SARS-CoV-2 Infection Promotes p38/MAPK Signaling Activity and Cell Cycle Arrest
Kinase activity analysis of SARS-CoV-2 phosphorylation profiles predicted upregulation of several components of the p38/mitogen-activated protein kinase (MAPK) signaling pathway, including MAP2K3, MAP2K6, MAPK12, MAPKAPK2 (MK2), and MAPKAPK3 (Figures 6A and 6B). Immunoblotting for activated phospho-p38 (T180/Y182), phospho-MK2 (T334), and phospho-cAMP response element-binding protein (CREB) and phospho-ATF-1 at their respective MAPKAPK2 sites (S133 in both) confirmed activation of the p38/MAPK pathway during SARS-CoV-2 infection in ACE2-expressing A549 human lung carcinoma cells (ACE2-A549) (Figure 6C). Furthermore, phosphoproteomics data depict increased phosphorylation of p38 pathway substrates such as negative elongation factor E (NELFE), heat shock protein beta-1 (HSPB1), and signal transducer and activator of transcription 1-alpha/beta (STAT1), among others (Figure 6D). Regulation of these sites occurs late in the time course (24 h after infection), likely reflecting a more advanced stage of viral infection, replication, and egress.
Figure 6 SARS-CoV-2 Activates the p38/MAPK Signaling Pathway and Causes Cell Cycle Arrest
(A) Diagram of the p38/MAPK signaling pathway.
(B) Kinase activity analysis for kinases in the p38/MAPK pathway.
(C) Western blot analysis of phosphorylated p38/MAPK signaling components in mock- and SARS-CoV-2-infected ACE2-A549 cells 24 h after infection.
(D) Log2 fold change profiles of indicated p38/MAPK substrates during SARS-CoV-2 infection in Vero E6 cells.
(E) Transcription factor activity analysis of SARS-CoV-2-infected A549, Calu-3, and NHBE cells, comparing p38/MAPK transcription factors with transcription factors not associated with the p38/MAPK pathway. Statistical test: Mann-Whitney test.
(F) qRT-PCR analysis of the indicated mRNA from ACE2-A549 cells pre-treated with the p38 inhibitor SB203580 at the indicated concentrations for 1 h prior to infection with SARS-CoV-2 for 24 h. Statistical test: Student’s t test. See also Figures S4 and S5.
(G) Heatmap of Pearson’s correlation coefficients comparing SARS-CoV-2-infected Vero E6 phosphorylation profiles with profiles of cells with induced DNA damage and cells arrested at the indicated cell cycle stages.
(H) Log2 fold change profiles of the indicated cell cycle and DNA damage substrates during SARS-CoV-2 infection in Vero E6 cells.
(I) DNA content analysis of cells infected with SARS-CoV-2 for 24 h compared with mock-infected cells.
The p38/MAPK pathway mediates the cellular response to environmental stress, pathogenic infection, and pro-inflammatory cytokine stimulation, whereas downstream effectors of the pathway include transcription factors and RNA binding proteins that promote inflammatory cytokine production (Cuadrado and Nebreda 2010; Wen et al., 2010). Analysis of estimated transcription factor activity from gene expression data (STAR Methods; Table S6) derived from the infection of a human lung carcinoma cell line (A549), a human epithelial lung cancer cell line (Calu3), and primary human bronchial epithelial (NHBE) cells demonstrated that transcription factors regulated by the p38/MAPK pathway were among the most highly activated upon infection (Figure 6E; Blanco-Melo et al., 2020).
To investigate the contribution of the p38/MAPK pathway to cytokine production, SARS-CoV-2-infected ACE2-A549 cells were treated with the p38 inhibitor SB203580. The mRNA of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and others increased during infection and were inhibited by p38 inhibition in a dose-dependent manner (Figures 6F, right, and S4A). Interestingly, p38 inhibition also reduced SARS-CoV-2 subgenomic mRNA (Figure 6F, left) in the absence of major cellular toxicity (Figure S5 ), indicative of reduced viral replication. The SB203580-induced decrease in virus production was further confirmed using an anti-SARS-CoV-2 N protein (anti-NP) antibody-based assay (Figure S5, New York Vero E6). Multiplexed ELISA analysis of supernatants of cells from the same experiment demonstrated strong upregulation of inflammatory cytokines at the protein level, including IL-6, CXCL8, CCL20, and CCL2, which were decreased upon p38 inhibition (Figure S4 B; Table S7). However, because SARS-CoV-2 replication is also inhibited by SB203580, we cannot deconvolve the contributions of p38/MAPK pathway activity and SARS-CoV-2 virus presence on cytokine production.
Figure S5 Pharmacological Profiling for Viral Titers and Cell Viability, Related to Figure 7
Dose response of phosphoproteomics-informed drugs and compounds. Assays performed in New York (N; red, anti-NP; blue TCID50) and Paris (P; red, RT-qPCR; purple, plaque assays) across two cell lines (A549-ACE2 and Vero E6). Cell viability shown in black. Mean of three biological replicates is shown. Error bars are SEM.
Figure S4 Cytokine Profiling upon Infection, p38 Inhibition, and Cell Cycle Analysis, Related to Figure 6
(A) RT-qPCR analysis of indicated mRNA from A549-ACE2 cells pre-treated with p38 inhibitor SB203580 at indicated concentrations for one hour prior to infection with SARS-CoV-2 for 24 hours. Statistical test is Student’s t test. Error bars are SD. (B) Same as in (A) but a Luminex-based quantification of indicated cytokines. Error bars are SD. (C) Cell cycle analysis of Vero E6 cells (same as in Figure 6I) upon SARS-CoV-2 infection at an MOI of 1. Cell stained with DAPI DNA stain prior to flow cytometry analysis. Statistical test is Mann-Whitney test. Error bars are SD.
Comparing phosphoproteomics profiles of SARS-CoV-2-infected cells with a database of phosphorylation profiles collected at specific cell cycle stages, viral infection was most highly correlated with cells arrested at the S/G2 transition and was negatively correlated with profiles of cells in mitosis (Figure 6G). We also observed SARS-CoV-2-dependent regulation of CDK2 T14/Y15 phosphorylation, initially increased in response to SARS-CoV-2 infection at 2 h, followed by a decrease over the remainder of the infection time course (Figure 6H, left). CDK2 activity promotes transition from the G2 phase of the cell cycle into mitosis and is inhibited by phosphorylation at positions T14 and Y15 by kinases WEE1 and MYT1, preventing premature entry into mitosis (Parker and Piwnica-Worms 1992; Mueller et al., 1995). CDK2 can also become phosphorylated when the cell cycle is arrested because of checkpoint failure or DNA damage. In addition, H2AX S140 phosphorylation (i.e., γ-H2AX), a hallmark of the DNA damage response, exhibited a profile similar to CDK2, suggesting that the DNA damage response may become activated early during infection (Rogakou et al., 1998; Figure 6H, right).
To more directly test whether SARS-CoV-2 infection affects cell cycle progression, cells were infected with SARS-CoV-2 for 24 h, and their DNA content was measured using DAPI DNA staining and flow cytometry. A significant increase in the fraction of cells in S phase and at the G2/M transition and a decrease in the fraction of cells in G0/G1 phase were observed (Figures 6I and S4C). This observation is consistent with arrest between S and G2 phases of the cell cycle. A relationship between p38 activity and cell cycle arrest has been described previously, and the two could be linked mechanistically during SARS-CoV-2 infection (Lee et al., 2002; Yee et al., 2004).