Integrin αvβ1 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 ␣v␤3 or ␣v␤5 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 ␣v␤1. Function-blocking antibodies directed against ␣v or ␤1, but not ␤3, ␤5, or ␣5, integrin subunits block Ad infection and viral endocytosis. Therefore, ␣v␤1 serves as a coreceptor for Ad infection, and the lack of ␤3 and/or ␤5 but the relatively high expression of ␣v␤1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.Adenovirus (Ad) host cell entry requires initial attachment to cells mediated by the fiber interaction with its cellular receptor CAR. The subsequent association of penton base with ␣v␤3 or ␣v␤5 integrins promotes Ad entry (1,21,23). Integrins are heterodimeric receptors for extracellular matrix proteins and cell surface counterreceptors. ␣v integrins (␣v␤1, ␣v␤3, ␣v␤5, ␣v␤6, and ␣v␤8) mediate cell adhesion to various matrix proteins, including fibronectin, vitronectin, and fibrinogen, that contain an arginine-glycine-aspartic acid (RGD) sequence (8,19,24). The interaction of the Ad penton base and ␣v integrin triggers the activation of several signaling molecules (14) that promote actin cytoskeletal reorganization and enhance Ad internalization. Although HEK293 cells have been widely used for propagation of recombinant Ad vectors, the integrin repertoire of these cells has not been clearly established. For example, several reports indicate that HEK293 cells do not express ␣v␤3 and ␣v␤5 integrins (10, 18), while another report indicates that they do (6). Furthermore, ␤5 integrin-deficient mouse fibroblast cells support Ad infection, suggesting that other integrins play a role in Ad infection (11). HEK293 cells were also reported to express ␣5␤1 and ␣v␤1 integrins (2), which have been identified as RGD-dependent receptors for both fibronectin and vitronectin (2,15,22). Considering the fact that soluble fibronectin or RGD-containing peptide reduced Ad infection (23) and that an ␣5␤1-activating antibody has been reported to enhance Ad-mediated gene delivery to certain melanoma cells (4), it was of interest to determine whether Ad uses either ␣v␤1 and/or ␣5␤1 integrins as alternative receptors for virus internalization.The role of CAR and ␣v integrins in Ad infection of HEK293 cells. To investigate whether CAR or integrins promote Ad attachment, we preincubated HEK293 cells with an excess of recombinant Ad type 2 (Ad2) fiber protein or with anti-CAR monoclonal antibody (MAb ) (RmcB), with a function-blocking Fab fragment of a penton base MAb (DAV-1) (20), or with function-blocking ␣v␤5 (P1F6), ␣5 (P1D6), or nonfunctionblocking ␣v (LM142) control MAb prior to measuring the specific binding of 125 I-labeled Ad5 particles (Fig. 1A) as previously described (13). Pretreatment of cells with recombi...

Insulin-like Growth Factor-independent Effects Mediated by a C-terminal Metal-binding Domain of Insulin-like Growth Factor Binding Protein-3

“…In particular, it might be important to establish whether such discrimination, if it exists, could lead to the development of therapeutic molecules that can target cell environments characteristic of disease processes such as cancer or atherosclerosis. ␣ v ␤ 1 integrin is a major integrin displayed by HEK293 cells in culture and serves as a coreceptor for adenovirus (43). ␣ v ␤ 1 integrins, in general, are preferentially displayed by invasive tumors, as well as in inflammatory disease, retinopathy, and other disease states (12, 13); a cationic nanoparticle coupled to an integrin ␣ v ␤ 3 -targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice (14).…”

Interaction of penton base Arg-Gly-Asp motifs with integrins is crucial for adenovirus serotype 35 vector transduction in human hematopoietic cells

“…28 Several types of integrins, such as a v b 3 , a v b 5 and a v b 1 , mediate efficient transduction with Ad5 vectors. [2][3][4][5][6] The structures of the Ad5 fiber and penton base allow easy access of penton base RGD motifs to integrins on the cell surface. Ad5 possess a long (22 b-repeats), and flexible fiber shaft.…”

Molecular basis of the inflammatory response to adenovirus vectors

“…19 Following the highaffinity binding, a lower affinity interaction between a v integrins (a v b 3 , a v b 5 , a v b 1 ) and an arginine-glycineaspartic acid (RGD) motif on the adenovirus penton base capsid protein mediates virus internalization by receptor-mediated endocytosis through clathrin-coated vesicles. 20,21 At 10 min following internalization, pHdependent penetration of the endosome occurs. 22 Adenovirus penetration into the cytoplasm is believed to involve a pH-dependent conformational change in the adenovirus penton base and an interaction with a v b 5 integrins.…”