Comparative analysis of N-linked sialic acid-binding requirements for various recombinant adeno-associated virus (rAAV) serotypes. ALI cultures from human (HAE), pig (PAE), ferret (FAE) and mouse (MAE) were treated with neuraminidase (NA-treated) or Ultraser G control medium (untreated) prior to infection. This was followed by application of rAAV luciferase virus to the apical surface at a multiplicity of infection of 2.0 × 10³ particles per cell (2 × 10⁹ total particles per well) at 4 °C for 1 h (as described in Figure 2). The infected cultures were washed following virus binding, and cells were harvested for analysis of the viral genome. (a) The extracted viral DNA was detected by Southern blotting using a ³²P-labeled luciferase DNA probe as described previously.22,31 (b) Quantitative analyses of rAAV genomes following the above infection protocol used Taqman PCR. Plotted are the means (± s.e.m.) of the numbers of rAAV viral genomes (per well) from three independent experiments (N = 12). The forward primer (5′-TTTTTGAAGCGAAGGTTGTGG-3′), and the reverse primer (5′-CACACACAGTTCGCCTCTTTG-3′) were designed to amplify a 32-bp fragment in the luciferase-coding region of rAAVLuc. The Taqman probe (5′-ATCTGGATACCG GGAAAACGCTGGGCGTTAAT-3′) was tagged with FAM dye (6-carboxyfluorescein) at its 5′-end to serve as the reporter, and with TAMRA dye (6-carboxytetramethylrhodamine) at the 3′-end to serve as the quencher.32 Series dilution of rAAVLuc was used to generate the standard curve, and was only accepted as linear when the slope fell to between −3.74 and −3.32 and the correlation coefficient was above 0.99.32

Comparative biology of recombinant adeno-associated virus (rAAV) transduction in human, ferret and pig airway epithelia. (a–c) Polarized air–liquid interface (ALI) airway epithelial cultures from human (HAE), ferret (FAE) and pig (PAE) were generated from tracheal tissue as described previously.21 Differentiated cultures were infected with rAAV luciferase virus pseudotyped in serotype 1, 2 or 5 capsid containing an rAAV2 viral genome (rAAV2/1, rAAV2/2, rAAV2/5). Viruses were generated using a triple plasmid transfection method as described previously.22 Infections were performed by applying 2.0 × 10³ particles per cell (2 × 10⁹ total particles per well) to either the apical or basal surface for 16 h as described previously.11 Relative luciferase activity was measured at 3 days post-infection. (d, e) ALI cultures were apically infected with 2.0 × 10³ particles per cell in the presence or absence of 40 μM N-acetyl-L-leucyl-L-leucyl-L-norleucine and 5 μM doxorubicin for 16 h, as described previously22 and luciferase activity was measured at 3 days post-infection. Proteasome inhibitors (PI) were removed after 16 h following viral infection. Numbers above each data set represent the fold induction in the presence of proteasome inhibitor. Data in all panels represent the mean (± s.e.m.) relative luciferase activity (per well) from three independent experiments (N = 12 transwells for each experimental point). †,* marked comparisons indicate signficant difference as determined by the Student’s t-test.

Comparative analysis of candidate viral-binding receptors in human, mouse, ferret and pig airway epithelia. (a, b) Immunostaining for heparan sulfate proteoglycan (HSPG) and 2,3-linked sialic acid using the Maackia amurensis (MAA) lectin on sections from ferret and pig airway epithelial ALI cultures (left panel) and native tracheas (right panel), as described previously for human and mouse ALI cultures and tracheal tissue.11 Arrowheads mark the basolateral side of each epithelium and 4’,6-diamidino-2-phenylindole demarcates nuclei. Antibodies used included rat anti-HSPG monoclonal antibody followed by fluoroisothiocyanate (FITC)-labeled anti-rat secondary antibody, and FITC-labeled MAA lectin. (c–f) ALI epithelial cultures from human, mouse, ferret and pig were pretreated with neuraminidase (NA-treated) to remove sialic acid from the apical surfaces of airway epithelial cultures, or with Ultraser G (USG) medium as a control (untreated), prior to infection with virus. Specifically, 200 μl of either USG medium containing 50 mU ml⁻¹ of type III NA from Vibrio cholerae (Sigma, Santa Louis, MO, USA), or USG medium alone, was added to the apical side of the cultures and left there for 2 h (at 37 °C). NA was then removed and cultures were washed three times with phosphate-buffered saline. This was followed by apical infection at a multiplicity of infection of 2.0 × 10³ particles per cell (2 × 10⁹ total particles per well) with the indicated serotypes recombinant adeno-associated virus luciferase virus for 1 h at 4 °C. Cultures were then shifted to a 37 °C incubator and the efficiency of transduction was evaluated by measuring luciferase activity at 7 days post-infection. Data represent the means (± s.e.m.) of the relative luciferase activity (per well) from three independent experiments (N = 12). †,* marked comparisons indicate significant difference as determined by the student’s t-test.