During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia.
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The authors thank S. Snapper (Harvard Medical School) and C. Brakebusch (University of Copenhagen) for providing N-WASP and Cdc42 deficient cells, respectively. The authors also thank N. O'Reilly (Francis Crick Institute) for synthesizing peptides, M. Matsuda (Kyoto University) for the iRFP670 clone and S. Caplan (University of Nebraska Medical Center) for GFP-tagged EHD1–4 clones. The authors acknowledge members of the Way Laboratory and H. Walden (University of Dundee) and D. Stephens (University of Bristol) for comments on the manuscript. Research was supported by Cancer Research UK and the Francis Crick Institute.
The authors declare no competing financial interests.
Supplementary Figures 1-5, Supplementary Video 1-4 legends (PDF 232610 kb)
Intersectin-1 and Eps15 are simultaneously recruited to CEV inducing actin tails (AVI 1171 kb)
NPF motifs are required for viral recruitment of intersectin-1 (AVI 816 kb)
NPF motifs are required for viral recruitment of Eps15 (AVI 796 kb)
Loss of all three A36 NPF motifs results in significantly longer actin tails and a faster velocity of the virus (AVI 3238 kb)
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Snetkov, X., Weisswange, I., Pfanzelter, J. et al. NPF motifs in the vaccinia virus protein A36 recruit intersectin-1 to promote Cdc42:N-WASP-mediated viral release from infected cells. Nat Microbiol 1, 16141 (2016). https://doi.org/10.1038/nmicrobiol.2016.141
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