Endocytosis assay of M78 mutants.

<p>Hela cells were transfected with plasmids expressing HA-tagged M78, either wild type (wt), C-terminally truncated (CΔ6—CΔ155) or with mutations of the acidic cluster (AC) or the acidic dileucine motif (DD-AA or LL-AA). One day post-transfection, cells were analysed for endocytosis via antibody feeding (rabbit anti-HA, 1hr), then fixed and processed for immunofluorescence either before (AF⁵⁹⁴ conjugated anti-rabbit) or after (AF⁴⁸⁸ conjugated anti-rabbit) permeabilisation. Panels A and B show results for two independent experiments. Coverslips were mounted and images of random fields captured using the 594 and 488 fluorescence filters. Images were analysed using ImageJ software to determine the fluorescence intensity of both the 594 and 488 channels for each transfected cell (>50 cells analysed for each group). The endocytosis index (EI) is plotted as the ratio of intensities (488/594), expressed as log₁₀ values. Box and whiskers plots of the endocytosis index are shown, with bars indicating the group median and whiskers the 5^th and 95^th percentiles. Asterisks indicate statistical significance (Kruskal-Wallis, with Dunn’s post-test) comparing mutants with wild type M78 (***<i>P<</i>0.001). The various constructs and effects upon endocytosis are depicted schematically in Panel C. Solid shading indicates a median EI >2, hatched shading indicates 2>EI>1, and stippled shading EI<1.</p

The M78 cytoplasmic C-tail is required for rapid, constitutive endocytosis.

<p>Hela cells were transfected with plasmids expressing HA-tagged M78, either wild type (wt) or C-terminally truncated (CΔ155). 1 day post-transfection, cells were fed primary rabbit anti-HA antibody (1hr, 37degs) then stained with secondary anti-rabbit IgG either before (pre-perm. AF⁵⁹⁴ conjugate) or after (post-perm. AF⁴⁸⁸ conjugate) permeabilisation. Confocal images are shown for AF⁵⁹⁴ (red), AF⁴⁸⁸ (green) or the merged channels.</p

Effects of M78 mutation upon replication kinetics in different cell types.

<p>Multi-step growth curves of wild type (wt), M78_null and M78_ CΔ155 in fibroblast (MEF), endothelial (SVEC), epithelial (NMuMG) and macrophage (BMM) cell types. Cells were infected in 24 well trays at low multiplicity (0.01 pfu/cell) and supernatant harvested at various times post-infection for titration of infectious virus by plaque assay (mean and standard error shown; n = 3). Statistical significance was determined via 2-way ANOVA with Bonferroni post-test and is reported in the text.</p

Western blot detection of mutated M78 constructs.

<p>Panel A shows a schematic depiction of M78 with boxes indicating positions of the seven predicted transmembrane domains (grey) and putative 8^th membrane-associated helix (white); positions of the various truncation mutants are also shown. Secondary structure predictions made via the PredictProtein server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165066#pone.0165066.ref032" target="_blank">32</a>].</p> <p>Panel B shows the M78 C-tail amino acid sequence, numbering beneath indicates amino acid position. Vertical lines, with their designations above, indicate positions of truncations. Positions of amino acid substitutions are underlined, namely of the acidic cluster (DEDDDD) and the acidic di-leucine (DDvsaLL). The position corresponding to the SalI site used for certain plasmid constructs is boxed in grey.</p> <p>Panel C shows immunoblot analysis of the various constructs. Hela cells were transfected with plasmids expressing HA-tagged M78, either wild type (wt), C-terminally truncated (CΔ6—CΔ155) or amino acid substitutions disrupting the acidic cluster ₃₈₆DEDDDD₃₉₁ (AC: VQNAAA) or the acidic dileucine motif ₄₅₈DDxxxLL₄₆₄ (DD-AA or LL-AA). One day post-transfection, cells were harvested and analysed by western blotting, using a mouse monoclonal anti-HA antibody. The marker band sizes are shown (in kDa). The truncation and substitution mutants were analysed on different gels; vertical black lines indicate where the gel images were spliced to remove irrelevant lanes.</p

Development of neutralising antibodies against MVA following vaccination.

<p>MVA plaque reduction neutralisation titre of sera taken from ponies following initial vaccination with recombinant MVA and two subsequent boosts. Arrows denote days of vaccination.</p

Detection of MVAVP2 or MVAVP7-expressed VP2 and VP7 protein, respectively, within QT35 and ESF cells.

<p>Cell lysates of uninfected cells (lanes 1,3,5 and 7) and cells infected at high MOI with MVA-VP2 (lanes 2 and 4) or MVA-VP7 (lanes 6 and 8), and harvested at 24 hours post-infection, were separated by SDS-PAGE on 10% gels. Immunoblotting was conducted with either anti-VP2 mAb (lanes 1–4) or anti-VP7 mAb (lanes 5–8).</p

Detection of immunoprecipitated, recombinant-baculovirus-expressed VP2V5 with MVAVP2-vaccinated pony serum.

<p>Lysates of uninfected Sf9 cells (lanes 1,3,5,7 and 9) and Sf9 cells infected with recombinant baculovirus FBVP2-V5 were immunoprecipitated with anti-V5tag mAb and Protein G agarose. The immunoprecipitates were separated by SDS-PAGE on 10% gels, and immunoblotted with MVAVP2 vaccinated pony sera (4256, 5483) (lanes 1–8) or anti-V5tag mAb (lanes 9 & 10). The pony sera tested were derived from a pre-vaccination control bleed (lanes 1 & 2) and three post-vaccination bleeds (lanes 3–8) from days 21, 42, and 84, respectively. A non-specific band was present to some extent in each lane at ∼50 KDa.</p

Detection of recombinant baculovirus-expressed VP7 with MVA-VP7-vaccinated pony serum.

<p>A semi-purified preparation of recombinant baculovirus FBVP7-expressed VP7 was separated by by SDS-PAGE on 10% gels, and immunoblotted with MVA-VP7-vaccinated pony sera (lanes 1–4) or anti-VP7 mAb (lane 5). The pony sera tested were derived from a pre-vaccination control bleed (lane 1) and three post-vaccination bleeds (lanes 2–4) from days 21, 42, and 84, respectively.</p

AHSV-neutralising antibody responses in ponies vaccinated with MVAVP2.

*<p>Expressed as the reciprocal of the highest dilution that provided >50% protection of the Vero cell monolayer.</p