Binding of Adeno-associated Virus Type 5 to 2,3-Linked Sialic Acid Is Required for Gene Transfer*

Robert W. WaltersDagger §, Su Min P. Yi||, Shaf Keshavjee**, Kevin E. BrownDagger Dagger , Michael J. WelshDagger §§§, John A. Chiorini¶¶, and Joseph ZabnerDagger ||||

From the Departments of Dagger  Internal Medicine, § Physiology and Biophysics, and || Otolaryngology,  Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242, the ** Toronto Lung Transplant Program, University of Toronto, Toronto, Ontario M5G 2C4, Canada, and the Dagger Dagger  Hematology Branch, NHLBI and ¶¶ Gene Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, February 20, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant adeno-associated viruses (AAV)1 have been used widely for gene transfer to a variety of cells in vitro and several organs in vivo (1-7). Several advantages make adeno-associated viruses promising vectors for gene therapy including the following: the lack of human pathology associated with wild-type adeno-associated viruses, prolonged expression of the transgene, and the lack of a cell-mediated immune response. Adeno-associated viruses are members of the parvovirus family, and have in common a similar size, structure, and dependence on a helper virus for replication and gene expression. To date six primate isolates have been reported, and their genomes appear to be organized in a similar manner (8-13).

AAV2 was the first primate AAV to be cloned into a plasmid and has been studied extensively (14). In animal experiments, AAV2 vectors have been used to target the liver, muscle, eyes, and the central nervous system (4, 5, 15, 16). More importantly, this vector has been shown recently to mediate factor-IX gene transfer to the muscle of humans, making hemophilia one of the first human genetic diseases that has been partially corrected by gene transfer (7). AAV2 has also been investigated for gene transfer of the cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia in cystic fibrosis in vitro and in vivo (2, 3, 17-20). However, compared with the efficiency of AAV2 gene transfer to muscle, eye, and liver, gene transfer to human airway epithelia from the apical surface is inefficient (18, 19, 21-25). The receptors and coreceptors for AAV2 include heparan sulfate proteoglycan, fibroblast growth factor receptor-1, and alpha vbeta 5 integrins (26-30). In human airway epithelia, basolateral localization of these molecules explains why AAV2 is inefficient when applied to the apical surface (18).

Recently, adeno-associated viruses from the other five serotypes have been cloned, and their tropism has been studied in laboratory cell lines, in differentiated human airway epithelia in vitro, and in murine tissues in vivo (9-11, 13, 25, 31-33). Sequence and biochemical comparisons of all AAV serotypes indicate that AAV5 is the most divergent (11). Not only is the capsid protein markedly different between AAV2 and AAV5 but also the inverted terminal repeats and Rep protein of AAV5 are sufficiently divergent to be unable to complement the replication of AAV2 (11).

In earlier work, we found that AAV5 was 50 times more efficient than AAV2 at gene transfer from the apical surface of human airway epithelia in vitro, and it was 20 times more efficient than AAV2 at gene transfer to the murine airway epithelia in vivo (25). Moreover, AAV5 has been shown to be more efficient than AAV2 at gene transfer to the ependymal cells of the ventricles, the cerebral hemispheres, and muscle (32, 34).

These data suggest that AAV5 might use a receptor distinct from that for AAV2. Furthermore, the improved efficiency in airway epithelia suggests that the receptor is located on the apical surface. The first goal of this work was to test directly the hypothesis that AAV5 has an apical membrane receptor in differentiated human airway epithelia.

Several observations hint at sialic acid as a candidate receptor for AAV5. For instance, influenza virus infects airway epithelia from the apical surface, and it uses sialic acid as a receptor (35-37). Also, some parvoviruses such as canine parvovirus and feline panleukopenia virus bind sialic acid; however, the significance of this interaction is not well understood (38-41). Based on these observations we also tested the hypothesis that sialic acid is required for binding and gene transfer with AAV5.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Culture-- COS-7 and 293 cells were cultured on 24-well plates (Corning Costar) in Eagle's minimal essential media (EMEM; Sigma) supplemented with 10% fetal calf serum (Sigma), 1% nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin.

A parental CHO cell line (Pro-5) and a Pro-5 mutant (Lec-2) were obtained from the American Type Culture Collection. The Lec-2 mutant was selected from the parental line based on resistance to wheat germ agglutinin (WGA). Specifically, the Lec-2 cells are deficient in transport of CMP-sialic acid into the Golgi compartment and hence do not efficiently process sialic acid onto their cell surface. Both cell lines were cultured on 24-well plates (Corning Costar) in alpha -minimum essential media (Sigma) supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 µg/ml streptomycin.

Airway epithelial cells were obtained from trachea and bronchi of lungs removed for organ donation. Cells were isolated by enzyme digestion as described previously (42, 43). Freshly isolated cells were seeded at a density of 5 × 105 cells/cm2 onto collagen-coated 0.6-cm2 area Millicell polycarbonate filters (Millipore Corp.). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and air. Twenty four hours after plating, the mucosal media were removed, and the cells were allowed to grow at the air-liquid interface (42-44). The culture media consisted of a 1:1 mix of Dulbecco's modified Eagle's medium/Ham's F-12, 5% Ultraser G (Biosepra SA), 100 units/ml penicillin, 100 µg/ml streptomycin, 1% nonessential amino acids, and 0.12 units/ml insulin. Airway epithelia were allowed to reach confluence and develop a transepithelial electrical resistance, indicating the development of tight junctions and an intact barrier. Epithelia were allowed to differentiate by culturing for at least 14 days after seeding, and the presence of a ciliated surface was tested by scanning electron microscopy (43, 45).

Adeno-associated Viruses-- Recombinant AAV2 and AAV5 were produced as described previously (33). Briefly, AAV2/beta -galactosidase and AAV5/beta -galactosidase were prepared by triple plasmid cotransfection of COS cells using a calcium phosphate cotransfection system (Life Technologies, Inc.). COS cells were grown to 50% confluence on 150-mm plates. For every 5 plates, 6.1 µg of vector plasmid (p2LacZ or p5LacZ), 6.1 µg of helper plasmid (p2RepCap or p5RepCap), and 12.8 µg of pAd12 were precipitated with calcium phosphate. Precipitants were slowly added to COS cells, and the cells were cultured at 37 °C before harvesting.

Wild-type AAV2 and AAV5 were produced by coinfecting COS cells with wild-type AAV2 or AAV5 and wild-type adenovirus. Prior to their use, COS cells and seed stocks of wild-type adenovirus were screened for wild-type AAV contamination by polymerase chain reaction. COS cells grown to 50% confluence were infected for 1 h at 37 °C with 1 multiplicity of infection of wild-type AAV2 or wild-type AAV5 and 1 multiplicity of infection of wild-type adenovirus delivered in EMEM. Serum-containing media were then added, and the cells were cultured for an additional 72 h.

72 hours post-transfection or infection, cells were harvested by scraping and pelleted by low speed centrifugation. For every 10 plates, the pellet was resuspended in 5 ml of tissue dissociation buffer (140 mM NaCl, 5 mM KCl, 0.7 mM K2HPO4, 25 mM Tris/HCl, pH 7.4) and stored at -70 °C. The cell pellet was thawed at 37 °C, and benzonase (Sigma) was added to a final concentration of 20 units/ml. Sodium deoxycholate was then added to a final concentration of 0.5%, and the suspension was incubated for 1 h. The suspension was homogenized thoroughly (20 strokes in a Wheaton B homogenizer). Next, CsCl was added to a final density of 1.4 g/cm3, and the homogenate was centrifuged in polyallomer tubes using an SW40 rotor at 38,000 rpm for 65 h at 20 °C. The gradients were fractionated by side puncture. Fractions with a refractive index of 1.373-1.371 were pooled, centrifuged again using an SW50.1 rotor, and fractionated as described. Refractive indices were determined using a refractometer (Fisher).

Recombinant and wild-type AAV2 and AAV5 viruses were titered by Southern blot and transmission electron microscopy. The viral titers for recombinant preparations ranged between 1 × 1012 and 1 × 1013 pt/ml and for wild-type preparations between 1 × 1012 and 5 × 1014 pt/ml. The viruses were screened for wild-type adenovirus by a serial dilution assay using a FITC-hexon antibody (sensitivity, 1 particle in 105) (45).

Binding Assays-- Binding of AAV2/beta -galactosidase or AAV5/beta -galactosidase to COS cells, 293 cells, CHO cells, and the apical surface of human airway epithelia was measured using a quantitative dot blot assay (24, 25). Kinetic experiments were carried out by binding AAV at 2 × 109 pt/ml for up to 150 min at 4 °C. Once equilibrium conditions were defined, they were used in all additional binding experiments. Competition experiments were carried out by competing binding of recombinant AAV5 or AAV2 (4 × 109 pt/ml for COS or 293 cells, and 2 × 1010 pt/ml for airway epithelia) with 100-fold excess of wild-type AAV5 or AAV2. Binding to sialidase-treated cells and sialic acid-deficient cells was studied using 4 × 109 pt/ml of AAV on COS, 293, or CHO cells and 2 × 1010 pt/ml on airway epithelia. Cells were maintained at 4 °C for the duration of the experiment. Following binding, cells were rinsed 3 times with EMEM. Cell-associated AAV viral DNA from cell lysates of three epithelia were pooled for each dot analysis. Samples were subjected to 3 freeze/thaw cycles and then blotted onto a nylon membrane (Ambion). A dilution series of AAV was used to ensure that the assay was linear. AAV viral DNA was detected by hybridizing with a 32P-labeled beta -galactosidase cDNA probe. Unhybridized probe was washed as follows: two washes with 2× SSC and 0.1% SDS at room temperature for 15 min, one wash with 0.5× SSC and 0.1% SDS at 55 °C for 1 h, and finally one wash with 0.5× SSC and 0.1% SDS at 65 °C for 30 min. Dot blots were developed and quantitated using a PhosphorImager (Molecular Dynamics) (24, 25).

Gene Transfer Assays-- Gene transfer was measured as described previously (25). Briefly, 500 particles of recombinant AAV per cell (in EMEM) were added to either COS cells, 293 cells, CHO cells, or the apical surface of human airway epithelia. Following a 1-h incubation at 37 °C, the viral suspension was removed, and the cells or epithelia were rinsed twice with EMEM. After infection, the cultures were incubated at 37 °C for an additional 2 days in the case of COS, 293, and CHO cells, and 14 days in the case of airway epithelia.

We measured total beta -galactosidase activity using a commercially available method (Galacto-Light, Tropix Inc.). Briefly, after being rinsed with phosphate-buffered saline, cells were removed from filters or plastic by incubation with 120 µl of lysis buffer (25 mM Tris phosphate, pH 7.8; 2 mM dithiothreitol; 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; 10% glycerol; and 1% Triton X-100) for 15 min. Light emission was quantified in a luminometer (Analytical Luminescence Laboratory).

Hemagglutination-- Rhesus monkey erythrocytes were collected by phlebotomy and washed 3 times in phosphate-buffered saline by centrifugation (5000 rpm for 5 min). Virions (1 × 1010 particles) were diluted in serial 2-fold steps in 96-well plates containing 75 µl of phosphate-buffered saline, pH 7.8. To each dilution, 25 µl of a 2% suspension of erythrocytes was added. The sedimentation pattern was determined after incubation for 1 h at 4 °C.

Lectin Staining and Competition-- Lectin staining of COS cells, 293 cells, and the apical surface of human airway epithelia was performed by incubating cells in the presence of FITC-labeled WGA, maackia amurensis lectin (MAA), sambucus nigra lectin (SNA), or concanavalin A (ConA) (Vector Laboratories Inc.). Briefly, cells were chilled to 4 °C, and then 10 µg/ml lectin in EMEM was added to cells or the apical surface of epithelia. Cultures were incubated at 4 °C for 20 min, rinsed three times with EMEM, and then fixed with 4% paraformaldehyde in phosphate-buffered saline. Binding of lectin to cells or the apical membrane of epithelia was detected with fluorescence microscopy.

Lectin competition experiments were done by preincubating cells or epithelia at 4 °C with 100 µg/ml of either WGA, MAA, SNA, or ConA (Vector Laboratories Inc.) in EMEM for 10 min. The preincubation solution was removed, and EMEM containing 100 µg/ml lectin and either 4 × 109 pt/ml virus (in the case of COS and 293 cells) or 2 × 1010 pt/ml virus (in the case of airway epithelia) was added. Cultures were maintained at 4 °C for 1 h and then rinsed and either assayed for binding or cultured and then assayed for infection.

Cell Surface Modifications-- Sialic acid was biochemically removed from the surface of erythrocytes, COS cells, 293 cells, and from the apical surface of epithelia by sialidase treatment. Cells and epithelia were rinsed with EMEM and then incubated with the bacterial sialidase NA type III from Vibrio cholerae (Sigma). Cells were treated with 50 milliunits/ml enzyme, and epithelia were treated with 200 milliunits/ml enzyme diluted in EMEM for 2 h at 37 °C, and washed with EMEM before binding or infection (46).

Sialic acid was restored to the surface of erythrocytes in defined linkages using purified sialyltransferases (47). Erythrocytes were incubated with either 2,3-sialytransferase (Calbiochem) or 2,6-sialytransferase (Roche Molecular Biochemicals) in the continued presence of 1 mM CMP-sialic acid (Roche Molecular Biochemicals). Resialylation was carried out with 100 milliunits/ml sialyltransferase in EMEM for 1 h at 37 °C. The resialylation was confirmed by specific lectin adsorption.

Data Analysis-- Concentration binding curves of AAV2 and AAV5 on COS cells, 293 cells, and differentiated human airway epithelia were analyzed to obtain the apparent affinity (KD) and the number of binding sites per cell (48). Binding curves were separated into nonspecific and specific components by fitting the total binding data to a single-site binding isotherm using nonlinear regression (Prism, Graphpad Software). We chose nonlinear regression analysis because unlike linear regression, it does not distort the experimental error (48). The specific component of binding was further analyzed to obtain the maximal binding and the apparent affinity (virus concentration required to reach half-maximal binding).

We assessed the validity of this model by analyzing the competition binding data. Specifically, we found similar results when we compared the measured nonspecific binding from competition experiments, and the calculated nonspecific binding from the curve fits. For instance, 34% of the total binding of AAV5 to airway epithelia did not compete; at the same concentration of ligand, the calculated nonspecific binding was 28% of the total binding. Evaluation of competition data from COS cells and 293 cells was also consistent with calculated values of nonspecific binding.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Receptor-mediated Binding of AAV5-- To determine if AAV5 binds to the apical surface of differentiated human airway epithelia and to learn if binding is receptor-mediated, we asked if binding fulfills classical criteria for a receptor-mediated event. That is, is there a saturating component of the concentration-binding curve and is binding competed on cells permissive for AAV5 infection? Because COS cells are easily infected with AAV2 and AAV5, and 293 cells are permissive for AAV2 but are infected less efficiently with AAV5, we also studied these cells.

Previous work (49, 50) has shown that the sensitivity of detection for radioligand binding with virus is below that required to detect receptor-mediated binding to primary epithelia. This, plus the fact that the apical surface of our model of well differentiated human airway epithelia has a limited apical surface area, led us to develop a more sensitive binding assay based on dot blot hybridization.

Since saturation, specificity, and competition should be tested under equilibrium conditions, we measured binding of AAV2 and AAV5 as a function of incubation time using a dot blot assay. Binding of both viruses to COS cells, 293 cells, and differentiated human airway epithelia reached equilibrium by 90 min at 4 °C (Fig. 1, A, C, and E). We found the transepithelial resistance of airway epithelia did not change over the duration of the experiment (data not shown). This suggests that the junctional integrity of the epithelia remained unchanged and that the viruses did not have access to the basolateral surface. Therefore, we measured binding only to the apical membrane.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Kinetics and dose-response of AAV2 and AAV5 binding to COS cells, 293 cells, and the apical surface of differentiated human airway epithelia. Binding of AAV to COS cells (A), 293 cells (C), and airway epithelia (E) measured as a function of incubation time. Equilibrium binding of AAV to COS cells (B), 293 cells (D), and human airway epithelia (F) as a function of virus concentration. Data are mean binding ± S.E. (n = 3-9). In some instances the error bars are hidden by the symbols.

To assess saturation, we measured binding with increasing concentrations of AAV under equilibrium conditions (90 min incubation at 4 °C). Binding to COS cells was relatively similar for AAV2 and AAV5 (Fig. 1B). However, on 293 cells we observed more binding with AAV2 than AAV5 (Fig. 1D). Unlike 293 cells, we observed more binding with AAV5 than AAV2 on the apical surface of airway epithelia (Fig. 1F). We separated total binding, which is shown in Fig. 1, into nonspecific and specific components by fitting the total binding curve to a single-site binding isotherm based on the assumption that nonspecific binding is directly proportional to the ligand or virus concentration (nonspecific and specific binding curves are not shown) (30, 48, 51, 52). Analysis of the specific component of binding revealed high affinity, saturable binding for AAV2 on COS and 293 cells (Table I). However, analysis of AAV2 binding to airway epithelia did not reveal specific binding suggesting either no apical receptors for AAV2 or the affinity and binding site number are below the sensitivity of this assay. These results are consistent with previous reports that heparan sulfate proteoglycan and the coreceptors for AAV2 are not present on the apical surface (18). In contrast, the specific component of AAV5 binding to COS cells and airway epithelia was saturable. Analysis of the specific binding of AAV5 revealed a high affinity interaction (apparent KD = 89 pM). Saturation and high affinity binding of AAV5 suggest that human airway epithelia have high affinity receptors for AAV5 on their apical surface.

                              
View this table:
[in this window]
[in a new window]
 
Table I

To test for specific competition, we measured binding of AAV2/beta -galactosidase or AAV5/beta -galactosidase in the presence of competing wild-type AAV2 or AAV5 using a dot blot assay. Because the binding assay detects the beta -galactosidase cDNA, we were able to use wild-type AAV to test competition. Wild-type AAV2 competed binding of AAV2/beta -galactosidase to COS and 293 cells, suggesting a specific receptor-mediated interaction but not to human airway epithelia, indicating a nonspecific or very low affinity interaction (Fig. 2). In contrast, wild-type AAV5 competed binding of AAV5/beta -galactosidase to COS cells but not to 293 cells, suggesting that 293 cells have very few receptors for AAV5. Both AAV5 and AAV2 bound efficiently to COS cells. However, wild-type AAV5 did not compete binding of AAV2/beta -galactosidase, and wild-type AAV2 did not compete binding of AAV5/beta -galactosidase, indicating that the receptors for these two viruses are different. Finally, we found that wild-type AAV5 competed binding of AAV5/beta -galactosidase on the apical surface of human airway epithelia, suggesting the presence of specific receptors for AAV5.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Competition of AAV2 and AAV5 binding to COS cells, 293 cells, and the apical surface of differentiated human airway epithelia (HAE). Binding of AAV/beta -galactosidase in the presence of excess competing wild-type AAV on COS cells (A), 293 cells (B), and human airway epithelia (C). Data are mean binding ± S.E. (n = 3-9). Conditions denoted as ND were not done.

Saturation and competition studies suggest AAV2 and AAV5 use different receptors to bind to cells, and the number of receptors varies among cell lines. As previously shown, the apical surface of airway epithelia lack high affinity receptors for AAV2. In contrast, the apical surface of well differentiated human airway epithelia have high affinity receptors for AAV5.

Hemagglutination with AAV5 Requires Sialic Acid-- If binding molecules are present on erythrocytes, then addition of virus will result in hemagglutination. Hence, the hemagglutination assay is a simple way to assess virus binding to cells (35, 47). AAV5 has been shown to agglutinate erythrocytes.2 We observed hemagglutination of Rhesus monkey erythrocytes with AAV5 but not AAV2 (Fig. 3A). In some cases, hemagglutination requires glycosylation of the erythrocytes; for example, influenza virus requires sialic acid, and human parvovirus requires globoside (35-37, 53). It is not known if erythrocyte glycosylation is required for hemagglutination with the dependent parvoviruses. Therefore, we treated cells with glycosidases and assayed hemagglutination with AAV5. Fig. 3B shows that pretreating erythrocytes with sialidase inhibited AAV5-dependent hemagglutination (Fig. 3B). Hence, we hypothesized that sialic acid may play a role in binding and infection of cells or epithelia with AAV5.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3.   Hemagglutination with AAV5 and AAV2. Rhesus monkey erythrocytes treated with a dilution series of either AAV5 or AAV2 (A). Viral concentration decreased in 2-fold dilutions from left to right. Erythrocytes pellet to the bottom of round-bottom wells in the absence of agglutination. Hemagglutination with AAV5 following pretreatment of erythrocytes with sialidase (B).

Binding and Infection with AAV5 Requires Sialic Acid-- Several parvoviruses agglutinate erythrocytes, and some of these viruses require sialic acid for hemagglutination (38-41, 54). Interestingly, not all parvoviruses that require sialic acid for hemagglutination also require sialic acid for binding and infection of host cells (39, 41). For instance, canine parvovirus requires sialic acid to hemagglutinate, but it does not require sialic acid to infect cells. Thus, the significance of sialic acid with respect to the biology of the parvoviruses is not well understood. Therefore, we tested the hypothesis that binding and gene transfer by AAV5 requires sialic acid. Sialidase treatment of COS cells, 293 cells, or airway epithelia did not reduce either binding or gene transfer with AAV2 (Fig. 4). In contrast, sialidase treatment markedly decreased both binding and gene transfer with AAV5. These data suggest that AAV5 may bind to sialic acid, and this interaction plays a role in gene transfer.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Sialidase treatment and binding and gene transfer with AAV. Binding of AAV2 and AAV5 to COS cells (A), 293 cells (C), and human airway epithelia (E) following sialidase pretreatment. Gene transfer with AAV2 and AAV5 in COS cells (B), 293 cells (D), and airway epithelia (F) following sialidase pretreatment. Data are mean binding or expression ± S.E. (n = 3-9).

To test this hypothesis further, we measured binding and gene transfer to CHO cells deficient in cell surface sialic acid. As shown in Fig. 5, AAV2 bound and infected both the parental cell line (Pro-5) and the sialic acid-deficient mutant (Lec-2). In contrast, AAV5 bound and infected the Pro-5 cells but failed to bind or infect the sialic acid-deficient mutant (Lec-2). Hence, sialic acid was required not only for AAV5-dependent hemagglutination but also binding and infection of cell lines and differentiated human airway epithelia. These data indicate that sialic acid plays an important role as a receptor for AAV5.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Binding and gene transfer in sialic acid-deficient cell lines. Binding of AAV2 and AAV5 to CHO cells (Pro-5) and sialic acid-deficient CHO cells (Lec-2) (A). Gene transfer with AAV2 and AAV5 in CHO cells (B). Data are mean binding or expression ± S.E. (n = 3-9).

Hemagglutination with AAV5 Requires 2,3-Linked Sialic Acid-- Cells process several different types of sialic acid onto their cell surface, and AAV5 may have specificity for a particular type. Since the sialic acid linkage determines the specificity of another virus, influenza virus, we focused on the importance of different linkages of sialic acid with respect to the specificity of AAV5 (55, 56). If AAV5 specifically binds to certain linkages of sialic acid, then resialylation of cells with the correct linkage might rescue AAV5 binding. To test this, we first treated Rhesus monkey erythrocytes with sialidase to remove all sialic acid. Then we applied 2,3- or 2,6-sialyltransferase to attach free sialic acid via a 2,3- or 2,6-linkage, respectively, to the terminal position of the glycosylation chain (47). Most important, the 2,3-sialyltransferase, but not the 2,6-sialyltransferase, rescued hemagglutination (Fig. 6). These data suggest that AAV5 specifically interacts with 2,3-linked sialic acid.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 6.   Sialic acid linkage specificity and hemagglutination with AAV5. Sialidase-treated Rhesus monkey erythrocytes were resialylated by treatment with either 2,3- or 2,6-sialyltransferase and incubated with AAV5. Viral concentration decreases in 2-fold dilutions from left to right. Erythrocytes pellet to the bottom of round-bottom wells in the absence of agglutination.

Specific Linkages of Sialic Acid on the Surface of Cells and Epithelia-- AAV5 binding and gene transfer to 293 cells was inefficient. However, these cells are of mammalian origin and thus unlikely to be devoid of sialic acid on their cell surface (57). Three possible explanations for this result include the following: 1) they may have very little sialic acid; 2) sialic acid may not be sufficient for infection with AAV5; or 3) they may efficiently process only a specific type of sialic acid onto their cell surface. Since the sialic acid linkage determines the specificity of AAV5-dependent hemagglutination, we focused on the importance of different linkages of sialic acid with respect to the tropism of AAV5. We asked if COS cells, 293 cells, and differentiated human airway epithelia display different linkages of sialic acid on their cell surface. We stained cells lines and the apical surface of epithelia with lectins that have been used to recognize four different epitopes as follows: 1) WGA recognizes all sialic acid; 2) MAA recognizes 2,3-linked sialic acid; 3) SNA recognizes 2,6-linked sialic acid; and 4) ConA recognizes alpha -mannose (58). WGA bound well to both of the cell lines and to the epithelial apical membrane (Fig. 7). Since all three culture systems display sialic acid, the presence or absence of sialic acid alone could not account for the differences in AAV5 permissiveness. SNA bound to 293 cells but not COS cells. Because AAV5 does not bind or infect 293 cells well, this finding suggests that 2,6-linked sialic acid is not the receptor for AAV5. In contrast, MAA bound COS cells but not 293 cells. Because AAV5 binds and infects COS cells more efficiently than 293 cells, this observation supports the hypothesis that AAV5 uses 2,3-linked sialic acid for binding and infection. This hypothesis was further supported because MAA also bound the apical surface of human airway epithelia.


View larger version (116K):
[in this window]
[in a new window]
 
Fig. 7.   Lectin binding to COS cells, 293 cells, and differentiated human airway epithelia. Cultures were stained with FITC-labeled lectins that bind to four different carbohydrates as follows: WGA binds sialic acid in any linkage, MAA binds 2,3-linked sialic acid, SNA binds 2,6-linked sialic acid, and ConA binds alpha -mannose.

Specificity of AAV5 for 2,3-Linked Sialic Acid-- If AAV5 binding and infection requires a 2,3-linked sialic acid, we predicted MAA should block AAV5 in COS cells. WGA and MAA, but not SNA or ConA, inhibited both binding and gene transfer by AAV5 on COS cells, indicating that 2,3-linked sialic acid is required for binding and gene transfer with AAV5 (Fig. 8). As a control, we studied the effect of lectins on AAV2. Neither MAA, SNA, or ConA had a significant impact on binding or gene transfer by AAV2 on COS cells. WGA did not decrease binding of AAV2 but had a small effect on infection. This is probably due to its ability to block nuclear pores (59). In human airway epithelia, the lectins had no impact on the minimal binding or gene transfer with AAV2, but as in COS cells, WGA and MAA inhibited both binding and infection with AAV5. These data confirm that 2,3-linked sialic acid is either a receptor for AAV5 or it is a key component of a receptor complex.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 8.   Specificity of sialic acid linkage with respect to binding and infection with AAV2 and AAV5. Binding of AAV in the continued presence of excess competing lectin on COS cells (A) and differentiated human airway epithelia (C). Infection with AAV in the continued presence of excess competing lectin on COS cells (B) and differentiated human airway epithelia (D). Data are mean binding or expression ± S.E. (n = 3-9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our data show that AAV5 requires 2,3-linked sialic acid for binding and gene transfer; this finding has significant implications for the use of AAV5 in gene transfer. Sialic acid is the most common terminal glycosyl residue on mammalian proteins; however, cells can present sialic acid in several different linkages, and some linkages are selectively restricted. For example, humans restrict 2,8-linked sialic acid to the central nervous system, and it is only present during certain stages of development (60, 61). In contrast, 2,3-linked sialic acid is the most abundant linkage, suggesting that AAV5 has the potential to transfer genes to a wide variety of tissues in addition to the airway epithelia.

Although our studies indicate that AAV5 binds to 2,3-linked sialic acid, we do not know if other molecular determinants or other coreceptors are involved in infection with AAV5. Specifically, AAV5 may require sialic acid on particular glycoproteins or glycolipids. In addition, AAV5 may also require additional molecules to act as coreceptors. Of interest, we have not identified cells that express 2,3-linked sialic acid but fail to bind or infect with AAV5, suggesting the identification of additional determinants might be difficult.

Influenza virus strains isolated from humans bind 2,6-linked sialic acid, whereas influenza strains isolated from equine and avian species bind 2,3-linked sialic acid (55, 56). Given this knowledge of influenza virus infection, we were surprised that AAV5 has specificity for 2,3-linked sialic acid. These observations suggest either AAV5 infects differently than influenza, sialic acid may not be sufficient for AAV5, or the influenza data could be misleading because human influenza viruses are passaged in eggs before they are studied.3

Previous studies suggest other parvoviruses adsorb to sialic acid. For instance, canine parvovirus, feline panleukopenia virus, and the minute virus of mice all require sialic acid for hemagglutination (38-41, 54). However, it is not known if they have specificity for particular linkages of sialic acid. Moreover, in the case of canine parvovirus and feline panleukopenia virus, the interaction with sialic acid is not required for infection, suggesting that for these viruses the hemagglutination assay may be misleading (38, 39, 41). In contrast, our work demonstrates that AAV5 requires sialic acid for hemagglutination, binding to the cell surface, and infection. In addition, AAV5 has specificity for 2,3-linked sialic acid. Hence, AAV5 may interact with sialic acid in a novel fashion.

In conclusion, these data show that binding of AAV5 to the apical surface of differentiated human airway epithelia involves a receptor. Moreover, 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Our findings have important implications for the use of AAV vectors in cystic fibrosis transmembrane conductance regulator gene transfer to the airway epithelia. However, the utility of AAV5 vectors may reach beyond their potential for targeting the airway. Given the prevalence of 2,3-linked sialic acid on mammalian cells, the use of AAV5 vectors may facilitate gene transfer to a variety of as yet inaccessible tissues.

    ACKNOWLEDGEMENTS

We thank Michael Seiler, Janice Launspach, Tom Moninger, Phil Karp, Pary Weber, Tamara Nesselhauf, Beverly Handelman, Theresa Mayhew, Christine McLennan, and Rosanna Smith for excellent assistance. We also thank Dr. John Engelhardt and Dr. Nick Kaludov for discussions and comments. We especially appreciate the help of ISOPO and IIAM for the human lungs. We appreciate the support of the University of Iowa Gene Transfer Vector Core and the In Vitro Cell Models Core.

    FOOTNOTES

* This work was supported by Center for Gene Therapy, NIDDKD, National Institutes of Health Grant T30DK54759, the Cystic Fibrosis Foundation, and the Roy J. Carver Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ Investigator of the Howard Hughes Medical Institute.

|||| To whom correspondence should be addressed: University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-353-5511; Fax: 319-335-7623; E-mail: joseph-zabner@uiowa.edu.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101559200

2 N. Kaludov, K. E. Brown, R. W. Walters, J. Zabner, and J. A. Chiorini, manuscript in preparation.

3 Slepushkin, V. A., Staber, P. D., Wang, G., McCray, P. B., and Davidson, B. L. (2001) Mol. Ther. 3, 395-402.

    ABBREVIATIONS

The abbreviations used are: AAV, adeno-associated viruses; WGA, wheat germ agglutinin; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; EMEM, Eagle's minimal essential media; MAA, maackia amurensis lectin; SNA, sambucus nigra lectin; ConA, concanavalin A; pt/ml, particles/ml.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Muzyczka, N. (1992) Curr. Top Microbiol. Immunol. 158, 97-129[Medline] [Order article via Infotrieve]
2. Flotte, T. R., Afione, S. A., Solow, R., Drumm, M. L., Markakis, D., Guggino, W. B., Zeitlin, P. L., and Carter, B. J. (1993) J. Biol. Chem. 268, 3781-3790[Abstract/Free Full Text]
3. Flotte, T. R., and Carter, B. J. (1995) Gene Ther. 2, 357-362[Medline] [Order article via Infotrieve]
4. Fisher, K. J., Jooss, K., Alston, J., Yang, Y., Haecker, S. E., High, K., Pathak, R., Raper, S. E., and Wilson, J. M. (1997) Nat. Med. 3, 306-312[Medline] [Order article via Infotrieve]
5. Snyder, R. O., Miao, C. H., Patijn, G. A., Spratt, S. K., Danos, O., Nagy, D., Gown, A. M., Winther, B., Meuse, L., Cohen, L. K., Thompson, A. R., and Kay, M. A. (1997) Nat. Genet. 16, 270-276[Medline] [Order article via Infotrieve]
6. Kurpad, C., Mukherjee, P., Wang, X. S., Ponnazhagan, S., Li, L., Yoder, M. C., and Srivastava, A. (1999) J. Hematother. Stem Cell Res. 8, 585-592[CrossRef][Medline] [Order article via Infotrieve]
7. Kay, M. A., Manno, C. S., Ragni, M. V., Larson, P. J., Couto, L. B., McClelland, A., Glader, B., Chew, A. J., Tai, S. J., Herzog, R. W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A. W., and High, K. A. (2000) Nat. Genet. 24, 257-261[CrossRef][Medline] [Order article via Infotrieve]
8. Muramatsu, S., Mizukami, H., Young, N. S., and Brown, K. E. (1996) Virology 221, 208-217[CrossRef][Medline] [Order article via Infotrieve]
9. Chiorini, J. A., Yang, L., Liu, Y., Safer, B., and Kotin, R. M. (1997) J. Virol. 71, 6823-6833[Abstract]
10. Rutledge, E. A., Halbert, C. L., and Russell, D. W. (1998) J. Virol. 72, 309-319[Abstract/Free Full Text]
11. Chiorini, J. A., Kim, F., Yang, L., and Kotin, R. M. (1999) J. Virol. 73, 1309-1319[Abstract/Free Full Text]
12. Bantel-Schaal, U., Delius, H., Schmidt, R., and zur Hausen, H. (1999) J. Virol. 73, 939-947[Abstract/Free Full Text]
13. Xiao, W., Chirmule, N., Berta, S. C., McCullough, B., Gao, G., and Wilson, J. M. (1999) J. Virol. 73, 3994-4003[Abstract/Free Full Text]
14. Samulski, R. J., Berns, K. I., Tan, M., and Muzyczka, N. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2077-2081[Abstract]
15. McCown, T. J., Xiao, X., Li, J., Breese, G. R., and Samulski, R. J. (1996) Brain Res. 713, 99-107[CrossRef][Medline] [Order article via Infotrieve]
16. Guy, J., Qi, X., Muzyczka, N., and Hauswirth, W. W. (1999) Arch. Ophthalmol. 117, 929-937[Abstract/Free Full Text]
17. Wagner, J. A., Reynolds, T., Moran, M. L., Moss, R. B., Wine, J. J., Flotte, T. R., and Gardner, P. (1998) Lancet 351, 1702-1703[Medline] [Order article via Infotrieve]
18. Duan, D., Yue, Y., Yan, Z., McCray, P. B., Jr., and Engelhardt, J. F. (1998) Hum. Gene Ther. 9, 2761-2776[Medline] [Order article via Infotrieve]
19. Teramoto, S., Bartlett, J. S., McCarty, D., Xiao, X., Samulski, R. J., and Boucher, R. C. (1998) J. Virol. 72, 8904-8912[Abstract/Free Full Text]
20. Beck, S. E., Jones, L. A., Chestnut, K., Walsh, S. M., Reynolds, T. C., Carter, B. J., Askin, F. B., Flotte, T. R., and Guggino, W. B. (1999) J. Virol. 73, 9446-9455[Abstract/Free Full Text]
21. Halbert, C. L., Alexander, I. E., Wolgamot, G. M., and Miller, A. D. (1995) J. Virol. 69, 1473-1479[Abstract]
22. Halbert, C. L., Standaert, T. A., Wilson, C. B., and Miller, A. D. (1998) J. Virol. 72, 9795-9805[Abstract/Free Full Text]
23. Bals, R., Xiao, W., Sang, N., Weiner, D. J., Meegalla, R. L., and Wilson, J. M. (1999) J. Virol. 73, 6085-6086[Abstract/Free Full Text]
24. Walters, R. W., Duan, D., Engelhardt, J. F., and Welsh, M. J. (2000) J. Virol. 74, 535-540[Abstract/Free Full Text]
25. Zabner, J., Seiler, M., Walters, R., Kotin, R. M., Fulgeras, W., Davidson, B. L., and Chiorini, J. A. (2000) J. Virol. 74, 3852-3858[Abstract/Free Full Text]
26. Summerford, C., and Samulski, R. J. (1998) J. Virol. 72, 1438-1445[Abstract/Free Full Text]
27. Summerford, C., Bartlett, J. S., and Samulski, R. J. (1999) Nat. Med. 5, 78-82[CrossRef][Medline] [Order article via Infotrieve]
28. Qing, K., Mah, C., Hansen, J., Zhou, S., Dwarki, V., and Srivastava, A. (1999) Nat. Med. 5, 71-77[CrossRef][Medline] [Order article via Infotrieve]
29. Qiu, J., Mizukami, H., and Brown, K. E. (1999) Nat. Med. 5, 467-468[CrossRef][Medline] [Order article via Infotrieve]
30. Qiu, J., Handa, A., Kirby, M., and Brown, K. E. (2000) Virology 269, 137-147[CrossRef][Medline] [Order article via Infotrieve]
31. Handa, A., Muramatsu, S., Qui, J., Mizukami, H., and Brown, K. E. (2000) J. Gen. Virol. 81, 2077-2084[Abstract/Free Full Text]
32. Davidson, B. L., Stein, C. S., Heth, J. A., Martins, I., Kotin, R. M., Derksen, T. A., Zabner, J., Ghodsi, A., and Chiorini, J. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3428-3432[Abstract/Free Full Text]
33. Alisky, J. M., Hughes, S. M., Sauter, S. L., Jolly, D., Dubensky, T. W., Jr., Staber, P. D., Chiorini, J. A., and Davidson, B. L. (2000) Neuroreport 11, 2669-2673[Medline] [Order article via Infotrieve]
34. Chao, H., Liu, Y., Rabinowitz, J., Li, C., Samulski, R. J., and Walsh, C. E. (2000) Mol. Ther. 2, 619-623[CrossRef][Medline] [Order article via Infotrieve]
35. Hirst, G. K. (1950) J. Exp. Med. 91, 161-176
36. Herrler, G., Rott, R., and Klenk, H. D. (1985) Virology 141, 144-147[Medline] [Order article via Infotrieve]
37. Rogers, G. N., Herrler, G., Paulson, J. C., and Klenk, H. D. (1986) J. Biol. Chem. 261, 5947-5951[Abstract/Free Full Text]
38. Goto, H., Yachida, S., Shirahata, T., and Shimizu, K. (1974) Nippon Juigaku Zasshi 36, 203-211[Medline] [Order article via Infotrieve]
39. Goto, H. (1975) Nippon Juigaku Zasshi 37, 239-245[Medline] [Order article via Infotrieve]
40. Carmichael, L. E., Jourbert, J. C., and Pollock, R. V. (1980) Am. J. Vet. Res. 41, 784-791[Medline] [Order article via Infotrieve]
41. Barbis, D. P., Chang, S. F., and Parrish, C. R. (1992) Virology 191, 301-308[Medline] [Order article via Infotrieve]
42. Kondo, M., Finkbeiner, W. E., and Widdicombe, J. H. (1991) Am. J. Physiol. 261, L106-L117[Abstract/Free Full Text]
43. Karp, P. P., Moninger, T. O., Weber, S. P., Nesselhaug, T. S., Launspach, J. L., Zabner, J., and Welsh, M. J. (2001) in Epithelial Cell Culture Protocols (Wise, C., ed) , Humana Press, Inc., Totowa, NJ, in press
44. Yamaya, M., Finkbeiner, W. E., Chun, S. Y., and Widdicombe, J. H. (1992) Am. J. Physiol. 262, L713-L724[Abstract/Free Full Text]
45. Zabner, J., Zeiher, B. G., Friedman, E., and Welsh, M. J. (1996) J. Virol. 70, 6994-7003[Abstract]
46. Pickles, R. J., Fahrner, J. A., Petrella, J. M., Boucher, R. C., and Bergelson, J. M. (2000) J. Virol. 74, 6050-6057[Abstract/Free Full Text]
47. Paulson, J. C., and Rogers, G. N. (1987) Methods Enzymol. 138, 162-168[Medline] [Order article via Infotrieve]
48. Limbird, L. E. (1996) Cell Surface Receptors: A Short Course on Theory and Methods , pp. 73-122, Kluwer Academic Publishers, Norwell, MA
49. Zabner, J., Freimuth, P., Puga, A., Fabrega, A., and Welsh, M. J. (1997) J. Clin. Invest. 100, 1144-1149[Abstract/Free Full Text]
50. Duan, D., Yue, Y., Yan, Z., Yang, J., and Engelhardt, J. F. (2000) J. Clin. Invest. 105, 1573-1587[Abstract/Free Full Text]
51. Tardieu, M., Epstein, R. L., and Weiner, H. L. (1982) Int. Rev. Cytol. 80, 27-61[Medline] [Order article via Infotrieve]
52. Persson, R., Wohlfart, C., Svensson, U., and Everitt, E. (1985) J. Virol. 54, 92-97[Medline] [Order article via Infotrieve]
53. Brown, K. E., Anderson, S. M., and Young, N. S. (1993) Science 262, 114-117[Medline] [Order article via Infotrieve]
54. Cotmore, S. F., and Tattersall, P. (1989) J. Virol. 63, 3902-3911[Medline] [Order article via Infotrieve]
55. Rogers, G. N., and Paulson, J. C. (1983) Virology 127, 361-373[Medline] [Order article via Infotrieve]
56. Rogers, G. N., Pritchett, T. J., Lane, J. L., and Paulson, J. C. (1983) Virology 131, 394-408[Medline] [Order article via Infotrieve]
57. Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G., and Marth, J. (1999) Essentials of Glycobiology , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
58. Vierbuchen, M. J., Fruechtnicht, W., Brackrock, S., Krause, K. T., and Zienkiewicz, T. J. (1995) Cancer (Phila.) 76, 727-735[Medline] [Order article via Infotrieve]
59. Tsuchiya, E., Hiraga, K., Fukui, S., and Miyakawa, T. (1989) FEBS Lett. 250, 285-288[CrossRef][Medline] [Order article via Infotrieve]
60. Finne, J. (1982) J. Biol. Chem. 257, 11966-11970[Abstract/Free Full Text]
61. Zuber, C., Lackie, P. M., Catterall, W. A., and Roth, J. (1992) J. Biol. Chem. 267, 9965-9971[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.