SARS-CoV-2 hijacks p38β/MAPK11 to promote virus replication

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38β is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38β substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38β and supports exploring p38β inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

all error bars represent one standard deviation from the mean for three biological replicates; p-value annotations were calculated using a one-way ANOVA test with post hoc testing using Tukey's method comparing each condition to siNTC for three biological replicates; "****" = p-value < 0.0001, "***" = 0.0001 < p-value < 0.001, "**" = 0.001 < p-value < 0.01, "*" = 0.01 < p-value < 0.05, "NS" = p-value > 0.05 Extracellular matrix organization genes (GO: 0030198)  C-D) Volcano plot of differentially expressed genes for the indicated condition comparisons; grey is grey is not differentially expressed, red is upregulated and blue is downregulated; E-F) Plot of log10(transcripts per million) for each gene represented in the indicated GO term for each condition, from same analysis as 3D.

Pre-treatment
Figure S5: A-B) Heatmap of Pearson's correlation analysis of protein abundance (A) or phospho-enriched (B) mass spectrometry samples from "terminal treatment" experiment arm; C-D) Heatmap of Pearson's correlation analysis of protein abundance (A) or phospho-enriched (B) mass spectrometry samples from "pre-treatment" experiment arm; E-F) Plot of peptide abundance (log2(mass spectrometry signal intensity)) for each replicate of protein abundance (E) or phospho-enriched (F) samples from "terminal treatment" experiment arm; E-F) Plot of peptide abundance (log2(signal intensity)) for each replicate of protein abundance (G) or phospho-enriched (H) samples from "pre-treatment" experiment arm; I) Plot of log2(signal intensity)of known p38β substrates from terminal-treatment experiment for each comparison; J) Plot log2(signal intensity) of known p38 β substrates from pre-treatment experiment for each comparison; K) Plot log2(signal intensity) of detected SARS-CoV-2 proteins from pre-treatment experiment for each comparison; L) Schematic of kinase perturbation phospho-profile clustering (KiPPC) pipeline; M) Gene ontology terms enriched from cluster-of-interest proteins using GSEA; N) Plot of A549-ACE2 cell viability normalized to siNTC for each cluster-of-interest-gene siRNA transfection; error bars represent one standard deviation from the mean for three biological replicates; p-values were calculated using a one-way ANOVA test with post hoc testing using Tukey's method comparing each condition to the infected control condition, for three biologicals replicates; "****" = p-value < 0.0001, "***" = 0.0001 < p-value < 0.001, "**" = 0.001 < p-value < 0.01, "*" = 0.01 < p-value < 0.05, "NS" = p-value > 0.05.5A).

Figure S1 :Figure S2 :
Figure S1: A) Heatmap of pairwise Pearson coefficients for protein group log2(fold-change) profiles from this study and published studies indicated; B) Bubble plot of kinase activity analysis of phosphosite group log2(fold-change) profiles from this study and published studies indicated; the absolute value of the normalized enrichment score (NES) is indicated by node sizes and the -log10(p-value) is indicated by the color scale.

Figure S3 :
FigureS3: A) Plot of principal component analysis of mRNA-Seq samples; B) Heatmap of Pearson correlation analysis of mRNA-Seq samples; C-D) Volcano plot of differentially expressed genes for the indicated condition comparisons; grey is grey is not differentially expressed, red is upregulated and blue is downregulated; E-F) Plot of log10(transcripts per million) for each gene represented in the indicated GO term for each condition, from same analysis as 3D.
Figure S4: A) Heatmap of Pearson's correlation analysis of protein abundance mass spectrometry samples; B) Heatmap of Pearson's correlation analysis of phosphopeptide-enriched mass spectrometry samples; C) Plot of peptide abundance (mass spectrometry signal intensity) for each biological replicate of protein abundance samples; D) Plot of phosphopeptide abundance (mass spectrometry signal intensity) for each biological replicate of phosphopeptide-enriched samples; E) Western blot of lysates from cells transfected with siRNA targeting each indicated gene and infected with SARS-CoV-2 MOI 0.1 or mock-infected for 30h in A549-ACE2 cells; F) Plot of the percent of SARS-CoV-2 N-positive cells analyzed using immunofluorescence cytometry for each indicated transfection condition after SARS-CoV-2 infection at an MOI of 0.1 for 30h in A549-ACE2 cells in the presence of DMSO or ruxolitinib; error bars represent one standard deviation from the mean for three biological replicates; p-values were calculated using a one-way ANOVA test with post hoc testing using Tukey's method comparing each condition between each cell type for three biological replicates; "NS" = p-value > 0.05.
Figure S6: A) Plot of log2(signal intensity) of each significantly differentially abundant phosphosite group on SARS-CoV-2 N from the terminal-treatment experiment arm (Figure