Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling

Abstract

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.

FIG. 1.

EGFR expression and HCMV infection in EGFR-positive and EGFR-negative cell lines. (A) Immunoblot detection of EGFR expression in NHDF and mammary breast cancer cell lines MB468 (EGFR positive) and MB453 (EGFR negative). (B) HCMV AD169 was unable to infect EGFR-negative or -positive mammary breast cancer cell lines as detected by immunoblotting. The IE1 viral protein was detected in NHDF, MB468, and MB453 cells infected with HCMV strain AD169 at an MOI of 10 PFU/ml. (C) HCMV Towne was unable to infect EGFR-negative or -positive mammary breast cancer cell lines as detected by immunoblotting. The IE1 viral protein was detected in NHDF, MB468, and MB453 cells infected with HCMV strain Towne at an MOI of 5.0 PFU/ml. h.p.i., hours postinfection. M, mock infection.

FIG. 2.

HCMV infection in EGFR neutralizing antibody-treated fibroblast cells. (A) Increasing concentrations of an EGFR neutralizing antibody were unable to inhibit HCMV AD169 entry into fibroblast cells. IE2-GFP gene expression was detected by flow cytometry in NHDF and HEL fibroblasts treated with EGFR neutralizing antibody, followed by infection with HCMV AD169-GFP at an MOI of 0.5 PFU/ml. (B) Increasing concentrations of an EGFR neutralizing antibody were unable to inhibit HCMV Towne entry into fibroblast cells. IE1 and IE2 gene expression was detected by immunofluorescence in NHDF and HEL cells treated with an EGFR neutralizing antibody, followed by infection with HCMV Towne at an MOI of 0.5 PFU/ml. (C) Positive control verifying that EGFR phosphorylation is inhibited in fibroblast cells treated with an EGFR neutralizing antibody (Ab.). NHDF and HEL cells were treated with EGFR neutralizing antibody, followed by stimulation with EGF. P-EGFR (1173) was detected by immunoblotting. Blot was stripped and reprobed for total EGFR.

FIG. 3.

HCMV infection of AG1478-treated fibroblast cells. (A) Increasing concentrations of AG1478 were unable to inhibit HCMV AD169 entry into fibroblast cells. IE2-GFP gene expression was detected by flow cytometry in NHDF and HEL cells treated with AG1478, followed by infection with HCMV AD169-GFP at an MOI of 0.5 PFU/ml. (B) Increasing concentrations of AG1478 were unable to inhibit HCMV Towne entry into fibroblast cells. IE1 and IE2 gene expression was detected by immunofluorescence in NHDF and HEL cells treated with AG1478, followed by infection with HCMV Towne at an MOI of 0.5 PFU/ml. (C) Positive control verifying that EGFR phosphorylation is inhibited in fibroblast cells treated with AG1478. NHDF and HEL cells were treated with increasing concentrations of AG1478, followed by stimulation with EGF. P-EGFR (1173) was detected by immunoblotting. The blot was stripped and reprobed for total EGFR.

FIG. 4.

EGF and HCMV stimulation of fibroblast cells. (A) HCMV is unable to stimulate EGFR phosphorylation in NHDF cells. NHDF cells were stimulated with EGF, HCMV AD169, or HCMV Towne. P-EGFR (Tyr-1173) was detected by immunoblotting. The blot was stripped and reprobed for EGFR. (B) HCMV is unable to stimulate EGFR phosphorylation in HEL cells. HEL cells were stimulated with EGF, HCMV AD169, or HCMV Towne. P-EGFR (Tyr-1173) was detected by immunoblotting. The blot was stripped and reprobed for EGFR. (C) HCMV is unable to stimulate EGFR phosphorylation in NHDF or HEL cells. Cells were stimulated with EGF, HCMV AD169, or HCMV Towne. All phospho-tyrosine proteins were immunoprecipitated (IP), followed by detection of EGFR by Western blotting (WB). M, mock infection.

FIG. 5.

HCMV VR1418 and BADrUL131 infection in EGFR neutralizing antibody-treated epithelial and endothelial cells. (A) An EGFR neutralizing antibody (Ab) at a concentration of 10 μg/ml was unable to inhibit HCMV VR1418 entry into epithelial and endothelial cells. IE2 gene expression was detected by immunofluorescence in ARPE-19 cells, UtMVEC, and HUVEC treated with EGFR neutralizing antibody, followed by infection with VR1814 at an MOI of 1.0 PFU/ml for 4 h. (B) Increasing concentrations of an EGFR neutralizing antibody were unable to inhibit HCMV BADrUL131 entry into epithelial and endothelial cells. GFP was detected by flow cytometry in ARPE-19 cells, UtMVEC, and HUVEC treated with an EGFR neutralizing antibody, followed by infection with HCMV BADrUL131 at an MOI of 1.0 PFU/ml for 2 h.