SARS-CoV-2 Infection Regulates Host Kinase Signaling
To study global changes in kinase signaling and their effect on host protein phosphorylation, regulated phosphorylation sites were grouped in five clusters based on their dynamics using a data-driven clustering approach (Figure 4 A; STAR Methods). For each of the groups, an enrichment analysis was performed for functions and pathways (Figure 4A; Table S3). The dynamics of these changes can be linked to the viral life cycle: entry (0–2 h), replication (4–12 h), and egress (24 h). Clusters 1 and 2 include phosphorylation sites that are, on average, upregulated during infection. Cluster 1 sites tended to be upregulated within 2 h (i.e., linked to viral entry) and were enriched in mRNA processing, cell cycle, apoptosis, and proteins involved in HIV infection. Cluster 2 included apoptosis proteins with later onset of phosphorylation, associated with replication and/or egress. Phosphorylation sites in clusters 3 and 4 were downregulated and enriched in RNA-processing functions. Sites within cluster 5 possessed a dynamic response to infection, with immediate downregulation followed by a rise during the middle and renewed downregulation at late time points. This cluster was enriched for DNA replication and the cell cycle, among others. These observations are corroborated by standard Gene Ontology (GO) enrichment analyses of biological processes regulated by phosphorylation (Figure S1G; Table S3; STAR Methods).
Figure 4 Signaling Changes in Host Cells in Response to SARS-CoV-2 Infection
(A) Clusters of significantly changing PHs (abs(log2FC) > 1 and adjusted p < 0.05) across the time course of infection with non-redundant enriched Reactome pathway terms (adjusted p value [q] < 0.01) for each cluster. Horizontal red lines below each pathway term correspond to phosphorylated proteins belonging to the pathway, and a black-bordered rectangle is indicative of a significantly enriched term.
(B) Kinases depicting a strong change in activity upon infection (abs(log10(p)) > 2.5) in at least one time point, with predicted activity in at least 5 of 6 time points.
(C) Schematic representation of interaction between host kinases and SARS-CoV-2 viral proteins from Gordon et al. (2020). Substrate PHs for each kinase are color-coded as blue (down) and red (up) based on the direction of change during infection. Only PHs corresponding to the kinase activity direction are shown.
(D) Correlation of kinase activity profiles of each time point with other biological conditions with at least one significantly changing kinase (abs(log10(p)) > 2.5) and having significant correlation with at least one time-point (false discovery rate [FDR] < 5%).
(E) Overall phosphorylation change (−log10(p)) of a protein complex, estimated as the change in phosphorylation on member proteins. Only non-redundant protein complexes with a significant change in phosphorylation (abs(log10(p)) > 2.5) in at least one time point are shown.
See also Figure S2.
We estimated activity regulation for 97 kinases based on the regulation of their known substrates (Ochoa et al., 2016; Hernandez-Armenta et al., 2017; Table S4), with the strongest regulation linked to viral entry (0–2 h) and late replication/egress (24 h). The kinases predicted to be most strongly activated (Figures 4B and S2A) include several members of the p38 pathway, including p38ɣ (MAPK12), CK2 (CSNK2A1/2), Ca2+/calmodulin-dependent protein kinase (CAMK2G), and the guanosine monophosphate (GMP)-dependent protein kinases PRKG1/2, which can inhibit Rho signaling. Kinases predicted to be downregulated include several cell cycle kinases (CDK1/2/5 and AURKA), cell growth-related signaling pathway kinases (PRKACA, AKT1/2, MAPK1/3, and PIM1), and the cytoskeleton regulators (PAK1), among others. Kinase activity estimates based on the 24-h mock control gave highly correlated results (r = 0.81), identifying the same set of highly regulated kinases (Figure S2D). Some of the changes in kinase activity can be directly linked to host-viral protein interactions (Figure 4C). Among the 10 interacting kinases detected in a virus-host protein-protein interaction map (Gordon et al., 2020), an increase in activity for CK2 and a decrease for MARK2 and PRKACA were observed (Figure 4C). Of note, although we predict decreased activity for PRKACA based on phosphorylation of its substrates, we simultaneously detected a significant increase in T198 phosphorylation (8, 12, and 24 h post infection) within its activation loop, suggesting an increase in PRKACA activity. It is possible that Nsp13 is sequestering active PRKACA away from its typical substrates.
Figure S2 Full Kinase Activities, Correlated Conditions, and Regulated Complexes, Related to Figure 4
(A) Changes in predicted kinase activities across different time points post-infection. (B) Correlation of kinase activity profiles of each time point with other biological conditions. Kinase activities were estimated for a wide-range of biological conditions obtained from previously published phosphoproteomics datasets (Ochoa et al., 2016). (C) Changes in phosphorylation in protein complexes. Overall phosphorylation change (-log10 (p value) of a protein complex was derived from change in phosphorylation of sites in member proteins. (D) Kinase activity estimates when using either the 0- or 24-h mock controls for those top regulated kinase activities from the 0-h control comparison.
To better understand the signaling states of cells over the course of infection, we compared our data with a compilation of public phosphoproteomics datasets of other conditions (Ochoa et al., 2016; Figures 4D and S2B). The first and last time point of infection resembled a kinase activation state induced by inhibition of mTOR, ERK, AKT, and EGFR, consistent with the estimated kinase activities of these growth-related pathways. Between 2 and 12 h after infection, kinase activity states resembling inhibition of phosphatidylinositol 3-kinase (PI3K), p70S6K, and Rho-associated protein kinases (ROCKs) were observed. Finally, several of the time points resembled S/G2 cell cycle state, suggestive of a cell cycle block. Conversely, some conditions were anticorrelated with kinase activity profiles (Figures 4D and S2B). In line with an S/G2 cell-cycle block, infection signaling appeared opposite to that of a mitotic cell. In addition, inhibitors of histone deacetylases (HDACs) (scriptaid and trichostatin A), the proteasome (bortezomib), Hsp90 (geldanamycin), and voltage-gated sodium channels (valproic acid) were also anticorrelated. These drugs, or drugs targeting these protein activities, could induce a signaling state that inhibits viral replication.
To further link kinase activities to downstream protein complexes, enrichment of up- or downregulated phosphorylation sites was determined within a curated set of human protein complexes defined by CORUM (Giurgiu et al., 2019; Figures 4E and S2C). This analysis revealed significant changes in phosphorylation of splicing related complexes (spliceosome), the proteasome (PA700-20S-PA28), and chromatin remodeling complexes (HuCHRAC and MLL2). In addition, a subset of regulated phosphorylation sites were detected that have known regulatory functions or high predicted functional scores (Ochoa et al., 2020) that are linked to regulation of protein activities (Table S5). Consistent with the observed signaling changes described above, these regulatory phosphorylation sites are involved in activation of chaperones (including HSP90), proteasome activity, inhibition of the anaphase-promoting complex (APC), and regulation of HDACs and cytoskeleton proteins, among others.