S (n = 10 mice) or Omicron (n = 10 mice) virus. Two independently generated virus stocks were used in this experiment. Body weight (a), clinical score (b), and survival (c) were monitored daily for 14 days. Mice that lost 20% of their initial body weight were euthanized. d,e, K18-hACE2 mice were intranasally inoculated with 1 × 104 PFU of WT (n = 14 mice), Omi-S (n = 14 mice) and Omicron (n = 14 mice). Lung samples of the infected mice were collected at 2 or 4 dpi to determine the viral titer (n = 4 mice) (d) or for immunohistochemistry (IHC) detection of the N protein (n = 3 mice) (e). In e, representative IHC images showing SARS-CoV-2 N (brown color) in alveoli (arrows) and bronchioles (arrowheads) in mice lungs at 2 dpi are presented. Scale bars, 100 µm. f, Percentage of N-positive bronchioles in the lungs of infected mice (n = 3 mice) at 2 dpi. Each dot represents an infected mouse. Statistical significance was determined using a two-tailed, unpaired t-test with Welch's correction (a,b,d,f) and log-rank (Mantel-Cox) test (c). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; NS, not significant.

In lungs of K18-hACE2 mice, whereas Omi-S caused extensive bronchiolar infection and injury, both Omicron and Omi-S/nsp6 showed decreased infection with no evidence of epithelial damage (Fig. 3f). Consistent with these findings, lungs of Omi-S/nsp6-infected mice produced viral titers equivalent to those seen for rOmicron and Omicron isolate (Fig. 3g). Finally, 71% of mice infected with Omi-S/nsp6 survived (Fig. 3h)-in contrast with the survival rates of only 20% that were observed in mice infected with Omi-S (Fig. 2c). Overall, these results indicate that mutations in S and nsp6 are sufficient to define Omicron's attenuated virulence. These observations support and further extend the findings of a previous study showing that mutations in the 5′-UTR–nsp12 region, in which nsp6 resides, contribute to Omicron's attenuation in K18-hACE2 mice30.

Fig. 3 | Mutations in S and nsp6 drive Omicron pathogenicity.a–d, Replication kinetics of indicated mNeonGreen reporter viruses in ACE2/TMPRSS2/Caco-2 cells (MOI = 0.01) determined by flow cytometry (n = 3 replicates) (a,c) and plaque assay (n = 3 replicates) (b,d). Experiments were repeated twice. e, ACE2/ TMPRSS2/Caco-2 cells were infected with virus mixtures at a 1:1 ratio to obtain the final MOI of 0.005 for each virus. The cells were fixed at the indicated times and analyzed by flow cytometry. Percentage of uninfected, singly infected, and doubly infected cells is shown. Singly infected cells were used for compensation. Individual data points are plotted along with the mean ± s.d. (n = 3 replicates). The experiment was repeated twice. f–h, K18-hACE2 mice were intranasally inoculated with 1 × 104 PFU of viruses. Lung samples of infected mice were collected at 2 dpi for IHC detection of the N protein (n = 3 mice) (f) or for the determination of viral titers (n = 4 mice) (g). In f, representative images of H&E staining of N-positive bronchioles are shown in insets. Bronchiolar epithelial necrosis is indicated with arrows. No evidence of necrosis was seen in the bronchioles of mice infected with Omicron or Omi-S/nsp6. Scale bar, 100 µm. The right graph in f shows the percentage of N-positive bronchioles in the lungs of infected mice. Each dot represents an infected mouse. h, Survival of infected mice monitored daily for 14 days. Mice that lost 20% of their initial body weight were euthanized. Statistical significance was determined using a two-tailed, unpaired t-test with Welch's correction (a–g) and log-rank (Mantel-Cox) test (h). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; NS, not significant.

The S protein RBM drives Omicron's vaccine escape 

A large body of literature has provided evidence of the extensive escape of Omicron from vaccine-induced humoral immunity14,19. To define S regions that are associated with the immune-escape phenotype of Omicron, we first compared the in vitro neutralization activity of sera from vaccinated individuals against WT SARS-CoV-2 (USA-WA1/2020), Omi-S, and Omicron. Sera collected within two months of the second dose of mRNA-1273 (Moderna mRNA vaccine; n = 12) or BNT162b2 (Pfizer-BioNTech mRNA vaccine; n = 12) vaccine was included (Extended Data Table 1). We performed a multicycle neutralization assay using a setting in which the virus and neutralizing sera were present at all times, mimicking the situation in a seropositive individual. All sera poorly neutralized Omicron, with an 11.1-fold (range: 4.4-fold to 81.2-fold; P < 0.0001) lower half-maximal neutralizing dilution (ND50) for Omicron compared with WA1 (Fig. 4a,b). In fact, around 80% of samples did not completely neutralize Omicron at the highest tested concentration (Extended Data Fig. 10). Notably, Omi-S exhibited identical ND50 values to Omicron (11.5-fold lower than that of WA1; P < 0.0001) (Fig. 4a,b), suggesting that the Omicron S protein, when incorporated into a WT virus, behaves the same way as it does in Omicron.

Fig. 4 | Role of S in the immune resistance of Omicron. ND50 values for WA1, Omi-S, and Omicron in sera from individuals who received two shots of Moderna (donors 1–12) or Pfizer (donors 13–24) vaccine (further details of sera are provided in Extended Data Table 1; individual curves are shown in Extended Data Fig. 10). b, Trajectories of ND50 values against WA1, Omi-S and Omicron (the data from a are plotted). The fold change in ND50 values is indicated (n = 24 serum samples). c–f, Schematic of the chimeric (left; c,d) and mutant (left; e,f) viruses. The amino acid numbering for WA1 mutants in e is based on the WA1 S sequence, whereas the numbering for Omicron mutants in f is based on the Omicron S sequence. Six of the 24 sera (3 from Moderna and 3 from Pfizer) were tested. Each serum sample is represented by a dot of a specific color. The data are plotted as the fold change of the parental virus. Statistical significance was determined using a two-tailed, unpaired t-test with Welch's correction. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; NS, not significant.

The SARS-CoV-2 S protein comprises two domains: the S1 domain, which interacts with the ACE2 receptor, and the S2 domain, which is responsible for membrane fusion32. Within the S1 domain lie an N-terminal domain (NTD) and a receptor-binding domain (RBD), which contains the receptor-binding motif (RBM) that makes direct contact with the ACE2 receptor33. The NTD of Omicron S has 11 amino acid changes, including 6 deletions and one 3-amino-acid-long insertion, whereas the RBD contains 15 mutations, 10 of which are concentrated in the RBM (Extended Data Fig. 1). Both the NTD and the RBD host neutralizing epitopes34–37, but the RBD is immunodominant and represents the primary target of the neutralizing activity that is present in SARS-CoV-2 immune sera37,38. To determine whether the neutralization resistance phenotype of Omicron is caused by mutations in a particular domain of the S protein, we generated two groups of chimeric viruses. The first group comprised the WA1 virus carrying the NTD, RBD or RBM of Omicron (Fig. 4c), and the second group consisted of Omi-S virus bearing the NTD, RBD or RBM of WA1 (Fig. 4d). The neutralization assay showed that mutations in the RBM were the main cause of Omicron's resistance to vaccine-induced humoral immunity: replacing the RBM of WA1 with that of Omicron decreased the ND50 by 5.4-fold (P < 0.0001), and, conversely, substituting the RBM of Omi-S with that of WA1 increased the ND50 by 5.6-fold (P = 0.0003) (Fig. 4c,d). The fact that none of the RBM-swap viruses achieved the difference of around 11-fold that was seen between WA1 and Omi-S suggests that mutations in other parts of S also contribute to vaccine resistance.

To investigate whether specific mutations in Omicron RBM drive vaccine escape, we generated two additional panels of recombinant viruses, one with WA1 S carrying Omicron RBM mutations, either singly or in combination (Fig. 4e), and the other with Omicron S lacking the same set of mutations (Fig. 4f). Two WA1 mutants-mutant 3 (with an E484A substitution) and mutant 4 (bearing a cluster of five substitutions Q493R, G496S, Q498R, N501Y and Y505H)-exhibited a moderate but statistically significant decrease of 1.4-fold (P = 0.0002) and 1.8-fold (P = 0.0003) in ND50 values, respectively, compared with WA1 (Fig. 4e). The opposite was observed when these mutations were removed from Omicron S; the Omicron mutant 3 (lacking the E484A substitution) and mutant 4 (lacking Q493R, G496S, Q498R, N501Y and Y505H) had 1.9-fold (P = 0.0082) and 3.1-fold (P = 0.0025) higher ND50 values compared with Omicron (Fig. 4f). As none of the mutants captured the overall phenotype of Omicron, we assume that the vaccine escape is a cumulative effect of mutations distributed along the length of the S protein. It is possible that mutations alter the conformation of Omicron S in such a manner that most of the immunodominant neutralizing epitopes are disrupted and become unavailable for neutralization. 

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Discussion 

This study provides key insights into viral proteins that contribute to SARS-CoV-2 pathogenicity. We show that S, the most mutated protein in Omicron, has an incomplete role in Omicron attenuation. In cell-based infection assays, the Omi-S virus exhibits an intermediate replication efficiency between the ancestral virus and Omicron. Similarly, in K18-hACE2 mice, Omi-S contrasts with non-fatal Omicron and leads to 80% mortality; the ancestral virus causes 100% mortality in these mice. Notably, when we combined S mutations with mutations in nsp6, the virus exhibited an attenuated phenotype largely resembling that of Omicron, indicating that these two proteins are major determinants of Omicron pathogenicity. Future studies will decipher the mechanism(s) by which nsp6 mutations affect viral replication.

One potential limitation of our study is the use of K18-hACE2 mice for pathogenesis studies instead of primate models that have more similarities with humans39. It should however be noted that K18-hACE2 mice are a well-established model for investigating the lethal phenotype of SARS-CoV-24,31. Although these mice develop lung pathology after SARS-CoV-2 infection, mortality has been associated with the involvement of the central nervous system owing to viral neuroinvasion and dissemination31,40. The fact that infection of K18-hACE2 mice with Omi-S, but not with Omicron, elicits neurological signs (for example, hunched posture and a lack of responsiveness) suggests that the neuroinvasion property is preserved in Omi-S, probably as a result of its higher replication efficiency, and that the determinants of this property lie outside of the S protein. These findings are consistent with a previous study showing that hamsters infected with Omi-S shed significantly more virus and lost more weight than those infected with Omicron, suggesting that mutations outside of S contribute to the attenuated pathogenicity of Omicron41.

We found that although the ancestral virus mainly replicates in lung alveoli and causes only rare infection of bronchioles in K18-hACE2 mice, both Omi-S and Omicron exhibit an increased propensity to replicate in the bronchiolar epithelium, indicating that the S protein is accountable for the changed tropism. The mechanism behind this switch is unknown, but it is possible that Omicron S is more efficient than WT S in using cathepsin B or cathepsin L (refs. 25,42,43), which form an active viral entry pathway in bronchioles and other airway cells41. By contrast, the entry of SARS-CoV-2 into alveolar epithelial cells is mainly driven by TMPRSS2 (refs. 28,44), which Omicron S is deficient in utilizing25,45, leading to poor infection of these cells4,5,25,43. These findings may explain the attenuated lung pathology caused by Omicron.

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Omicron nsp6 has two altered sites relative to the prototype SARS CoV-2 Wuhan-Hu-1 isolate: a three-amino-acid deletion (LSG, positions 105–107) and an I189V substitution (Extended Data Fig. 9). Several functions of nsp6 in coronavirus replication have been described; chief among them is the biogenesis of double-membrane vesicles (DMVs), which represent the site of viral RNA synthesis46–50. A previous study showed that SARS-CoV-2 DMVs are mainly generated by the concerted action of three viral proteins: nsp3, nsp4, and nsp6; although nsp3 and nsp4 are sufficient for the formation of DMVs, nsp6 connects these DMVs with the endoplasmic reticulum and channelizes the essential communication between these structures46. Whether the constellation of mutations in Omicron nsp6 affects the formation or functions of DMV needs further investigation. Nsp6 also activates NLR3-dependent cytokine production and pyroptosis in the lungs of patients with COVID-19, serving as a key virulence factor47. Of note, an nsp6 variant that is associated with asymptomatic COVID-19 exhibited a reduced ability to induce pyroptosis47, prompting speculation that mutations in Omicron nsp6 may also influence pyroptosis. Detailed mechanistic studies will be required to dissect the effect of Omicron mutations on the functions of nsp6.

It is at present unknown whether mutations in S and nsp6 work in concert with each other to drive Omicron attenuation. Given that Omicron S showed a higher predilection for bronchioles, it is possible that S is responsible for the altered viral tropism, whereas non-spike mutations-including those in nsp6-are mere adaptations to the changed tissue environment. It is worth mentioning that although nsp6 seems to be the major non-spike protein behind Omicron attenuation, the contribution of other viral proteins cannot be completely ruled out. In vitro experiments examining the role of non-spike mutations were all carried out in ACE2/TMPRSS2/Caco-2 cells. Using other, more immune-competent cell types could reveal the effect of other non-spike mutations as well. In addition, our chimeric viruses contained Omicron S paired with only one non-spike protein at a time, which limited long-range epistatic interactions between mutations in multiple viral proteins.

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Our study shows that mutations in the RBM of Omicron S are the main determinants of Omicron's escape from neutralizing antibodies, although mutations in other regions of S also contribute. Within the RBM, we identify two hotspots of mutations, which give Omicron S the ability to resist neutralization: one bearing the E484A substitution and the other containing a cluster of five substitutions-Q493R, G496S, Q498R, N501Y, and Y505H. The E484A substitution has been shown to escape neutralization by convalescent sera51. Moreover, structural modeling suggests that some therapeutic monoclonal antibodies establish highly stable salt bridges with the E484 residue, entirely losing their binding when this residue is changed to A or after Q493K and Y505H changes52. Similarly, mapping of RBM residues that directly interact with 49 known neutralizing antibodies revealed N440, G446, S477, and T478 as low-frequency interactors, N501, Y505, and Q498 as medium-frequency interactors, and E484 and Q493 as high-frequency interactors53, in line with our neutralization assay results. Notably, although the antibody-binding potential of Omicron S is impaired54, its receptor-binding capacity is intact. In fact, the Omicron RBD has a higher affinity for ACE2 relative to the Wuhan-Hu-1 and Delta RBDs25. This indicates that mutations in Omicron S have evolved in such a manner that they hinder antibody binding but preserve receptor engagement. This opens up the possibility of targeting the conserved and structurally constrained regions of S that are involved in ACE2 recognition for the design of broad-spectrum vaccines to control the COVID-19 pandemic.

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