(A) CLUSTALW [47] alignment of h-GAAP and v-GAAP with related proteins from other species: VACV strain Evans, CMLV strain CM-S, human, orangutan, dog, mouse, X. laevis, zebrafish, A. thaliana, and D. melanogaster. Identical residues are shown in black, and residues conserved in six to eight or nine out of ten sequences are in light or dark grey, respectively. Horizontal bars show predicted transmembrane domains (I–VII) based on the Kyte–Doolittle hydropathy model. An asterisk denotes a block of 20 aa.
(B) Hydrophobicity profiles of human, VACV, and A. thaliana GAAPs according to Kyte and Doolittle, plotted using the program WinPep [48]. Putative transmembrane domains (I–VII) are outlined.

(A) Time of v-GAAP expression during infection. Semi-quantitative RT-PCR (30 cycles) analysis of CMLV-infected TK⁻143 cells for presence of mRNA for v-GAAP and CMLV gene 110L (equivalent to VACV gene D8L) (left panel). The right panel shows an immunoblot analysis of v-GAAP RevHA–infected TK⁻143 cells for expression of v-GAAP and the late VACV protein A36 using α-HA and α-A36 mAbs, respectively. m, mock.
(B) v-GAAP localises to the Golgi. HeLa cells were infected with v-GAAP WT, v-ΔGAAP, and v-GAAP Rev for 6 h and stained for v-GAAP using anti-GAAP Ab and for Golgi using anti-GM130 Ab. Scale bars, 20 μm. The right panel shows an immunoblot analysis of v-GAAP mutant viruses 16 h p.i. using the α-GAAP Ab. The black arrow (left panel) indicates the GAAP protein.
(C) Tissue expression profile of h-GAAP. The 714-bp h-GAAP gene (35 cycles, top panel) or 983-bp glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene fragment (30 cycles, bottom panel) were amplified by PCR from human cDNA master panels and analyzed by agarose gel electrophoresis. intestine, small intestine; PBL, peripheral blood leukocyte; sk. muscle, skeletal muscles.
(D) h-GAAP localises to the Golgi. HeLa cells were stained for endogenous h-GAAP and Golgi marker GM130 using anti-GAAP and anti-GM130–specific Abs. Scale bars, 20 μm. Pre-immune serum was used as a control (right panel).

(A) U2OS-neo (top row), U2OS-v-GAAP (middle row), or U2OS-h-GAAP (lower row) cells were stained using an anti-HA mAb together with an α-GM130 Ab. All primary Abs were detected with secondary Abs conjugated to fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. Scale bars, 20 μm. The right panel shows an immunoblot analysis of U2OS stable cell lines using an α-HA mAb.
(B) Top left panel: Cryo-immunoelectron microscopy was used to label the v-GAAP HA-tagged protein in U2OS v-GAAP cells with anti-HA mAb (Covance, diluted 1/10), followed by rabbit anti-mouse (Cappel) and 6-nm protein-A Au (scale bars 100 nm, top left panel). Golgi stack morphology was compared in the different cell lines by conventional thin sections of Epon-embedded samples and was examined by electron microscopy in U2OS-neo (top right panel), US-OS-v-GAAP (lower left panel), and U2OS h-GAAP (lower right panel). Scale bars, 200 nm.
(C) HeLa cells were transfected with pCI-h-GAAPHA or pCI-v-GAAPHA and fixed 4 h post transfection. GAAPs were detected using an α-HA mAb, and cells were co-stained with the Golgi marker α-GM130. Scale bars, 20 μm.

HeLa cells (A–D) or U2OS-h-GAAP cells (E and F) were transfected with siRNA oligos directed to h-GAAP (siRNAs 1–3) or GFP (GFP-siRNA) for 36 h (A and E), or 56 h (B–D and F).
(A) mRNA level of h-GAAP in HeLa cells was assessed by semi-quantitative RT-PCR (35 cycles) using h-GAAP- or actin-specific primers, respectively. −RT indicates negative control.
(B) HeLa cell morphology was recorded using a Zeiss Axiovert 200M Live Imaging System with an AxioCam HRc CCD. Images shown are representative of at least three independent experiments.
(C) HeLa cells were loaded with the fluorescent dye TMRE and loss of the inner mitochondrial membrane potential was assessed by TMRE fluorescence two-color flow cytometry. The graph shows the percentage of cells with decreased TMRE uptake compared to mock-transfected cells. Data are mean percentages from at least three independent experiments (one of which is shown in Figure S2). *, p < 0.05; ** p < 0.005.
(D) HeLa cells were treated with GFP-siRNA or h-GAAP siRNA1 or were mock-treated and assessed for cleavage of PARP by immunoblotting.
(E and F) h-GAAP protein expression in U2OS-h-GAAP cells was assessed by immunofluorescence (E) or immunoblotting (F) using an α-HA mAb. In (E), the upper row of panels show the fluorescent images, and the bottom row shows a merged image of the top panels and an image of the same cells taken by differential interference contrast microscopy. Data are representative of three independent experiments. Scale bars, 20 μm.

(A) Transient assay of Bax-induced apoptosis. Saos-2 cells were transfected with different plasmids as indicated, and representative flow cytometry profiles of CD20-positive cells are shown. The bar graph represents the percentage of sub-G1 cells (labeled M1 in the left panel). Data are mean percentages from at least two independent experiments. Protein expression was detected by immunoblotting using α-Bax or α-HA antibodies, respectively.
(B–D) U2OS-neo, U2OS-v-GAAP, or U2OS-h-GAAP cells were mock-treated or treated with 3 μM staurosporine for 6 h (B), 10 μg/ml cisplatin for 24 h (C), or 10 μM C₂-ceramide for 36 h (D), and apoptosis was assessed as above. The graphs show the relative percentage of apoptosis induced by staurosporine (B) or cisplatin (C). Data are mean percentages (± 1 standard deviation [SD]) from at least two independent experiments done in triplicate.
(D) After 24 h treatment with C₂-ceramide, the cell morphology was recorded as for Figure 3B. The graphs show the percentage of sub-G1 cells induced by C₂-ceramide. Data are mean percentages (± 1 SD) from at least two independent experiments done in triplicate. Statistically significant differences are shown.

(A) U2OS-neo, U2OS-v-GAAP, or U2OS-h-GAAP cells were treated with 10 μg/ml of CHX alone or together with 500 ng/ml of anti-Fas Ab or 10 ng/ml of TNF-α. The bar graph represents the percentage of apoptotic cells (± 1 SD) after 24 h.
(B) Relative inhibition of Fas and TNF-α–induced apoptosis in U2OS-neo (designated as 100%), U2OS-v-GAAP, and U2OS-h-GAAP cells. Data are mean percentages (± 1 SD) from at least three independent experiments. Statistically significant differences are shown.
(C and D) After 2, 4, or 6 h incubation with TNF-α and cycloheximide, proteolytic cleavage of caspase-8 into p43/p41 and p18 fragments (C) and PARP (D) were assessed by immunoblotting.

(A) U2OS-v-GAAP or U2OS-h-GAAP cells were transfected with siRNA oligonucleotides directed against h-GAAP (siRNAs 1–3) or v-GAAP (siRNA1) for 36 h. v-GAAP and h-GAAP expression was assessed by immunofluorescence using an α-HA mAb, followed by secondary Ab conjugated to FITC. The upper and third rows of panels show the fluorescent images, and the second and bottom rows show merged images of the fluorescent panel (immediately above) and an image of the same cells taken by differential interference contrast microscopy. Images shown are representative for at least three independent experiments. Scale bars, 20 μm.
(B) Cell morphology of U2OS-h-GAAP or U2OS-v-GAAP cells 56 h after transfection with GAAP siRNAs 1–3 was assessed as for Figure 4B.

Groups of four BALB/c mice aged between 6 and 8 wk were mock-infected with PBS (•) or infected with 10⁷ pfu of v-GAAP WT (♦), v-ΔGAAP (), or v-GAAP Rev (▴).
(A) Mice were weighed daily, and results are the mean percentage weight change of each group ± standard error of the mean (SEM) compared with the weight on the day of infection.
(B) Animals from (A) were monitored daily for signs of illness, which was scored as described [27]. Data are expressed as the mean ± SEM. p-Values were determined by using Student's t-test and indicate mean weight loss (A) or mean signs of illness. *, p-values of mice infected with v-ΔGAAP that were significantly different from those of mice infected with v-GAAP WT and v-GAAP Rev (B).
(C) Mice infected intranasally with 10⁷ pfu of v-GAAP WT, v-ΔGAAP, or v-GAAP Rev were sacrificed at various days p.i. as indicated. BAL cells were recovered and counted. Columns represent the mean cell yield per mouse ± SEM. Those marked with an asterisk indicate mean cell numbers recovered with v-ΔGAAP that were significantly different (p < 0.05) from those recovered from v-GAAP WT and v-GAAP Rev (three independent experiments with groups of four to five mice).