To orchestrate context-dependent signalling programmes, poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signalling mediators are essential for poxvirus production, yet their substrate profiles and systems-level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wild-type, F10 and H1 mutant vaccinia viruses, we systematically defined the viral signalling 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 harbour a hidden hypercleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phosphoproteomic profiling 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 proteotype–phenotype–genotype relationships that are masked by classical genetic studies.
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We would like to thank P. Traktman, R. Condit, B. Moss and P. H. Verardi for generously donating VACV mutants for this study. We greatly acknowledge A. Frei, S. Götze and A. Leitner for maintenance of the mass spectrometers. We are grateful to all members of the Mercer and Wollscheid laboratories for critical comments and suggestions throughout this project. This work was supported by the Swiss National Science Foundation (31003A_160259 to B.W.) and the InfectX project from the Swiss Initiative in Systems Biology SystemsX.ch (to B.W.), the MRC Programme Grant (MC_UU12018/7) (J.M.), the European Research Council (649101 UbiProPox) (J.M.) and the Swiss National Foundation Ambizione (PZ00P3_131988) (J.M.).
J.V. is an employee of Biognosys AG and helped with the Spectronaut DIA analysis in the revised version of the manuscript. K.N. joined Biognosys AG during the revision process of the manuscript upon finishing his PhD at ETH Zurich.
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Supplementary Figures 1–8, Supplementary Reference.
Label-free quantification phosphoproteome data of HeLa cells infected with VACV WT, H1 and F10, and uninfected cells.
Label-free quantification of MV WT versus H1 proteomes.
Label-free quantification phosphoproteome data of MV WT versus H1.
Label-free quantification data of MV WT versus H1 phosphotyrosine enrichment.
Primers used in this study.
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Novy, K., Kilcher, S., Omasits, U. et al. Proteotype profiling unmasks a viral signalling network essential for poxvirus assembly and transcriptional competence. Nat Microbiol 3, 588–599 (2018). https://doi.org/10.1038/s41564-018-0142-6