Proteotype profiling unmasks a viral signaling network essential for poxvirus assembly and transcriptional competence

To orchestrate context-dependent signaling programs poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signaling mediators are essential for poxvirus production, yet their substrate profiles and systems level functions remain enigmatic. Using a phospho-proteomic screen of cells infected with wildtype, F10, and H1 mutant viruses we systematically defined the viral signaling network controlled by these enzymes. Quantitative cross comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships we found that H1-deficient virions harbor a hidden hyper-cleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phospho-proteotyping further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutant virions. Together these results highlight the utility of combining quantitative proteotype screens with mutant viruses to uncover novel proteotype-phenotype-genotype relationships that are masked by classical genetic studies.