Adenovirus endocytosis via alpha(v) integrins requires phosphoinositide-3-OH kinase

Abstract

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

FIG. 1

Phosphorylation events occurring during adenovirus interaction with cells. SW480 cells were incubated with adenovirus at an MOI of 200 PFU or with 1 μg of recombinant penton base or fiber for 10 min at 37°C. Cell lysates were then immunoprecipitated with an antiphosphotyrosine antibody followed by Western blotting with an anti-FAK (upper panel) or anti-p85/PI3K antibody (lower panel) as described in Materials and Methods. The protein bands were quantitated by densitometry. Control cell samples were incubated in medium alone.

FIG. 2

PI3K activation during adenovirus entry into cells. SW480 cells were incubated with adenovirus at an MOI of 200 PFU for various times at 37°C, and cell extracts were immunoprecipitated with an antiphosphotyrosine antibody. The kinase reaction was performed with the immune precipitates with [γ-³²P]ATP and PI(4,5)-bisphosphate as a substrate. Following solvent extractions and washes, the reaction product, PIP₃, was detected by thin-layer chromatography (top) and quantitated by phosphorimager densitometry (bottom). Control cell samples were incubated in medium without adenovirus. Cells were also incubated with EGF for 10 min at 37°C as a positive control for PI3K activation. The data are representative of four experiments.

FIG. 3

Adenovirus penton base interaction with α_v integrins promotes PI3K activation. (A) SW480 epithelial cells were incubated with Ad2, recombinant penton base (PB), fiber proteins, or EGF for 10 min at 37°C and then analyzed for PI3K activation as described in the legend to Fig. 2. The results represent the average of duplicate samples (+ standard deviation) and are representative of at least three separate experiments. (B) α_v integrin-expressing M21-L4 cells or α_v integrin-negative M21-L12 cells were incubated with adenovirus particles for 10 min at 37°C prior to analyzing cell lysates for PI3K activation by using phosphatidylinositol as a substrate.

FIG. 4

Effect of wortmannin on adenovirus-induced PI3K activation (A), virus internalization (B), and gene delivery (C). SW480 cells were treated with various concentrations of wortmannin for 10 min at 37°C and then incubated with adenovirus particles for an additional 10 min at 37°C. PI3K activation was analyzed as described in the legend to Fig. 2 by using PI(4,5)-bisphosphate as a substrate. Cells were also treated with various concentrations of wortmannin at 4°C for 10 min followed by preincubation with ¹²⁵I-labeled adenovirus for 30 min at 4°C. After removing unbound virus by washing, the cells were resuspended in PBS and then warmed to 37°C for 10 min. Wortmannin was present throughout the internalization process. Internalization was measured by resistance to trypsin digestion as described in Materials and Methods. In separate studies to analyze virus infection, SW480 cells were pretreated with various amounts of wortmannin or with a 2 μM concentration of the myosin light chain kinase inhibitor ML7 or with 20 μM of the ERK1/ERK2 MAP kinase inhibitor PD98059 (C, inset). β-Galactosidase activity in virally transduced cells was measured 48 h postinfection by use of a colorimetric assay (A₆₀₀). The data are the average of duplicate samples (± standard deviation) and are representative of two experiments.

FIG. 5

Effect of different lipid and protein kinase inhibitors on adenovirus internalization. SW480 cells were pretreated in suspension for 10 min at 4°C with 100 nM wortmannin (WTM) or were treated with 100 μM LY294002, 2 μM ML7, or 20 μM PD98095 or in medium alone (control) for 2 h at 37°C before detachment. The cells were then incubated with ¹²⁵I-labeled adenovirus for 30 min at 4°C, washed, and warmed to 37°C for various times prior to assaying for virus internalization as measured by resistance to trypsin treatment.

FIG. 6

Expression of the iSH2 domain of the p85 subunit of PI3K inhibits adenovirus internalization and gene delivery. SW480 cells were transfected with a control plasmid lacking a foreign gene or with a plasmid encoding p85/iSH2. The transfection efficiency was approximately 50 to 70%, as judged by delivery of the lacZ-containing plasmid pcDNA3. (A) The cells were assayed 48 h posttransfection for adenovirus-induced PI3K activation as described in the legend to Fig. 2 and for expression of the iSH2 protein by immunoblotting with an anti-c-myc tag antibody. Orig., origin of sample application. (B) Transfected cells were also analyzed for binding of soluble penton base by flow cytometry (Control, cells incubated with FITC-streptavidin alone; PB, cells incubated with biotinylated penton base followed by incubation with FITC-streptavidin; PB*, cells incubated with unlabeled penton base followed by incubation with biotinylated penton base and FITC-streptavidin). (C to E) Control transfected (stippled bars) or p85/iSH2-transfected (solid bars) cells were also assayed for their ability to adhere to plastic tissue culture wells coated with immobilized penton base (PB) or fibronectin (FN) (C) and for susceptibility to adenovirus-mediated gene delivery (D) and virus internalization (E). Tfn, transferrin. The data are the average of duplicate samples (+ standard deviation) and are representative of two experiments.

FIG. 7

Effect of transient expression of p85/iSH2 on cell motility. A549 cells were transfected with p85/iSH2 or a control plasmid and examined 48 h posttransfection for cell migration on fibronectin-coated membrane filters (A), for adenovirus-mediated gene delivery (B), and for virus internalization (C). In parallel studies, A549 cells were treated with the MAP kinase inhibitor PD98059 prior to assaying cell functions.