Integrin alpha(v)beta1 is an adenovirus coreceptor

Abstract

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

FIG. 1

Adenovirus infection of HEK293 cells is mediated by fiber and penton base proteins. (A) Fiber protein mediates Ad binding to HEK293 cells. HEK293 cells (1.5 × 10⁶ cells/sample, in duplicate) in suspension were preincubated with recombinant Ad2 fiber, penton base (10 μg/ml), the anti-CAR antibody (RmcB), anti-penton base antibody (DAV-1), or anti-integrin antibodies at 20 μg/ml at 4°C for 60 min. Specific Ad binding to cells was determined by using ¹²⁵I-labeled Ad2 as previously described (13). (B) Penton base interaction with αv integrins promotes Ad-mediated gene delivery. HEK293 cells (10⁶ cells/sample) were preincubated with fiber protein, anti-CAR, anti-penton base, or anti-integrin antibodies (20 μg/ml) as described above, followed by incubation with Ad.CMV.LacZ at an MOI of 1 particle/cell at 4°C for another 30 min. The cells were then warmed to 37°C for 15 min. Noninternalized virus was removed by trypsinization. Ad-mediated gene delivery was determined at 20 h postinfection by expression of β-galactosidase. Representative data from two independent experiments were plotted as the mean ± standard deviation.

FIG. 2

Flow cytometric analysis of integrin expression on HEK293 cells. HEK293 or A549 cells were incubated with 20 μg of anti-integrin antibodies/ml at 4°C for 60 min. Cell surface-bound antibody was detected by incubation with an R-phycoerythrin-labeled goat anti-mouse secondary antibody. Dashed line, secondary antibody control; solid line, indicated integrin antibodies.

FIG. 3

Immunoprecipitation analyses of integrin expression on HEK293 cells. HEK293 (A) and A549 (B) cells (10⁷ cells) were detached with 1 mM EDTA and then biotinylated with 1 mg of sulfo-N-hydroxysuccinimide-biotin. After being washed with phosphate-buffered saline four times, labeled cells were then lysed in 1 ml of lysis buffer containing 1% Nonidet P-40 and protease inhibitors. Lysates from biotinylated cells (equal to 10⁶ cells) were immunoprecipitated with 2 μg of anti-integrin antibodies followed by capture with protein A/G beads. The immunoprecipitates were then separated on 6% sodium dodecyl sulfate gels under reducing conditions. After transfer to a polyvinylidene difluoride membrane, the filter was probed with a horseradish peroxidase-conjugated anti-biotin antibody.

FIG. 4

Integrin-dependent cell adhesion to fibronectin (A) or penton base (B). Non-tissue culture-treated cluster plates were precoated with 10 μg of recombinant penton base or fibronectin/ml. HEK293 cells were preincubated with 20 μg of anti-integrin antibodies/ml before the cells were allowed to attach to coated plates for 15 min at 37°C. Attached cells were quantitated after fixation and stained with crystal violet. Data are presented as the mean ± standard deviation of duplicate samples. Antibodies: LM609, anti-αvβ3; P1F6, anti-αvβ5; L230, anti-αv; P4C10, anti-β1; P1D6, anti-α5; 1973z, anti-α1.

FIG. 5

Integrin αvβ1 promotes Ad-mediated gene delivery and virus internalization. (A) HEK293 cells in suspension were preincubated with anti-integrin antibodies for 60 min at 4°C, followed by incubation with Ad.CMV.LacZ at an MOI of 1 viral particle/cell. After being warmed to 37°C for 15 min, noninternalized virus was removed by trypsinization. Cells were plated in 6-well plates, and β-galactosidase expression was determined at 20 h postinfection (mean ± standard deviation). (B) HEK293 cells were pretreated with 20 μg of anti-integrin antibodies/ml at 4°C for 60 min, followed by the addition of ¹²⁵I-labeled Ad2 (2 × 10⁵ cpm/cell). Bound virus particles were then allowed to internalize by warming the cells at 37°C for 15 min. Internalized virions were determined by measuring their resistance to trypsinization. The data represent the percentage of trysin-resistant cpm/total cpm of specifically bound Ad ± standard deviation of duplicate samples.