Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions - PubMed (original) (raw)

Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions

C Summerford et al. J Virol. 1998 Feb.

Abstract

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.

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Figures

FIG. 1

Inhibition of AAV infection by various GAGs. (A) rAAV was incubated with the indicated concentrations of heparin (▪), chondroitin sulfate B (dermatan sulfate) (○), chondroitin sulfate A (◊), or chondroitin sulfate C (▵) for 1 h at 37°C prior to a 1-h adsorption of the virus-GAG mixture to HeLa cells for infection. β-Galactosidase activity was assayed 44 h postinfection by using a Galacto-Light Plus kit (Tropix Inc.) and measured in a luminometer. Each point represents the average percent decrease in RLU per microgram of protein relative to the maximum level obtained in experiments without GAG. (B) HeLa cells were preincubated with increasing concentrations of heparin at 37°C for 1 h. After thorough washing, cells were infected with rAAV as described above. Data points represent the average percent maximum RLU/microgram of protein obtained without heparin preincubation.

FIG. 2

Soluble heparin inhibits binding of AAV to the cell surface. After preincubation of 3H-labeled wt AAV-2 with increasing concentrations of the indicated GAGs or the GAG analog dextran sulfate, labeled virus was adsorbed to HeLa cells for 90 min at 4°C. Unbound virus was removed by three washes with ice-cold binding buffer, and radioactivity was quantitated as described in Materials and Methods. Data are represented as the average percent inhibition relative to the counts per minute bound in the absence of soluble GAG.

FIG. 3

Effect of enzymatic digestion of cell surface GAGs on AAV binding and infection. (A) HeLa cells were treated with the indicated concentrations of the GAG lyase heparitinase, heparinase I (▪), heparinase I (◊), chondroitinase ABC (○), or chondroitinase AC (▵) as described in Materials and Methods. After thorough washing, the ability of AAV to bind the cell surface was assessed as described for Fig. 2. Data points represent the average percent reduction in AAV binding relative to AAV binding obtained without enzymatic treatment. (B) HeLa cells were treated with heparitinase or heparinase I as described in Materials and Methods. After thorough washing, rAAV was incubated with cells for a 1-h adsorption period at 37°C. Cells were harvested 44 h postinfection and assayed for β-galactosidase activity. Results are graphed as the average percent reduction in AAV transduction relative to transduction observed in the absence of enzymatic treatment. Data points represent the mean and standard deviation of experiments performed in triplicate.

FIG. 4

HS proteoglycan serves as a primary attachment receptor for AAV-2. Wild-type CHO-K1 cells and CHO-K1 mutants defective in proteoglycan synthesis were assessed for the ability to bind AAV-2. Cell line pgsA-745 lacks HS and chondroitin sulfate proteoglycans; pgsD-677 lacks HS proteoglycan and produces a threefold excess of chondroitin sulfate proteoglycans; pgsB-618 produces 15% of normal proteoglycans; pgsE-606 produces an undersulfated form of HS proteoglycan and normal levels of chondroitin sulfate proteoglycans. (A) Cy3-labeled AAV-2 was bound to wt CHO cells (I) and the pgsA-745 mutant that lacks proteoglycans (II) as described in Materials and Methods. Images were captured by confocal microscopy. (B) Binding of 3H-AAV to parental and mutant CHO cells. Binding assays were performed at 4°C in Eppendorf tubes. A total of 3 × 105 cells were incubated with 4 × 1011 particles of 3H-AAV for 90 min in HBSB. After thorough washing, cells were pelleted and solubilized, and radioactivity was quantitated as described in Materials and Methods. Nonspecific binding was determined by parallel binding studies done in the presence of a 100-fold excess of unlabeled virus. Data represent the mean specific binding and standard deviation obtained from experiments performed in triplicate.

FIG. 5

HS proteoglycan mediates AAV infection. (A) AAV infection of wt and mutant CHO cells deficient in proteoglycan synthesis (described in the legend to Fig. 4). rAAV-LacZ virus was incubated with cells at an MOI of 10 for 1 h at 37°C. Cells were harvested 44 h postinfection and assayed for β-galactosidase activity. Data represent the mean and standard deviation of triplicate experiments. (B) UV treatment of wild-type and mutant CHO cells and its effect on rAAV transduction. Cells were treated with UV (45 J/m2) in a UV Stratalinker (Stratagene) prior to infection with rAAV-LacZ as described above. β-Galactosidase activity was measured as described for non-UV-treated cells.

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Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions - PubMed (original) (raw)