Vaccinia Protein F12 Has Structural Similarity to Kinesin Light Chain and Contains a Motor Binding Motif Required for Virion Export
Figure 4
Full length F12 is required for IEV transport.
(A) Illustration of HA-tagged F12 mutant proteins analysed. (B) HeLa cells were infected with vΔF12L at 5 pfu/cell and were transfected with plasmids encoding F12-HA at 2 h post infection. After 10 h cells were lysed and lysates were immunoblotted with anti-HA mAb. The blot was stripped and reprobed with anti-D8 mAb (a VACV protein) to confirm the equivalence of loading. (C, D) Rescue assay demonstrating that the full length F12-HA protein is required for the formation of cell surface virions and actin tails. HeLa cells were infected with vΔF12L-EGFPA5L at 5 pfu/cell for 2 h and then were transfected with plasmids expressing F12 for 10 h. Cells were stained with anti-B5 mAb (red) prior to fixation and staining with anti-HA mAb (cyan) and phalloidin (green). Inset shows B5-positive virus-tipped actin tails. Loss of surface B5 labelling and actin tail formation in cells transfected with the F12-HA truncations demonstrated virions have not reached the cell surface. Actin tails were counted and data were expressed as the percentage of actin tails formed by each mutant compared to WT. Data are from a single experiment that is representative of three. (E) A36 is localised to vΔF12L IEV. Cells infected as in (C) were permeabilised and stained for A36 (red) or visualized for EGFP. The merged image shows A36-positive IEV particles (arrows). Scale bars C = 10 µm, E = 5 µm.