Volume 15, Issue 11, Pages 1963-1972 (November 2007) Intracellular Trafficking of a Fiber-modified Adenovirus Using Lipid Raft/Caveolae Endocytosis  Sophie Rogée, Elodie Grellier, Claudine Bernard, Anne Loyens, Jean-Claude Beauvillain, Jean-Claude D'halluin, Morvane Colin  Molecular Therapy  Volume 15, Issue 11, Pages 1963-1972 (November 2007) DOI: 10.1038/sj.mt.6300283 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Ability of HAdV2/BAdV4 to infect various cell lines. A549, Hep G2, MCF7, and Chinese hamster ovary (CHO) cells were incubated for 2 hours at 37 °C in serum-free medium with 2,000 physical particles/cell of HAdV5βgal (closed square) or HAdV2/BAdV4βgal (open square). After an additional 24 hours, the β-galactosidase expression was evaluated by flow cytometry after incubation with fluorescein-β-D-galactopyranoside. The results are expressed as (a) a percentage of positive cells and (b) relative fluorescence intensity (RFI). (**P < 0.01, ***P < 0.001). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Differences in uptake and gene expression kinetics between HAdV5 and HAdV2/BAdV4. Chinese hamster ovary (CHO), A549, Hep G2, or MCF7 cells were incubated with 104 physical particles/cell of labeled or non-labeled HAdV2/BAdV4 (open square) or HAdV5 (closed triangle) respectively at 37 °C. (a) At the indicated time points, the cells were treated with trypsin to remove cell-bound particles, and uptake of vectors was determined using flow cytometry. (b) The β-galactosidase activity was determined by flow cytometry 24 hours after infection. The results are expressed as a percentage of positive cells (left panel) and relative fluorescence intensity (RFI) (right panel). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Co-localization of HAdV2/BAdV4 particles and caveolin-1 in various cell lines. (a) Chinese hamster ovary (CHO) cells were incubated with 6-carboxyfluorescein (FAM)-labeled HAdV2/BAdV4 (upper panel) or HAdV5 (lower panel) with 104 physical particles/cell for 90 minutes at 4 °C, followed by a chase period of 30 minutes at 37 °C. Confocal microscopy analyses were performed, caveolin-1 was visualized in red (A, D) and viruses in green (B, E). Co-localization (C, F, white arrows) of caveolin-1 with viruses is shown. Nuclei were stained with TOPRO-3. The bar represents 10 μm. (b) The same treatment was applied to A549 (upper panel) and Hep G2 (lower panel). Co-localization between caveolin-1 and HAdV2/BAdV4 (left panel), and caveolin-1 and HAdV5 (right panel) are shown. The bar represents 10 μm. (c) Cells were treated as described in a and the percentage of co-localization with caveolin-1 was calculated after specific software analysis of several confocal microscopy slides. (***P < 0.001). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Moderate clathrin-coated pit vesicles and phagocytosis uptake of HAdV2/BAdV4 in Chinese hamster ovary (CHO) cells. (a) Cells were either depleted of intracellular potassium (closed square) or not (open square). Subsequently, fluorescein isothiocyanate-labeled transferrin or FP485-labeled viruses were added. After 15 minutes at 37 °C, the cells were rinsed with trypsin, and the fluorescence intensity analyzed by flow cytometry. (NS, non-significant; *P < 0.05, ***P < 0.001). (b) A549 (left panel), CHO (middle panel), and Hep G2 (right panel) cells were incubated with 6-carboxyfluorescein (FAM)-labeled HAdV2/BAdV4 or HAdV5 at 104 physical particles (PPs)/cell for 1 hour at 4 °C followed by a chase period of 5 minutes at 37 °C. Co-localization (white arrows) of clathrin (in red) with viruses (in green) is shown. Nuclei were stained with TOPRO-3. The bar represents 10 μm. The percentage of co-localization with clathrin was calculated after specific software analysis of several confocal microscopy slides. (**P < 0.01, ***P < 0.001). (c) CHO and A549 cells were incubated with FAM-labeled HAdV2/BAdV4 and HAdV5, respectively, at 104 PPs/cell with 1 μm Nile Red-labeled microspheres (10/cells) for 1 hour at 4 °C followed by a chase period of 20 minutes at 37 °C. Co-localization (white arrows) of microspheres with viruses is shown (left panel). The bar represents 10 μm. The percentage of co-localization is determined as described above (right panel). (NS, non-significant). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Reduced binding of HAdV2/BAdV4 to CHO-CAVdom– cell lines. (a) Chinese hamster ovary (CHO) cells were stably transfected with N-terminally green fluorescent protein (GFP)-tagged dominant-negative mutant of caveolin-1. Two clones, CHO-CAVdom– cl1(hatched bars) and CHO-CAVdom– cl2 (white bars), were selected. The GFP expression was measured using flow cytometry and compared against the expression in CHO cells (black bars). The results are expressed as relative fluorescence intensity (RFI). (b) GM1 labeling of CHO and CHO-CAVdom– cl2 cells after incubation with cholera toxin subunit B (CTB) (10 μg/ml) for 30 minutes on ice. CTB is visualized in red and CHO-CAVdom– cl2-expressing GFP in green. The bar represents 10 μm. (c, d) 106 CHO (black bars), CHO-CAVdom– cl1(hatched bars) and CHO-CAVdom– cl2 (white bars) cells were incubated with 104 physical particles/cell of FP485-labeled HAdV2/BAdV4 at 4 °C for 90 minutes. The cells were rinsed, and the binding of FP485-labeled HAdV2/BAdV4 was quantified using flow cytometry. In order to determine uptake, the cells were treated as described previously, except that after incubation at 4 °C, an incubation period of 30 minutes at 37 °C was added. The cells were rinsed with trypsin and the uptake of FP 485-labeled HAdV2/BAdV4 was quantified using flow cytometry. The results are expressed as a (c) percentage of positive cells and (d) as RFI (NS, non-significant; *P < 0.05, ***P < 0.001). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Transgene expression is strongly reduced in the CHO-CAVdom– cell lines. (a) 106 Chinese hamster ovary (CHO) (black bars), CHO-CAVdom– cl1(hatched bars) and CHO-CAVdom– cl2 (white bars) cells were infected with various concentrations of HAdV2/BAdV4 for 2 hours at 37 °C in a serum-free medium. Twenty-four hours after infection, cells were lysed and the β-galactosidase activity was determined. (NS, non-significant, *P < 0.05). (b) A549 (upper panel) and CHO (lower panel) cells were treated with methyl-β-cyclodextrin (MβCD) (2–5 mmol/l) for 2 hours at 37 °C. GM1 labeling of cells was performed after incubation with Alexa Fluor 555-CTB (10 μg/ml) for 30 minutes on ice. The bar represents 10 μm. (c) CHO and A549 cells were treated with 0 (black bars), 2 (hatched bars), or 5 mmol/l (white bars) of MβCD for 30 minutes at 37 °C. HAdVs (human adenoviruses) (104 physical particles (PPs)/cell) were then added, and the cells were incubated for a further 2 hours at 37 °C. They were rinsed once with trypsin to remove cell surface-bound virus. Twenty four hours after infection, the β-galactosidase activity analysis was performed using fluorescein-β-D-galactopyranoside. (*P < 0.05, **P < 0.01). Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Detection of HAdV2/BAdV4 in subcellular fractions containing GM1 and caveolin-1/2 proteins in CHO cells. Non-infected (left panel) and HAdV2/BAdV4-infected (right panel) (2 × 1011 physical particles/ml, 90 minutes at 4 °C) cells were solubilized in 1% Triton X-100 for 1 hour at 4 °C and the solution adjusted to 40% (wt/vol) sucrose. The samples were then layered under a 4–30% (wt/vol) sucrose gradient and centrifuged overnight at 100,000g. One hundred microliters of each sucrose gradient fraction (F1–F8) were spotted onto a nitrocellulose membrane and GM1, caveolin-2, caveolin-1, and HAdV2/BAdV4 were detected using monoclonal antibodies. Purified virus was spotted onto a nitrocellulose membrane and used as a positive control (V). HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Electron microscopy analysis of intracellular virions of HAdV5 and HAdV5/BAdV4. (a) A549 (A–D) and Chinese hamster ovary (CHO) (E–H) cells were incubated with either HAdV5 or HAdV2/BAdV4 for 1 hour at 4 °C, and then for 30 minutes at 37 °C at an input multiplicity of 104 physical particles/cell (A and E: binding, B/C and F/G: uptake, D: multivesicular body, H: megavesicle). The bar represents 200 nm. (b) CHO cells were treated as described in a. Megavesicles formed in CHO cells are detected (B and C). Immunolabeling (long arrow) of vesicles was performed using a monoclonal antibody against caveolin-1 (C). The presence of proteins was then detected using a 12 nm colloidal Gold-AffiniPure goat anti-mouse antibody. CHO cells, incubated without virus for 90 minutes at 4 °C following a 30 minute period incubation at 37 °C were used as a negative control (A). For parts a and b, vesicles are shown with short arrows and viruses with arrow heads, bars represent 200 nm. (c) Vesicles were examined on the basis of 4 cluster sizes (Vesicle size (VS) <200 nm, 200 < VS < 600 nm, 600 < VS < 800 nm, VS > 800 nm). 36 micrographs were randomly examined for each condition. The percentage of vesicles was calculated as described in Material and Methods, 5 minutes (black bars), 15 minutes (hatched bars) or 30 (white bars) after infection at 37 °C. HAdV, human adenovirus. Molecular Therapy 2007 15, 1963-1972DOI: (10.1038/sj.mt.6300283) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions