Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells

Abstract

Most adenoviruses bind directly to the coxsackie and adenovirus receptor (CAR) on target cells in vitro, but recent research has shown that adenoviruses can also use soluble components in body fluids for indirect binding to target cells. These mechanisms have been identified upon addressing the questions of how to de- and retarget adenovirus-based vectors for human gene and cancer therapy, but the newly identified mechanisms also suggest that the role of body fluids and their components may also be of importance for natural, primary infections. Here we demonstrate that plasma, saliva, and tear fluid promote binding and infection of adenovirus type 5 (Ad5) in respiratory and ocular epithelial cells, which corresponds to the natural tropism of most adenoviruses, and that plasma promotes infection by Ad31. By using a set of binding and infection experiments, we also found that Ad5 and Ad31 require coagulation factors IX (FIX) or X (FX) or just FIX, respectively, for efficient binding and infection. The concentrations of these factors that were required for maximum binding were 1/100th of the physiological concentrations. Preincubation of virions with heparin or pretreatment of cells with heparinase I indicated that the role of cell surface heparan sulfate during FIX- and FX-mediated adenovirus binding and infection is mechanistically serotype specific. We conclude that the use of coagulation factors by adenoviruses may be of importance not only for the liver tropism seen when administering adenovirus vectors to the circulation but also during primary infections by wild-type viruses of their natural target cell types.

FIG. 1.

Physiological concentrations of FIX and FX promote efficient Ad5 infection of A549 cells (A) and HCE cells (B). Purified Ad5 virions were preincubated with or without physiological concentrations of coagulation factors, complement components, and/or 10 mM EDTA, as indicated, and then allowed to infect adherent cells. Forty-four hours postinfection, the cells were stained and quantified in a fluorescence microscope. The control infection level corresponds to the y axis value of 1. Values are means ± SD. *, P of <0.05 versus control. The bars indicate comparisons between FIX and FIX-EDTA or between FX and FX-EDTA.

FIG. 2.

Coagulation factors promote efficient Ad5 binding and gene delivery to epithelial cells in a dose-dependent manner. (A) ³⁵S-labeled CsCl-purified Ad5 virions were preincubated with or without increasing concentrations of FIX or FX and then incubated with A549 or HCE cells in suspension on ice for one hour. Unbound virions were removed by washing, and the cell-associated radioactivity was measured in a scintillation counter. (B) GFP-expressing, CsCl-purified Ad5 viruses were preincubated with or without increasing concentrations of FIX or FX and then incubated with adherent A549 or HCE cells on ice for one hour. Unbound viruses were removed by washing. The cells were incubated at 37°C for another forty-four hours. Next, GFP-expressing cells were quantified by flow cytometry. The control binding level corresponds to the y axis value of 1. Values are means ± SD. *, P of <0.05 versus control; **, P of <0.01 versus control.

FIG. 3.

Cell surface heparan sulfate is required for FIX- and FX-mediated binding of Ad5 to and infection of epithelial cells. Ad5 virions were preincubated with or without physiological concentrations of FIX or FX and increasing concentrations of heparin and then allowed to bind to A549 cells (A) or HCE cells (B) or to infect A549 cells (C) or HCE cells (D). Alternatively, Ad5 virions were preincubated with or without physiological concentrations of FIX or FX and then allowed to bind to A549 or HCE cells (E and G) or to infect A549 or HCE cells (F and H) pretreated with increasing concentrations of heparinase I. In binding assays, cell-associated radioactivity was measured with a scintillation counter, whereas in infection assays, infectivity was quantified by fluorescence microscopy. Values are means ± SD. *, P of <0.05 versus control; **, P of <0.01 versus control; ***, P of <0.001 versus control.

FIG. 4.

Low concentrations of FIX promote efficient binding of Ad31 to and infection of epithelial cells. Purified Ad31 virions were preincubated with or without physiological concentrations of complement components, or coagulation factors, with or without EDTA as indicated, and then allowed to infect adherent A549 cells (A) or HCE cells (B). Forty-four hours postinfection, infected cells were stained and quantified by fluorescence microscopy. (C) ³⁵S-labeled, CsCl-purified Ad31 virions were preincubated with or without increasing concentrations of FIX and then incubated with A549 or HCE cells in suspension on ice for one hour. Unbound virions were removed by washing, and cell-associated radioactivity was measured with a scintillation counter. The control binding level corresponds to the y axis value of 1. Values are means ± SD. *, P of <0.05 versus control; **, P of <0.01 versus control. The bars in panels A and B indicate comparisons between FIX and FIX-EDTA or between FX and FX-EDTA.

FIG. 5.

Preincubation of Ad31 with heparin inhibits FIX-mediated binding to and infection of epithelial cells. Ad31 virions were preincubated with or without physiological concentrations of FIX and increasing concentrations of heparin and then allowed to bind to A549 cells (A) or HCE cells (B) or to infect A549 cells or HCE cells (C). Cell-associated radioactivity (i.e., the extent of ³⁵S-labeled Ad31 virions) was measured with a scintillation counter, and the infectivity was quantified by fluorescence microscopy. Values are means ± SD. *, P of <0.05 versus control; **, P of <0.01 versus control.

FIG. 6.

Treatment of epithelial cells with heparinase I has no effect on FIX-mediated Ad31 binding and infection. Ad31 virions were preincubated with or without physiological concentrations of FIX and then allowed to bind to A549 cells (A) or HCE cells (B) or to infect A549 or HCE cells (C) pretreated with increasing concentrations of heparinase I. In binding assays, cell-associated radioactivity was measured with a scintillation counter, whereas in infection assays, infectivity was quantified by fluorescence microscopy.

FIG. 7.

FIX promotes efficient binding of Ad31 to and infection of intestinal cells, independently of cell surface heparan sulfate. (A) Ad31 virions were preincubated with coagulation factors, with or without EDTA as indicated, and allowed to infect adherent intestinal FHs74Int cells for forty-four hours. Infected cells were stained and quantified as described in Materials and Methods. (B to D) ³⁵S-labeled, CsCl-purified Ad31 virions were preincubated with increasing concentrations of FIX (B) or with/without fixed (physiological) concentrations of FIX and increasing concentrations of heparin (C), or with/without fixed concentrations of FIX, and then mixed with cells pretreated with increasing concentrations of heparinase I (D) for one hour on ice. Unbound virions were washed, and cell-associated radioactivity was measured in a scintillation counter. Values are means ± SD. *, P of <0.05 versus control; **, P of <0.01 versus control. Bars in panel A indicate comparisons between FIX and FIX-EDTA or between FX and FX-EDTA.