Interaction of adenovirus type 5 fiber with the coxsackievirus and adenovirus receptor activates inflammatory response in human respiratory cells

Abstract

The innate immune response to adenovirus (Ad)-derived gene transfer vectors has been shown to initiate immediately after interaction of Ad with respiratory epithelial cells, through the induction of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and JNK mitogen-activated protein kinase (MAPK), nuclear factor kappaB (NF-kappaB), and different proinflammatory genes. Ad serotypes 2 or 5 (Ad2/5) enter respiratory epithelia after initial binding of fiber with the coxsackievirus-adenovirus receptor (CAR) or, alternatively, with cell surface heparan sulfate glycosaminoglycans. Ad2/5 internalization is triggered by binding of penton base to cellular RGD-binding integrins. Here we investigated the role of the Ad5 surface domain proteins constituting the vector capsid, namely, the fiber, the penton base, and the hexon, on the transmembrane signals leading to the transcription of the different proinflammatory genes in the human respiratory A549 cell line. Interaction of Ad fiber with CAR activates both ERK1/2 and JNK MAPK and the nuclear translocation of NF-kappaB, whereas no activation was observed after exposing A549 cells to penton base and hexon proteins. Moreover, interaction of Ad fiber with CAR, but not heparan sulfate proteoglycans, promotes transcription of the chemokines interleukin-8, GRO-alpha, GRO-gamma, RANTES, and interferon-inducible protein 10. These results identify the binding of Ad5 fiber with the cellular CAR as a key proinflammatory activation event in epithelial respiratory cells that is independent of the transcription of Ad5 genes.

FIG. 1.

Purification of viral capsid proteins. (A) Separation of viral proteins on 10% SDS-PAGE. Ten to twenty μg of each purified viral protein was loaded. Lane 1, hexon (Hx; 130 kDa); lane 2, full-length fiber incubated in PAGE sample buffer at 94°C for 3 min, resulting in monomeric fiber (mF; 65 kDa); lane 3, full-length fiber incubated in PAGE sample buffer at room temperature to maintain the trimeric form (tF; 180 kDa); lane 4, penton base (Pb; 85 kDa). The molecular mass markers are indicated on the right. (B) Separation of recombinant knob on 15% SDS-PAGE. The knob domain as trimeric form (lane 1, tK; 75 kDa) and as monomeric form (lane 2, mK; 25 kDa) was obtained by incubating the recombinant fiber knob at room temperature or at 94°C for 3 min, respectively. Ten μg of purified knob was loaded in each lane. The molecular mass markers are indicated on the left. (C to E) A cell infection assay was performed as reported in Material and Methods by incubating A549 cells with buffer (C), purified fiber (1 μg/ml) (D), or Ad5 (MOI, 10) (E).

FIG. 2.

Effects of sCAR-D1 on TNF-α-dependent activation of MAP kinases and IL-8 mRNA. A549 cells were incubated with TNF-α (50 ng/ml) in the presence or absence of sCAR-D1 (200 μg/ml). (A) JNK activity (30 min); (B) ERK1/2 activity (30 min; mean ± standard error of the mean); (C) IL-8 mRNA (18 h; mean ± standard error of the mean).

FIG. 3.

Ad fiber induces ERK1/2 kinase activity. The ERK1/2-dependent phosphorylation of a specific peptide substrate containing a sequence of EGFR was measured in A549 cell lysates. A. Cells exposed to full-length fiber (1 μg/ml; filled circles) or full-length fiber preincubated with sCAR-D1 for 60 min (1 μg/ml and 200 μg/ml, respectively; open triangles). B. Cells exposed to the fiber knob domain (1 μg/ml; filled circles) alone or preincubated with sCAR-D1 (1 μg/ml and 200 μg/ml, respectively; open triangles). C. Cells exposed to penton base (1 μg/ml; filled circles) or an equivalent volume of buffer (open triangles). D. Cells exposed to hexon (1 μg/ml; filled circles) or an equivalent volume of buffer (open triangles). Radioactivity incorporated at each time is expressed as a percentage of the counts obtained at time zero. Data are means ± standard errors of the means of three independent experiments.

FIG. 4.

Ad fiber induces JNK1 kinase activity. Cell lysates were analyzed for JNK1 activity by an immunocomplex assay using c-Jun(1-169)-GST as phosphorylation substrate. Autoradiograms of phosphorylated substrate (c-jun-GST) and Western blots of total JNK1 (tJNK1) are shown. Cells were exposed to fiber knob (1 μg/ml) (A), fiber knob (1 μg/ml) preincubated with sCAR-D1 (200 μg/ml) (B), penton base (1 μg/ml) (C), or hexon (1 μg/ml) (D). TNF-α (50 ng/ml) was used as a positive control. Results are representative of three independent experiments.

FIG. 5.

Fiber knob induces the nuclear translocation of NF-κB. A549 cells were incubated for 4 h with full-length fiber (1 μg/ml) (A), full-length fiber (1 μg/ml) preincubated with sCAR-D1 (200 μg/ml) (B), fiber knob (1 μg/ml) (C), fiber knob (1 μg/ml) preincubated with sCAR-D1 (200 μg/ml) (D), hexon (1 μg/ml) (E), penton base (1 μg/ml) (F), suspension buffer (G), or sCAR-D1 alone (200 μg/ml) (H).

FIG. 6.

Effects of LPS and TNF-α on NF-κB translocation. A549 cells were incubated for 4 h with LPS, TNF-α with or without sCAR-D1, or buffer alone, as indicated. Cell autofluorescence (immunofluorescence with irrelevant rabbit immunoglobulin instead of primary antibody against the p65 subunit of NF-κB) (A), buffer alone (B), LPS (0.1 μg/ml) (C), LPS (10 μg/ml) (D), TNF-α (50 ng/ml) (E), or TNF-α (50 ng/ml) plus sCAR-D1 (200 μg/ml) (F).

FIG. 7.

Effect of heparin on induction of ERK1/2 kinase. The ERK1/2-dependent phosphorylation of a specific peptide substrate containing a sequence of EGFR was measured in A549 cell lysates obtained after exposure to Ad.CFTR (MOI of 25; filled circles) or Ad.CFTR preincubated with sCAR-D1 (MOI of 25 and 200 μg/ml, respectively; open triangles) (A), Ad.CFTR (MOI of 25; filled circles) or to Ad.CFTR preincubated with heparin (MOI of 25 and 1 μg/ml, respectively; open triangles) (B), or full-length fiber (1 μg/ml; filled circles) or full-length fiber preincubated with heparin (1 and 10 μg/ml, respectively; open triangles) (C). Data are means ± standard errors of the means of three independent experiments.

FIG. 8.

Effect of heparin on induction of NF-κB. A549 cells were incubated for 4 h with full-length fiber preincubated with heparin (1 and 10 μg/ml, respectively) (A), Ad.CFTR preincubated with heparin (MOI of 25 and 10 μg/ml, respectively) (B), Ad.CFTR preincubated with sCAR-D1 (MOI of 25 and 200 μg/ml, respectively) (C), or Ad.CFTR preincubated with heparin and sCAR-D1 (MOI of 25, 10 μg/ml, and 200 μg/ml, respectively) (D). Slides were incubated with a primary antibody directed against the NF-κB p65 subunit. Results are representative of three independent experiments.

FIG. 9.

Full-length fiber induces transcription of inflammatory genes. A. A549 cells (2 × 10⁵ cells) were grown in 2-cm-diameter wells and incubated with Ad.CFTR vector (MOI, 100), full-length fiber (1 and 0.1 μg/ml), full-length fiber (1 or 0.1 μg/ml) preincubated for 1 h at 37°C with sCAR-D1 (200 and 20 μg/ml), sCAR-D1 alone (200 μg/ml), full-length fiber (1 μg/ml) preincubated for 1 h at 37°C with heparin (10 or 1 or 0.1 μg/ml), or heparin alone (10, 1, or 0.1 μg/ml) for 18 h before RNA extraction, reverse transcription, and IL-8 quantitation, as described in Materials and Methods. Values are means ± standard errors of the means of four representative experiments. B. Quantitation of the indicated inflammatory genes was performed as for IL-8 by incubated full-length fiber (1 μg/ml) in the absence (open symbols) or presence (close symbols) of sCAR-D1 (200 μg/ml). Results are representative of three independent experiments. C. IL-8 induction by LPS. A549 cells were incubated for 18 h with the indicated concentrations of LPS, and IL-8 mRNA was quantified as specified above.