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To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins—and their distribution on virus particles—is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection. Super-resolution microscopy and single-particle analysis reveal that vaccinia virus membrane proteins are organized into functional domains whose polarization on the surface of mature virus particles is required for viral entry and virus–cell fusion.

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.

Introductory paragraph Cell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV induced cell motility, indicating that additional viral factors must be involved. Here we show that the VACV epidermal growth factor homolog, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10 whereupon it acts largely in a paracrine fashion to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR/MAPK/FAK/ADAM10 inhibitors, or with VGF and F11 deleted viruses, revealed defects in radial velocity and directional migration efficiency leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins