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. 2022 Sep 30:13:1016108.
doi: 10.3389/fimmu.2022.1016108. eCollection 2022.

SARS-CoV-2 variants Alpha, Beta, Delta and Omicron show a slower host cell interferon response compared to an early pandemic variant

Larissa Laine  1 , Marika Skön  1 , Elina Väisänen  1   2 , Ilkka Julkunen  2 , Pamela Österlund  1
Affiliations

SARS-CoV-2 variants Alpha, Beta, Delta and Omicron show a slower host cell interferon response compared to an early pandemic variant

Larissa Laine et al. Front Immunol. .

Abstract

Since the start of the pandemic at the end of 2019, arising mutations in SARS-CoV-2 have improved its transmission and ability to circumvent the immunity induced by vaccination and previous COVID-19 infection. Studies on the effects of SARS-CoV-2 genomic mutations on replication and innate immunity will give us valuable insight into the evolution of the virus which can aid in further development of vaccines and new treatment modalities. Here we systematically analyzed the kinetics of virus replication, innate immune activation, and host cell antiviral response patterns in Alpha, Beta, Delta, Kappa, Omicron and two early pandemic SARS-CoV-2 variant-infected human lung epithelial Calu-3 cells. We observed overall comparable replication patterns for these variants with modest variations. Particularly, the sublineages of Omicron BA.1, BA.2 and a recombinant sublineage, XJ, all showed attenuated replication in Calu-3 cells compared to Alpha and Delta. Furthermore, there was relatively weak activation of primary innate immune signaling pathways, however, all variants produced enough interferons to induce the activation of STAT2 and production of interferon stimulated genes (ISGs). While interferon mRNA expression and STAT2 activation correlated with cellular viral RNA levels, ISG production did not. Although clear cut effects of specific SARS-CoV-2 genomic mutations could not be concluded, the variants of concern, including Omicron, showed a lower replication efficiency and a slower interferon response compared to an early pandemic variant in the study.

Keywords: Omicron; SARS-CoV-2; innate immunity; interferon; mutations; replication; variants.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Mutations in the six pre-Omicron SARS-CoV-2 variants used to infect Calu-3 cells. The mutations are mapped against the hCoV-19/Wuhan/WIV04/2019 reference genome (EPI_ISL_402124). In green are the non-structural proteins located in Orf1ab and in blue are the structural proteins S, E, M, N, and accessory proteins. Receptor-binding domain (RBD, S protein amino acid residues 437-507, orange), multi-basic cleavage site (MBCS, S protein amino acid residues 681-685, (P-R-R-A-R), dark blue) are also marked.
Figure 2
Figure 2
Replication of six SARS-CoV-2 variants in Calu-3 cells. Cells were infected with each variant at a MOI of 1 TCID50/cell. Cell culture supernatant and total cellular RNA and protein samples were collected at 1, 6, 24, 48 and 72 hours post-infection (h p.i.). The figure shows the replication profiles for each variant. The relative cellular vRNA expression levels and the quantified vRNA copies/ml in cell culture supernatant (as determined by RT-qPCR) is shown in the graphs on the left Y-axis. The production of infectious virions (as determined by an end point dilution assay and shown as log TCID50/ml) is shown on the right Y-axis. The results are the mean values ± SEM of three independent experiments. Cellular protein samples were analyzed by immunoblotting with anti-SARS-CoV-2 S1 (S1) specific and cross-reactive anti-SARS-CoV N protein (N) specific antibodies to show the replication kinetics of the viruses at a protein level. Immunoblotting was carried out once for S protein and three times for N protein. Representative immunoblots are shown. The anti-SARS-CoV-2 S1 antibody recognizes both the full-length S protein (S) and the cleaved S1 fragment. GAPDH was used as a loading control. The blots were exposed with an equal exposure time. S protein amounts quantified using ImageJ are shown below the immunoblots.
Figure 3
Figure 3
Activation of signaling molecules involved in the induction of interferon, cytokine and ISG gene expression. Total cellular protein samples were collected at various time points following infection of Calu-3 cells with different SARS-CoV-2 variants at a MOI of 1 TCID50/cell. Representative immunoblots out of 3 repeated experiments are shown. (A) Immunoblots were probed with antibodies against phosphorylated interferon regulatory transcription factor 3 (p-IRF3) and total IRF3. Cellular protein samples collected at 8 h after Sendai virus infection in Calu-3 cells was used as a positive control (+). GAPDH was used as a loading control for p-IRF3. The immunoblot was carried out twice. (B) Immunoblots stained with antibodies against phosphorylated p38 (p-p38) and p38 (carried out once), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκB-α) and phosphorylated signal transducer (carried out twice) and activator of transcription 2 (p-STAT2) and STAT2 (carried out twice). p-STAT2 levels were quantified by ImageJ and the fold over mock values are seen below the p-STAT2 immunoblot. GAPDH was used as a loading control.
Figure 4
Figure 4
Interferon and CXCL10 mRNA expression levels in Calu-3 cells infected with SARS-CoV-2 variants. Cells were infected with each variant at a MOI of 1 TCID50/cell and total cellular RNA samples were collected at different time points during infection. (A) The kinetics of vRNA and host cell IFN-β1, IFN-λ1, IFN-λ2 and CXCL10 mRNA expression profiles were determined by RT-qPCR. Relative cytokine mRNA expression profiles in different SARS-CoV-2 variant infected cells are shown on the left Y axes and the vRNA expression levels on the right Y axes. The means of three independent experiments are shown. (B) Comparative analysis of relative cellular vRNA and IFN-β1, IFN-λ1, IFN-λ2 and CXCL10 mRNA expression at 24 h p.i. The results are the mean values ± SEM of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons test was used for the statistics. P < 0.05 (*), P < 0.01 (**), not significant (ns).
Figure 5
Figure 5
Kinetics of expression of antiviral ISGs in Calu-3 cells infected with different SARS-CoV-2 variants. Six different virus variants were used at MOI of 1 TCID50/cell to infect Calu-3 cells and during the 3-day infection cellular protein samples were collected. Representative immunoblots were probed with antibodies against interferon induced transmembrane protein 3 (IFITM3) (carried out once) and human Myxovirus resistance protein A (MxA) (carried out twice). GAPDH was used as a loading control.
Figure 6
Figure 6
Mutations in three Omicron sublineages, Fin55-BA.1, Fin58-BA.2 and recombinant Fin60-XJ. The hCoV-19/Wuhan/WIV04/2019 reference genome (EPI_ISL_402124) was used to map the mutations. In black are unique mutations and in red are mutations that are found in all the above Omicron sublineages. Mutations in the orange box are found in the RBD. Fin60-XJ is a recombinant of BA.1 and BA.2 with a cut off between nucleotides 13 296 (green) in Nsp10 and 15 240 (blue) in Nsp12. Receptor-binding domain (RBD, S protein amino acid residues 437-507, orange), multi-basic cleavage site (MBCS, S protein amino acid residues 681-685, (P-R-R-A-R), dark blue).
Figure 7
Figure 7
Replication of Omicron sublineages BA.1, BA.2 and XJ compared to Alpha and Delta. Calu-3 cells were challenged with three Omicron sublineages and Alpha and Delta variants at multiplicity of 1 for three days and different samples were collected for analysis of the viral molecules. (A) Relative cellular vRNA expression levels determined by RT-qPCR of Fin55-BA.1, Fin58-BA.2 and Fin60-XJ compared to Alpha (Fin34-α) and Delta (Fin37-δ). (B) Cell culture supernatant vRNA copies/ml were quantified by RT-qPCR. (C) An end point dilution assay was carried out to determine the production of infectious virions. Results shown as log TCID50/ml. (D) Immunoblot analysis of SARS-CoV-2 S protein expression by anti-SARS-CoV-2 S1 fragment antibody (S1) and N protein expression using a cross-reactive anti-SARS-CoV-N protein antibody (N) were carried out once. Full length S protein (S) and the cleaved S1 fragment (S1) are marked with arrows. S protein amounts quantified using ImageJ are shown in graphs below the immunoblots. GAPDH was used as a loading control. The qPCR and end point dilution assay results are the mean values ± SEM of three independent experiments.
Figure 8
Figure 8
Interferon and CXCL10 mRNA expression levels in Calu-3 cells with Omicron infection. The Omicron sublineages BA.1, BA.2 and recombinant XJ as well as the Alpha and Delta variants were used for infecting Calu-3 cells at a MOI of 1 TCID50/cell, and total RNA samples were collected at different time points for RT-qPCR analysis. (A) IFN-β1, IFN-λ1, IFN-λ2 and CXCL10 mRNA expression profiles determined by RT-qPCR (left Y axis) compared to the relative cellular vRNA expression profile (right Y axis) for cells infected with three Omicron variants (Fin55-BA.1, Fin58-BA.2 and Fin60-XJ), and with Alpha (Fin34-α) and Delta (Fin37-δ). The means of three independent experiments are shown. (B) Comparison of relative cellular vRNA and IFN-β1, IFN-λ1, IFN-λ2 and CXCL10 mRNA expression at 24 h p.i. The results are the mean values ± SEM of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons test was used for the statistics. P < 0.05 (*), P < 0.01 (**).
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References

    1. World Health Organization . WHO coronavirus (COVID-19) dashboard. Available at: https://covid19.who.int (Accessed August 8, 2022).
    1. Ilmjärv S, Abdul F, Acosta-Gutiérrez S, Estarellas C, Galdadas I, Casimir M, et al. . Concurrent mutations in RNA-dependent RNA polymerase and spike protein emerged as the epidemiologically most successful SARS-CoV-2 variant. Sci Rep (2021) 11:13705. doi: 10.1038/s41598-021-91662-w - DOI - PMC - PubMed
    1. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, et al. . Tracking changes in SARS-CoV-2 spike: Evidence that D614G increases infectivity of the COVID-19 virus. Cell (2020) 182:812–827.e19. doi: 10.1016/j.cell.2020.06.043 - DOI - PMC - PubMed
    1. Yurkovetskiy L, Wang X, Pascal KE, Tomkins-Tinch C, Nyalile TP, Wang Y, et al. . Structural and functional analysis of the D614G SARS-CoV-2 spike protein variant. Cell (2020) 183:739–751.e8. doi: 10.1016/j.cell.2020.09.032 - DOI - PMC - PubMed
    1. Zhou B, Thao TTN, Hoffmann D, Taddeo A, Ebert N, Labroussaa F, et al. . SARS-CoV-2 spike D614G change enhances replication and transmission. Nature (2021) 592:122–7. doi: 10.1038/s41586-021-03361-1 - DOI - PubMed

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