Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.

. Variation in phosphorylation status upon BTV infection. Scatter plots show the normalized log2 fold change upon BTV infection against log10 pvalue. Phosphosites which were significantly altered in abundance by t-test (p<0.05) are highlighted in green. The scatter plots represent phosphosites at A) 12h post-infection, B) 18h post-infection and C) 18h post-infection compared with 12h post-infection.

Figure S4. Identification of sequence motifs regulated by BTV infection.
Phosphosites showing a significant (t-test, p<0.05) increase or decrease in the abundance were submitted to the weblogo software for preparation of sequence logos. Data from A) 12h post-infection, B) 18h post-infection and C) 18h compared to 12h post-infection with BTV is shown. Figure S5. Inhibition of PKA reduces BTV replication while further stimulation of PKA enhances BTV replication. HeLa and sheep PT cells infected with BTV1 (MOI=5) were treated 1 h.p.i with DMSO, 40 µM H89 or 1 mM Dibutyryl-cAMP and harvested 12 h.p.i (A) and 18 h.p.i (B). Samples were analyzed by western blot with antibodies against the indicated antigens. Brackets at the side of the phosphorylated PKA substrate western blot denote region used for densitometry analysis.

Table S1. Phosphoproteins identified from BTV-infected HeLa cells.
Phosphoproteins identified from BTV-infected HeLa cells. A phosphoprotein was considered to be regulated by BTV infection if at least one of its corresponding phosphosites showed regulation. Only high confidence phosphosites identified in at least 2 replicates, with a localization score of ≥0.75 were used for this analysis. Phosphoproteins identified as regulated overall, or at a given timepoint are highlighted (t-test, p<0.05).

Table S2. Phosphopeptides identified from BTV-infected HeLa cells.
This table represents all the phosphopeptides identified within this experiment following removal of contaminants and reverse database hits. The first tab holds redundant information on all the phosphopeptides identified within each experiment and enrichment condition. The second tab holds non-redundant data on all the high confidence phosphopeptides. Phosphopeptides identified as regulated by BTV infection overall, or at a given time point are highlighted (t-test, p<0.05). Visualisation of all spectra is possible by download of PRIDE dataset PXD005550 and visualization in Maxquant version 1.5.7.4. For ease of viewing several columns are by default hidden which can be examined to reveal data on the ratio variability, and t-test difference and p-values for each peptide.

Table S3. Phosphosites identified from BTV-infected HeLa cells.
High confidence phosphosites, possessing a localization probability of >0.75, and identified in at least two of three replicates are presented. Phosphosites identified as regulated by BTV infection overall, or at a given time point are highlighted (ttest, p<0.05). Table S4. Differentially regulated phosphoproteins, phosphopeptides and phosphosites identified from BTV-infected HeLa cells. Phosphoproteins, phosphopeptides and phosphosites identified as differentially regulated by BTV infection are shown on separate tabs. A phosphoprotein was considered to be regulated if at least one phosphosite corresponding to this protein showed regulation. The relevant regulated phosphosites are show beneath each protein on the phosphoprotein tab. High confidence phosphosites, possessing a localization probability of >0.75, and identified in at least two of three replicates are presented. Phosphosites identified as regulated by BTV infection overall, or at a given time point are highlighted. In all cases regulation by BTV was determined by t-test (p<0.05). Table S5. Gene ontology analysis of differentially regulated phosphoproteins from BTV-infected HeLa cells. Analysis of differentially regulated phosphoproteins (Table S1) was performed with STRING 10.5 (https://string-db.org). A phosphoprotein was considered to be significantly regulated if at least one phosphosite corresponding to that phosphoprotein showed significantly altered abundance (t-test, p<0.05) Analyses corresponding to GO molecular function, GO biological process, GO cellular component and KEGG are given on separate tags. Table S6. Motif-X analysis of phosphosites significantly regulated during BTV infection. The Motif-X software was used to identify regulation of particular phosphorylation motifs by BTV infection. Those phosphosites showing significantly altered abundance by t-test (p<0.05) were input into the Motif-X software to look for overrepresentation compared to the human proteome at large. Fold-increase represents the degree of this overrepresentation. Different tabs hold data representing sequence motifs over-represented at 12h, 18h and 18h compared to 12h post-infection with BTV. A futher 'Total dataset' tab shows motifs identified from the whole high confidence phosphosite dataset (table S3) without prior selection of significantly altered sites. Table S7. Phoxtrack analysis to identify kinase regulation during BTVinfection. The Phoxtrack software was used to characterize kinase regulation at 12h, 18h and 18h compared to 12h post-infection with BTV. Both kinase regulation, and the annotation of individual phosphosites to the relevant kinase is shown. These data are presented on separate tabs within the file. On the kinase tables the columns correspond to the kinase of interest, database the kinase data was obtained from, enrichment and normalized enrichment values, p-value, false discovery rate and the Phoxtrack score which combines the directionality of the NEV with the FDR. One the phosphosite tabs, the experimental value represents the log2 fold-change observed with this phosphosite, and core-enrichment refers to whether a individual phosphosite was a primary contributor to a individual kinase score.