Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility

Abstract

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 homologue, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10, after which it acts largely in a paracrine manner to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR, MAPK, FAK and ADAM10 inhibitors or with VGF-deleted 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.

Figure 1. VGF is required for VACV-induced cell motility and virus spread.

a, Plaque formation by VACV WR and mutants (ΔVGF, ΔF11 and ΔVGF/ΔF11). Nuclei (red) and infected cells (green). b, c, 24 h MV and EEV yields from WR and mutant VACVs. d, e, CEVs and actin tails per cell during WR and mutant VACV infections. f, Representative images of cells infected with WR or VACV mutants for 10 h. g, Single cell tracking of WR and mutant VACV plaque formation (20 - 48 hpi). Tracks are colour-coded by time (hpi). h,i, The radial velocity and directional migration efficiency of cells migrating from the centre of plaques in g. Data represents 3 or more biological replicates (a-i). Images are representative of 3 biological replicates (a, f, g). Scale bars are 500 μm (a, g) or 20 μm (f). Bars represent means + SD (b, c), or means + SEM of n=5 plaques per condition/replicate (h, i). Lines represent means of 15-20 cells per condition/replicate (d, e). Paired (b, c) or unpaired (d, e, h, i) t-test was applied (**** P< 0.0001, ** P< 0.01, * P< 0.05, ns = not significant). See Supplementary Table 1 for exact statistics.

Figure 2. VGF activates cell motility through EGFR/MAPK/FAK signalling.

a-c, Immunoblot analysis of EGFR, MAPK and FAK phosphorylation during WR and ΔVGF infections. d, Plaque formation in the presence of VGF signalling inhibitors. e, Diameter of plaques from d. f, Single cell tracking of VACV plaque formation in the presence of EGFR, MEK or FAK inhibitors (24 - 48 hpi). Tracks are colour-coded by hpi. g, h, The radial velocity and directional migration efficiency of cells migrating from the centre of plaques in f. Data represent 3 or more biological replicates (a-h). Images are representative of 3 biological replicates (a-d, f). Lines represent means of 100 plaques per condition/replicate (e). Scale bar=500 μm (f). Bars represent means + SEM of n=5 plaques per condition/replicate (g, h). Unpaired t-test was applied (**** P< 0.0001). See Supplementary Table 1 for exact statistics.

Figure 3. ADAM10-mediated VGF release triggers cell motility in a paracrine fashion.

a, Analysis of directional motility by live cell imaging, single cell tracking and vector field analysis during plaque formation adjacent to wounds. b, Spatial analysis of VGF expression within a plaque using immunofluorescence. c, Supernatant and cell transfer assays to investigate paracrine and juxtacrine mediated activation of EGFR signalling by VGF. d, Metalloprotease inhibitors of ADAM10 (GI) and ADAM10/17 (GW), prevent VGF shedding and VGF paracrine signalling activity. e, RNAi-mediated silencing of ADAM10, but not ADAM17, prevents VGF shedding and VGF paracrine signalling activity. f, Single cell tracking of VACV plaque formation in the presence of ADAM10 inhibitor (GI) (24 - 48 hpi). Tracks are colour-coded by hpi. g, h, The radial velocity and directional migration efficiency of cells migrating from the centre of plaques in f. i, j, 24 h MV and EEV yields from DMSO- or GI-treated WR infected cells. k, l, CEVs and actin tails per cell during WR infections in the presence of GI for 10 h. Data represents 3 or more biological replicates (a-l). Images are representative of 3 biological replicates (a-f). Scale bars=500 μm (b, f). Bars represent means + SD (i, j), or means + SEM of n=5 plaques per condition/replicate (g, h). Lines represent means of 15-20 cells per condition/replicate (k, l). Paired (i, j) or unpaired (g, h, k, l) t-test was applied (**** P< 0.0001, ns = not significant). See Supplementary Table 1 for exact statistics.

Figure 4. VGF is required for lesion formation in vivo.

a, Multiphoton microscopy of WR and ΔVGF lesions in mice ear pinnae 6 days pi. Infected cells (green), collagen (blue), blood vessels (red). b, Confocal imaging of WR and ΔVGF lesions in cross section. Infected cells (green), nuclei (blue) c, d Quantification of lesion widths and depths from a and b, respectively. e, Model of VGF mediated VACV induced cell motility (refer to text for details). Representative data from 2 mice/virus in biological triplicates (a. b). Lines represent means of 10-15 lesions per condition from 5 mice/virus (3 cross-section, 2 frontal sections). Unpaired t-test was applied (**** P< 0.0001, ** P< 0.01) (c, d). See Supplementary Table 1 for exact statistics.