To determine how SARS-CoV-2 hijacks host-protein signaling, a global phosphoproteomics experiment was performed in Vero E6 cells, a cell line originating from the kidney of a female African green monkey (Chlorocebus sabaeus) (Osada et al., 2014). This cell line was selected because of its high susceptibility to SARS-CoV-2 infection (Harcourt et al., 2020). Cells were harvested in biological triplicate at 6 time points after SARS-CoV-2 infection (0, 2, 4, 8, 12, or 24 h) or after mock infection at 0 or 24 h (Figure 1 A). Using a data-independent acquisition (DIA) proteomics approach, each sample was then partitioned and analyzed for changes in global protein abundance or phosphorylation (data available in Table S1). Chlorocebus sabaeus and human protein sequences were aligned, and phosphorylation sites and protein identifiers were mapped to their respective human protein orthologs. Phosphorylation fold changes calculated using the 0- or 24-h mock control were highly comparable (correlation coefficient r = 0.77); therefore, the 0-h mock control was used for all subsequent comparisons.
Figure 1 Global Proteomics of Phosphorylation and Abundance Changes upon SARS-CoV-2 Infection
(A) Vero E6 cells were infected with SARS-CoV-2 (MOI 1.0). After 1 h of viral uptake, cells were harvested (0 h) or, subsequently, after 2, 4, 8, 12, or 24 h. As a control, Vero E6 cells were also mock infected for 1 h and harvested immediately thereafter (0 h) or after 24 h of mock infection. All conditions were performed in biological triplicate. Following cell harvest, cells were lysed, and proteins were digested into peptides. Aliquots of all samples were analyzed by mass spectrometry (MS) to measure changes in protein abundance upon infection, whereas the remaining sample was enriched for phosphorylated peptides and subsequently analyzed to measure changes in phosphorylation signaling. A DIA approach was used for all MS acquisitions. Last, all phosphorylation sites and protein identifiers were mapped to their respective human protein orthologs.
(B) Principal-component analysis (PCA) of phosphorylation replicates after removing outliers. See also Figure S1.
(C) Correlation of protein abundance (AB) and phosphorylation sites (PHs) between replicates within a biological condition (red) and across biological conditions (black). Boxplots depict median (horizonal lines), interquartile range (boxes), maximum and minimum values (vertical lines), and outliers (solid circles).
(D) Median AB of individual SARS-CoV-2 proteins in the protein AB analysis.
(E) The number of significantly regulated PH groups across the infection time course.
(F) Volcano plot of PH group quantification 24 h after infection.
(G) The number of significantly regulated proteins across the infection time course.
(H) Volcano plot of protein AB quantification 24 h after infection.
(I) Gene Ontology enrichment analysis of all significantly changing proteins in terms of AB divided into two sets: downregulated (blue) and upregulated (red).
(J) Proportion of significantly regulated PH groups with a correlated (i.e., same direction, AB match) or anticorrelated (i.e., opposite direction, AB mismatch) significant or insignificant (gray) change in protein AB.
In (E)–(H), all infection time points are compared with the mock infection at 0 h, and significantly regulated proteins are defined as (absolute value of log2(infection/mock) > 1 and adjusted p < 0.05 or when only detected in infected or mock based on replicate and MS feature counts; STAR Methods).
See also Figure S1.