Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion

Abstract

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.

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Fig. 1: VACV binding and fusion show orientation bias that reflects distinct binding and fusion domains on virions.
Fig. 2: VACV binding and fusion proteins are organized into nanoscale clusters.
Fig. 3: A27 regulates the protein architecture of MV membranes.
Fig. 4: VACV fusion machinery polarization is required for full-fusion efficiency.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding authors on reasonable request.

Code availability

All custom code used for analysis in the current study is available on GitHub at https://github.com/HenriquesLab/fusiontools.

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Acknowledgements

We thank B. Moss and M. Esteban for providing the mutant viruses that were used in this study, and M. Turmaine (UCL Biosciences EM facility) and A. Weston (UCL School of Pharmacy—Electron Microscopy Unit) for use of their coater and SEM, respectively. We also acknowledge Eisenberg members J. C. Whitbeck, C. H. Foo and L. Aldaz-Carrol, of the poxvirus research group, for their help producing and purifying the VACV EFC antibodies. This work was funded by MRC Programme grant (MC_UU_00012/7; to J.M.), the European Research Council (649101, UbiProPox; to J.M.), the MRC (MR/K015826/1; to J.M. and R.H.), Biotechnology and Biological Sciences Research Council (BB/M022374/1, BB/P027431/1 and BB/R000697/1; to R.H.) and the Wellcome Trust (203276/Z/16/Z; to R.H.). R.D.M.G. is funded by the Engineering and Physical Sciences Research Council (EP/M506448/1). D.A. is a Marie Skłodowska-Curie fellow funded by the European Union (750673). C.B. is funded by the MRC LMCB PhD program.

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R.D.M.G., D.A., R.H. and J.M. conceived the project, designed the experiments and wrote the manuscript. R.D.M.G., D.A., C.B. and M.H. performed the experiments. G.H.C. helped to produce and purify all of the VACV EFC antibodies. I.J.W. and J.J.B. performed the EM. R.D.M.G., D.A. R.H and J.M analysed the data. R.D.M.G., D.A., R.H. and J.M. discussed the results and implications of the findings. All authors discussed the manuscript and provided comments.

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Correspondence to Ricardo Henriques or Jason Mercer.

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Gray, R.D.M., Albrecht, D., Beerli, C. et al. Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion. Nat Microbiol 4, 1636–1644 (2019). https://doi.org/10.1038/s41564-019-0488-4

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