Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility

Abstract

Human cytomegalovirus (HCMV) rapidly induces a mobile and functionally unique proinflammatory monocyte following infection that is proposed to mediate viral spread. The cellular pathways used by HCMV to initiate these biological changes remain unknown. Here, we document the expression of the epidermal growth factor receptor (EGFR) on the surface of human peripheral blood monocytes but not on other blood leukocyte populations. Inhibition of EGFR signaling abrogated viral entry into monocytes, indicating that EGFR can serve as a cellular tropism receptor. Moreover, HCMV-activated EGFR was required for the induction of monocyte motility and transendothelial migration, two biological events required for monocyte extravasation into peripheral tissue, and thus viral spread. Transcriptome analysis revealed that HCMV-mediated EGFR signaling up-regulated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin nucleator whose expression and function are normally limited in leukocytes. Knockdown of N-WASP expression blocked HCMV-induced but not phorbol 12-myristate 13-acetate (PMA)-induced monocyte motility, suggesting that a switch to and/or the distinct use of a new actin nucleator controlling motility occurs during HCMV infection of monocytes. Together, these data provide evidence that EGFR plays an essential role in the immunopathobiology of HCMV by mediating viral entry into monocytes and stimulating the aberrant biological activity that promotes hematogenous dissemination.

Fig. 1.

HCMV and EGF activate EGFR found on primary human peripheral blood monocytes. (A) PBMCs were stained with anti-CD3-PE-Cy5 (T cells), anti-CD14-APC-Cy7 (monocytes), and anti-CD20-PE (B cells) antibodies and were then costained with either an anti-EGFR-GFP antibody (green) or a GFP isotype-matched control antibody (blue). The red line indicates unlabeled cells. MDA-MB-468 breast cancer cells were used as a positive control cell line. Examination of EGFR expression was performed by flow cytometry. (B) Isolated monocytes were treated with EGF or infected with HCMV (Towne/E or TB40/E) for 0, 10, 30, 45, and 60 min. Phosphorylated EGFR (pEGFR), total EGFR, phosphorylated Akt (pAkt), and total Akt were detected by immunoblotting. (C) Monocytes were treated with an anti-EGFR antibody or AG1478 before treatment with EGF or infection with Towne/E or TB40E. Total lysate was harvested, and pEGFR, total EGFR, phosphorylated PI(3)K [pPI(3)K], total PI(3)K, pAkt, and total Akt were determined by immunoblotting. Membranes were reprobed with antibody against β-actin (B and C).

Fig. 2.

EGFR acts as a tropism receptor directing HCMV internalization. PBMCs were mock-infected (red) or infected with a GFP tegument-tagged TB40/E virus (blue) and then labeled with anti-CD3-PE-Cy5 (T cells) (A), anti-CD14-APC-Cy7 (monocytes) (B), or anti-CD20-PE (B cells) (C) antibody and examined by flow cytometry to detect GFP-positive viral particles. (D) Purified CD14⁺ monocytes were mock infected (red), TB40/E infected (blue), or TB40/E infected in the presence of anti-EGFR antibody (orange) or soluble heparin (green) and then analyzed by flow cytometry. (E) Monocytes were pretreated with an anti-EGFR antibody or AG1478, cooled to 4 °C, and infected with TB40/E. Cells were then immediately fixed or temperature-shifted to 37 °C for 1 h and then fixed. Red fluorescence represents the labeled EGFR staining, green fluorescence represents TB40/E-GFP staining, blue fluorescence represents DAPI-stained nuclei, and the white dashed line represents the outer monocyte cell membrane. Images are representative of three independent experiments from different donors. (F and G) Monocytes were pretreated with an anti-EGFR antibody, an anti-IgG control antibody, AG1478, or LY294002; were cooled to 4 °C; and were infected with Towne/E. Cells were then treated with PK or incubated at 37 °C for 1 h before PK treatment. The presence of genomic HCMV DNA was determined by RT-PCR (F) and qRT-PCR (G) analysis using HCMV genomic immediate early-specific primers.

Fig. 3.

HCMV stimulates monocyte motility and transendothelial migration in an EGFR-dependent manner. Monocytes were pretreated with an anti-EGFR antibody, an IgG isotype-matched control antibody, or AG1478. (A) Monocytes were infected with Towne/E, and photomicrographs were captured at 6 hpi. Images are representative of three independent experiments from different donors. (B) Cells were plated on colloidal gold-coated coverslips and infected with Towne/E or treated with EGF for 24 h. The average area of colloidal gold cleared per monocyte was determined. (C) Monocytes were mock infected, Towne/E infected, or EGF treated for 6 h nonadherently, and they were subsequently labeled with CellTracker Green. Labeled monocytes were added to cell-culture inserts containing confluent monolayers of ECs and incubated for 24 h. Following incubation, the percentage of monocytes that underwent diapedesis was determined by fluorescence microscopy. (B and C) Results are from three independent experiments from different donors. Statistical significance between experimental means (P value) was determined using the Student's t test. *, P < 0.05.

Fig. 4.

HCMV up-regulation of N-WASP occurs in an EGFR-dependent manner and is required for HCMV-induced monocyte motility. (A) Monocytes were treated with an anti-EGFR antibody, AG1478, or LY294002 before infection with HCMV. At 24 hpi, total N-WASP expression was detected by immunoblotting. (B) Cells were transfected with N-WASP siRNA or control siRNA, and N-WASP protein levels were analyzed by immunoblotting. (C) Monocytes were mock-transfected (lanes 1–3), N-WASP siRNA-transfected (lane 4), or control siRNA-transfected (lane 5) and incubated for 24 h. Cells were then cooled to 4 °C for 30 min and mock-infected (lane 1) or Towne/E-infected (lanes 2–5) for 90 min at 4 °C. Cells were either immediately treated with PK (lane 3) or incubated for 1 h at 37 °C (lanes 1, 2, 4, and 5) before treatment with PK. The presence of genomic HCMV DNA was determined by RT-PCR using HCMV genomic immediate early-specific primers. RT-PCR analysis of GAPDH was done as a loading control. (D) N-WASP siRNA, control siRNA, or untransfected monocytes were infected with Towne/E, and photomicrographs captured at 6 hpi. Images are representative of three independent experiments from different donors. (E) N-WASP siRNA, control siRNA, or untransfected monocytes were plated on colloidal gold-coated coverslips and infected with Towne/E (multiplicity of infection of 5), treated with PMA (10 ng/mL), or treated with EGF (100 ng/mL). The average area of colloidal gold cleared per monocyte was determined. Results are from three independent experiments from different donors. Statistical significance between experimental means (P value) was determined using the Student's t test. *, P < 0.05.