Journal: bioRxiv

Article Title: Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

doi: 10.1101/2022.10.19.512927

Figure Lengend Snippet: (A) Global phosphoproteomics map of viral protein phosphorylation sites detected during infection with at least one virus (Alpha, Beta, Gamma, Delta, Omicron BA.1, VIC, or IC19) in Calu-3 cells (black lollipops). Bold site labels indicate phosphorylation sites detected in all infections and with total protein abundance-normalized phosphorylation site intensities unchanging (p>0.001) between viruses. Gray site labels indicate phosphorylation sites detected and changing (p≤0.001) between at least one pair of VOCs (see Table S10 and Methods). Colors delineate known protein domains. (B) The absolute value t-statistic from t-tests comparing phosphorylated peptide intensities between pairs of viruses. Each dot represents one phosphopeptide compared between two viruses. Phosphorylated peptide intensities are normalized by the corresponding total protein abundance. Comparisons were restricted to viral peptides with identical sequences between virus pairs, given that peptide intensities of peptides with different sequences are not directly comparable using mass spectrometry. If p≤0.001, dots are colored dark blue, comparisons that are not significant are colored gray, comparisons that are not significant but are detected for all viruses are colored black (conserved, as in A). (C) Top five kinase groups predicted to phosphorylate S53 in Orf9b using GPS 5.0 , ranked by GPS confidence score. (D) TBK1-mediated phosphorylation of Orf9b measured using an in vitro ADP-Glo kinase time course. An increase in luminescence indicates increased peptide phosphorylation. Peptides were synthesized to reproduce the area surrounding Orf9b S53, including 7 amino acids downstream of S50 and 7 amino acids upstream of S53. Orf9b 2xAla indicates positions S50 and S53 were converted to non-phosphorylatable alanine. Orf9b 3xAla indicates positions S50, S53, and T70 were converted to alanine. Myelin basic protein was used as a positive control. TBK1 kinase only indicates the addition of kinase without any peptide substrate. (E) Cartoon schematic of proposed Orf9b-TBK1 signaling pathway. It was previously known that viral RNA is detected by host RNA sensors, which activate the MAVS-Tom70 complex and TBK1, leading to the upregulation of interferon stimulated genes. We previously reported Orf9b to bind to Tom70 and antagonize innate immune activation, but only when not phosphorylated at S53 (black lines). Here, we report TBK1 is able to phosphorylate Orf9b S53, revealing a negative feedback loop between Orf9b and TBK1. (F) Significantly changed phosphorylation sites (p≤0.001) on N protein between pairs of viruses. Length of each lollipop depicts the abs(t-statistic) between the pairs of viruses. (G) Heatmap of significantly changed phosphorylated peptides (p≤0.001) mapping to N protein depicted in (F) relative to Beta and Omicron (see for full heatmap). Rows indicate the comparison (first virus is numerator, second is denominator) and columns indicate the phosphorylated peptides, and residues, on N. Color represents the t-statistic and black bounding boxes indicate p≤0.001. Gray boxes indicate peptides that were either not detected in one or both viruses or did not possess identical sequences between viruses. Numbers next to viruses indicate experiment 1 or 2.

Article Snippet: ADP-Glo kinase reactions (Promega #V6930) were carried out per the manufacturer’s instructions using 10 nM TBK1 kinase (Promega #V3991) and 10 μM peptide substrate with 100 μM ATP.

Techniques: Infection, Mass Spectrometry, IF-P, In Vitro, Synthesized, Positive Control, Activation Assay