VACV strain WR F12 (WRF12) was aligned with human KLCs 1 and 2 (HsKCL1, HsKCL2). Identical residues are boxed and conserved residues are shaded. Vertical bars and arrows indicate the positions of HsKLC TPRs. The red asterisk above the alignment denotes the conserved aromatic residues in TPRs. The F12 WD motif at aa residues 534 to 538 is underlined and residues 351–458 (blue) have been shown to bind A36 [56]. Accession numbers are WR F12 (AAO89330), HsKLC1 (AA01263), and HsKLC2 (Q9H0B6).

The TPRs of A36, E2 and F12 were aligned using Clustal-X and are displayed using the Clustal colour scheme (http://www.jalview.org/help/html/colourSchemes/clustal.html). The amino acid co-ordinates of each TRP domain are listed on the right. The TPR consensus sequence is shown above. Accession numbers are WR F12 (AAO89330), WR E2 (YP_232940) and WR A36 (YP_233041).

The three-dimensional structure of the KLC2 TPRs (A) compares favourably with the model of the F12 TPR domains 7–12 (B) illustrated by an overlay of the two structures (C). (D) KLC2 TPR 1 is overlaid with F12 TPR 6 illustrating the spatial conservation of key hydrophobic aa side-chains responsible for holding together the two alpha helices that comprise a single TPR domain.

(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.

Cryoimmunoelectron microscopy of ultrathin sections of HeLa cells infected with VACV WR (top) or vΔF12L (bottom) and stained for B5, A36 or KLC with mAbs followed by 6-nm gold particles. Note B5 and A36 are present on VACV WR and vΔF12L IEV, but KLC is recruited to VACV WR IEV (arrows) but not to vΔF12L IEV. Scale bar = 100 nm.

HeLa cells were infected with vEGFPA5L (top) or vΔF12-EGFPA5L (bottom) at 5 pfu/cell for 12 h. Cells were then fixed, permeabilised and stained for KHC (red) and visualized for EGFP (green). The merged image shows KHC recruitment to IEV particles at the periphery of the cell in vEGFPA5L-infected cells. Note KHC recruitment to IEV particles does not occur in cells infected vΔF12-EGFPA5L. Scale bar = 10 µm.

HeLa cells were either mock-infected or infected at 5 pfu/cell with vEGFPA5L or vΔF12-EGFPA5L. At 12 h p.i. cells were fixed, permeabilised and stained for KLC (red), DAPI (blue) or visualized for EGFP (green). The merged image is shown on the right. Note KLC is recruited to IEV particles at the periphery of the cell in vEGFPA5L-infected cells, but not in cells infected with vΔF12-EGFPA5L. Scale bar = 10 µm.

HeLa cells were infected at 5 pfu/cell with WR (A, D, G, J), vΔF12L (ΔF12) (B, E, H, K) or vVAC-BACΔE2L (ΔE2) (C, F, I, L) for 12 h. Cells were then processed for thin 70-nm serial-section analysis using conventional electron microscopy. Images show virions at different stages of morphogenesis: (A–C) IMV association with wrapping membranes, (D–F, K and L) complete IEV, (G–I) complete IEV in which part of the wrapping membrane is not tightly associated with the virions, and (J) CEV/EEV formed by WR. Micrographs A–J & L are shown at the same magnification. Scale bars = 200 nM.

HeLa cells were infected at 5 pfu/cell with WR, vΔF12L or vVAC-BACΔE2L for 12 h. Cells were then fixed and processed for conventional electron microscopy. Viral particles (n = 100) were counted in thin sections and the percentage of IMV, partially wrapped IEV, completely wrapped IEV and CEV were determined and are expressed as a percentage of total virions. Note: i) loss of F12 and E2 caused no difference in the number of partially wrapped IEV relative to wild type but there were three times as many IEV in cells infected with deletion viruses; ii) No CEV were seen for the deletion mutants but the sum of IEV/CEV were equivalent for mutants compared to wild type.

Cells were infected and processed for thin 90-nm serial section electron microscopy as described in Figure 8. Serial section analysis of vΔF12L (ΔF12, A, E, I, M) and or vVAC-BACΔE2L (ΔE2, C, G, K, O). Tilt series analysis of vΔF12L (B, F, J, N) (derived from a different IEV) or vΔ vVAC-BACΔE2L (D, H, L, P) IEV tilted the indicated degree in the electron beam. For vVAC-BACΔE2L, the section shown in (G) was tilted through 20° increments. Note the continuity of the double membrane forming the IEV particles. All micrographs are shown at the same magnification. Bar, 200 nm.

(A) Alignment of the WD kinesin-1 binding site in F12 orthologues in chordopoxviruses with the consensus (L-E-W-E-E/D-S-I) sequence shown below. (B) Alignment of the WD kinesin-binding site in other protein reported to bind KLC with the consensus (L-E/D-W-D-D-S-A) sequence shown below. Overall these sequences suggest the WD KBS conforms to the sequence L-E/D-W/ϕ-D/E-D/E-S/N (Where ϕ is another aromatic residue). The Human 14-3-3 protein and calsyntenin-1 and calsyntenin-2 contain two WD motifs shown as A and B in the alignment. Accession numbers are VACV WR F12 (AAO89330), Lister F12 (DQ121394.1) Myxoma virus (MYXV: NP_051735), Yaba-like disease virus (YLDV: NP_073411), Swinepox virus (SWPV: NP_570184.1), Molluscum contagiosum virus (MOCV: MC019L), Goatpox virus (GTPV: YP_001293217.1), Bovine papular stomatitis virus (BPSV: NP_957919.1), Yaba monkey tumor virus (YMTV: NP_938282.1), Orf virus (ORFV: AAR98105.1), Canarypox virus (CNPV: NP_955159.1), Fowlpox virus (FPV: NP_039072.1), VACV WR A36 (YP_233041), Human collapsin response mediator protein-2 (HsCRMP-2, Q16555), Human calsyntenin-1 (HsCal1: CLSTN1), Human calsyntenin-2 (HsCal2: CLSTN2), Human calsyntenin-3 (HsCal3: CLSTN3), Human 14-3-3 protein (Hs14-3-3: BAG35533), Human caytaxin (HsCaytaxin: NM_033064) and Human amyloid precursor protein (HsAPP: 351 APP).

(A) HeLa cells were infected at 5 pfu/cell with vΔF12L-EGFPA5L for 2 h. Cells were then transfected with plasmids expressing WT or mutant F12-HA, and 10 h later were stained with mAbs against HA (blue) or KHC (red), or visualized for EGFP. The overlay is shown in the Merge image. In cells transfected with F12-WT-HA the particles are widely distributed and can recruit both F12-HA and KHC (white in merged image). In cells transfected with the mutant F12-AAA-HA the particles remain centrally localized, and are unable to recruit either F12-HA or KHC. (B) Cells were infected and transfected as in (A) and then stained for HA (blue), and phalloidin (red) to identify actin tails. Top panels shows F12-WT-HA is targeted to widely distributed particles and actin tails are formed at the plasma membrane. Note virions on actin tails are negative for F12L-HA. Bottom panel show absence of F12 recruitment and lack of actin tail formation. Insets show ×2.5 enlargements of the boxed regions. Scale bar = 10 µm.

Quantification of actin tail formation by analysis of data shown in Figures 12 and S8. Data are the means +/− SEM of triplicate experiments. Note mutation of F12 WD motif inhibits actin tail formation, whereas mutation of the A36 WD motif does not.