Nat. Microbiol. 3, 588–599 (2018)

Vaccinia virus (VACV) is an enveloped virus containing ~80 different viral proteins. Several VACV enzymes, including F10 kinase, I7 protease, and H1 phosphatase, are required for proper viral assembly. To create a comprehensive network of functional viral phosphorylation events, Novy et al. used quantitative mass spectrometry-based proteotype analysis to define the role of F10 and H1 phosphorylation of viral proteins. In infected cells, the authors identified 43 phosphorylated proteins, 10 of which are shared F10/H1 substrates, and 9 of these are essential for VACV assembly. Comparing wild-type and H1-deficient virions led them to focus on two H1 substrates, F10 and I7, demonstrating that phosphorylation of F10 itself does not impact virus assembly, while dynamic phosphorylation of I7 S134 by F10 is required for I7 protease activity. F10 phosphorylation was required to regulate I7-mediated viral protein processing during both the initial viral membrane formation and the transition from immature to mature virions. Additionally, the authors showed that the transcription defect of H1-deficient mature virions is likely due to hyperphosphorylation of the transcription factor A7. These results uncover a key viral signaling network that controls assembly of infectious poxvirus particles and highlight the power of combining quantitative proteotype analysis with mutant viruses to unmask proteotype–phenotype–genotype relationships.