Redistribution of A36 during virus exit.

<p>(<b>A</b>) 3D-SIM fluorescent micrograph of A36-YFP/B5-RFP infected BSC-1 cell. Virus particles associated with F-actin (red, left panel; visualised with Lifeact-cerulean) show polarisation of A36 (green) whereas B5 (red, right panel) localises to the CEV circumference (close-ups). 3D-SIM fluorescent micrographs of (<b>B</b>) A36-YFP, (<b>C</b>) A36^YdF-YFP in non-permeabilised BSC-1 cells probed with rat anti-B5 primary antibody and Alexafluor 568 anti-rat secondary antibody. Close-ups show A36 (green) localisation in two representative intracellular (no B5 visible) and extracellular virus particles. (<b>D</b>) 3D-SIM fluorescent micrographs of representative intracellular and extracellular B5^P189S/A36-YFP, B5^P189S/A36^YdF-YFP, A34^K151E/A36-YFP or A34^K151E/A36^YdF-YFP viruses in non-permeabilised BSC-1 cells probed with rat anti-B5 primary antibody and Alexafluor 568 anti-rat secondary antibody. All close-ups from larger panels are arranged in order from top to bottom. Scale bar = 5 µm.</p

Plaque size and release of VACV is inhibited by mutation of Y112 and Y132 residues in A36.

<p>(<b>A</b>) BSC-1 cells were infected with the indicated viruses, incubated with a semi-solid (plaque assay, 1.5% CMC) or liquid (comet assay, DMEM) overlay, with either imatinib or carrier (DMSO), stained at 72 hpi. (<b>B</b>) Average plaque size of WR, A36^Y112F, A36^Y132F and A36^YdF in BSC-1 or (<b>C</b>) NIH 3T3 cells. Error bars represent s.e.m. from 50 plaques. (<b>D</b>) Single-step growth analysis of VACV strains. Monolayers of BSC-1 cells were infected with the indicated viruses at a MOI of 5. At 4, 8, 12 and 24 hpi cells and supernatants were harvested and virus titres were determined by titration with BSC-1s. The average titres of two independent experiments are plotted at each time point. (<b>E</b>) Levels of infectious EEV in supernatants collected 16 hpi from BSC-1 cells or (<b>F</b>) NIH3T3 cells infected at an MOI of 0.1 and overlayed with DMEM containing 0.01% DMSO (unfilled) or 10 µM imatinib (filled). Error bars represent s.e.m. from 3 replicate wells in 3 independent experiments. P values of <0.05, <0.01 and <0.001 are represented by *, ** and ***, respectively.</p

A second site mutation in B5 restores EEV release in A36^YdF.

<p>(<b>A</b>) Plaque (semi-solid overlay) and comet (liquid overlay) assays of A36^YdF, B5^P189S, B5^P189S/A36^YdF, A34^K151E or A34^K151E/A36^YdF stained at 72 hpi. (<b>B</b>) Levels of infectious EEV in supernatants collected at 16 hpi from BSC-1 cells infected at an MOI of 0.1. Error bars represent s.e.m. from 3 replicate wells in 3 independent experiments. P values of <0.05 and <0.01 are represented by * and **, respectively.</p

A36^YdF is invaginated at the cell membrane during egress.

<p>Representative transmission electron micrographs of BSC-1 cells infected with (<b>A</b>) WR and (<b>B</b>) A36^YdF at an MOI of 5 and fixed at 9 hpi. In contrast to WR, distinct membrane pits containing virus are apparent during A36^YdF virus egress. Scale bar = 0.2 µm. (<b>C</b>) Average percentage of viral membrane circumference in contact with cell membrane calculated from 14 (WR) or 16 (YdF) viruses randomly selected from the transmission electron micrographs. *** = P value of <0.001.</p

Actin polymerisation is required for efficient EEV release.

<p>Infectious EEV levels in supernatant collected at 16 hpi from (<b>A</b>) Nck-null cells infected with WR, A36^YdF B5^P189S, B5^P189S/A36^YdF, A34^K151E or A34^K151E/A36^YdF at an MOI of 0.1; (<b>B</b>) BSC-1 cells infected with WR, A36^YdF, B5^P189S or A34^K151E at an MOI of 0.1 and overlayed with DMEM containing 0.01% DMSO (unfilled) or 0.1 µg/ml Cyt D (filled). Error bars represent s.e.m. from 3 replicate wells in 3 independent experiments. P values of <0.01 and <0.001 are represented by ** and ***, respectively. (<b>C</b>) Fluorescent micrographs of HeLa cells infected with WR, A36^Y112F and A36^YdF. Robust F-actin comets are associated with WR and localised accumulation of actin is observed sporadically with A36^Y112F (insets). Extracellular virus is detected with B5 antibody (red, non-permeabilised cells) and actin with phalloidin (green). Scale bar = 10 µm. (<b>D</b>) Confocal micrographs of HeLa cells infected with B5-YFP, A36^Y112F/B5-YFP and A36^YdF/B5-YFP and expressing Lifeact-mRFP. Distinct F-actin comets are associated with both B5-YFP and A36^Y112F/B5-YFP, while colocalisation between F-actin and A36^YdF/B5-YFP occurs stochastically as viruses move over previously existent F-actin structures. Scale bar = 3 µm.</p