The SARS-CoV-2 Omicron variant has rapidly displaced the Delta variant as the major prevalent variant in most of the world. The Omicron variant harbors more than 60 mutations, and 15 of the mutations are located in the receptor-binding domain (RBD)1. The L452R mutation is one of the most common mutations, is the only RBD mutation that occurs in the Delta variant but not in the Omicron variant2. The SARS-CoV-2 variant with the L452R Spike protein mutation initially spread widely in California, USA, and showed enhanced transmissibility, infectivity, and immune escape. By early 2021, this variant has seen a more than 50% increase in California cases, an 18.6%-24% increase in infectivity, a 2-fold increase in body shedding, and reduced antibody neutralization in COVID-19 patients and vaccine recipients3. Compared with parental virus and previous variants, the Omicron variant is characterized by decreased hospitalization rates and less severe disease in patients. The research shows that the pathogenicity of SARS-CoV-2 patients is related to the fusion pathogenicity of the virus4, and the L452R mutation will lead to increased fusogenicity and infectivity of SARS-CoV-25.
Recently, the team of Dr. Qinong Ye published a high-scoring report. The report showed that if the L452R (Omicron-L452R) is carried by Omicron, it cannot only improve the fusogenicity, but also enhance the cleavage ability of the Spike protein, and the infectious ability of the virus to humanized mouse lung tissue. At the same time, sufficient energy and nucleotide substances are obtained by significantly enhancing the anaerobic glycolysis of host cells to complete the replication of virus strains in human cells. Pseudovirus wild strain (vazyme #DD1402), Delta mutant strain (vazyme #DD1454), Omicron mutant strain (vazyme #DD1768), luciferase substrate (vazyme #DD1204) and Mut Express II Fast Mutagenesis Kit V2 (vazyme #C214) used in this study are provided by Vazyme Biotech Co.,Ltd.
High fusogenicity of Omicron-L452R
The Omicron variant has exhibited diminished fusogenicity and pathogenicity compared with the Delta variant and parental virus. Due to the important role of the L452R mutation infusion and infectivity of SARS-CoV-2, Dr. Qinong Ye’s team investigated the effect of L452R on the fusion of Omicron variant. Consistent with a previous report, compared to the parental virus, the Omicron variant almost lost fusogenicity, and the Delta variant demonstrated greatly enhanced fusogenicity (Figure 1).
Figure 1. Relative fusogenicity of different SARS-CoV-2
Omicron-L452R enhances Spike protein cleavage
Compared with the Delta variant and the parental virus, the Omicron variant reduced the cleavage of the SARS-CoV-2 Spike protein (S protein), whereas the Omicron-L452R variant greatly enhanced the cleavage of the S protein (Figure 2). It has been reported that mutations in the Omicron spike protein introduce electrostatic contacts and enhance the interaction between the S1 and S2 subunits, resulting in reduced cleavage of the S protein cleavage. Whether the L452R-Omicron variant promotes cleavage of the S protein by decreasing the interaction of the S1 and S2 subunits remains to be investigated.
Figure 2. Western blot analysis of SARS-CoV-2 spike cleavage of the indicated variant pseudoviruses in 293T cells stably transfected with hACE2
Omicron-L452R enhances the replication of viruses
To obtain available energy and nucleotides for viral replication, SARS-CoV-2 induces metabolic reprogramming in host cells in a manner similar to the Warburg effect in cancer cells. Researchers found that the Delta and Omicron-L452R variants increased lactate production more than the Omicron variant in Huh-7, 293T-ACE2, and H1299-ACE2 cells, although the Omicron variant enhanced lactate production in H1299-ACE2 cells (Figure 3).
Figure 3. Lactate production assay in Huh-7 cells
Consistent with lactate production, the extracellular acidification rate (ECAR) assay indicated that the Delta and Omicron-L452R variants highly stimulated glycolysis in Huh7, H1299-ACE2, and 293T-ACE2 cells, while the Omicron and parental viruses slightly increased glycolysis flux (Figure 4).
Figure 4. Extracellular acidification rate (ECAR) analysis of glycolysis (following glucose injection), glycolytic capacity (following oligomycin injection), and glycolytic reserve (glycolytic capacity; glycolysis) in Huh-7 cells and ACE2 stably transfected 293T and H1299 cells infected with B.1, Delta, Omicron and the Omicron-L452R variant pseudoviruses
Omicron-L452R significantly enhanced infectivity
Consistent with previous data, the pseudovirus infection assay indicated that the Omicron variant demonstrated enhanced entry into Huh-7, 293T-ACE2, and H1299-ACE2 cells compared to the Delta variant and parental virus, and the Omicron-L452R variant further increased viral entry into these cells (Figure 5). Therefore, the Omicron-L452R variant not only rescued fusogenicity but also strengthened infectivity, which suggested that the Omicron-L452R variant can be very risky.
Figure 5. Luciferase activity of different variants into Huh-7, 293T-ACE2 and H1299-ACE2 cells
Furthermore, the infectivity of the Omicron-L452R variant is investigated in K18-hACE2 transgenic mice in vivo. The bioluminescence of infected mice was measured to indicate the pseudovirus infection severity. Consistent with the live SARS-CoV-2 virus, the Delta variant has the highest infectivity. The Omicron variant modestly inhibited infectivity, whereas Omicron-L452R significantly enhanced infectivity. qRT-PCR was performed to detect the infected pseudovirus copies. Compared to mice infected with the parental virus, the virus copies in the Omicron-infected mouse lung tissues were reduced, while the virus copies in the Omicron-L452R variant-infected mouse lung tissues were markedly enhanced. Therefore, the Omicron-L452R variant enhanced the ability of Omicron to infect lung tissues in vivo.
In this investigation, Dr. Qinong Ye’s team found that the further mutation of L452R in the Omicron variant not only rescued fusogenicity but also strengthened the high infectivity. Importantly, the Omicron-L452R variant enhanced the ability of Omicron to infect lung tissues of humanized mice. By dramatically enhancing the anaerobic glycolysis of host cells, the Omicron-L452R variant obtains enough energy and nucleotide material to replicate in human cells. In general, the L452R mutation dramatically increases the risk of the Omicron variant. The experimental data explain why the Omicron variant is less fusogenic and suggest that the Omicron variant should be closely monitored for the presence of the L452R mutation and that specific therapeutic antibodies and vaccines against the L452R mutation should be developed.
1. Cui, Z. et al. Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron. Cell 185, 1–12 (2022).
2. Yanan Z. et al. SARS-CoV-2 spike L452R mutation increases Omicron variant fusogenicity and infectivity as well as host glycolysis. Signal Transduction and Targeted Therapy (2022) 7:76.
3. Focosi, Daniele. SARS-CoV-2 Spike Protein Convergent Evolution. Springer Nature, 2021.
4. Rajah, M. M., Bernier, A., Buchrieser, J. & Schwartz, O. The Mechanism and Consequences of SARS-CoV-2 Spike-Mediated Fusion and Syncytia Formation. J. Mol.Biol. 1, 167280 (2021).
5. Motozono, C. et al. SARS-CoV-2 spike L452R variant evades cellular immunity andincreases infectivity. Cell Host Microbe 29, 1–13 (2021).