The Role of Epidermal Growth Factor Receptor Signaling Pathway during Bovine Herpesvirus 1 Productive Infection in Cell Culture

Abstract

Accumulating studies have shown that the epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in mediating cellular entry of numerous viruses. In this study, we report that bovine herpesvirus 1 (BoHV-1) productive infection in both the human lung carcinoma cell line A549 and bovine kidney (MDBK) cells leads to activation of EGFR, as demonstrated by the increased phosphorylation of EGFR at Tyr1068 (Y1068), which in turn plays important roles in virus infection. A time-of-addition assay supported that virus replication at post-entry stages was affected by the EGFR specific inhibitor Gefitinib. Interestingly, both phospholipase C-γ1 (PLC-γ1) and Akt, canonical downstream effectors of EGFR, were activated following virus infection in A549 cells, while Gefitinib could inhibit the activation of PLC-γ1 but not Akt. In addition, virus titers in A549 cells was inhibited by chemical inhibition of PLC-γ1, but not by the inhibition of Akt. However, the Akt specific inhibitor Ly294002 could significantly reduce the virus titer in MDBK cells. Taken together, our data suggest that PLC-γ1 is stimulated in part through EGFR for efficient replication in A549 cells, whereas Akt can be stimulated by virus infection independent of EGFR, and is not essential for virus productive infection, indicating that Akt modulates BoHV-1 replication in a cell type-dependent manner. This study provides novel insights on how BoHV-1 infection activates EGFR signaling transduction to facilitate virus replication.

Keywords: Akt; bovine herpesvirus 1; epidermal growth factor receptor; phospholipase C-γ1.

Figure 1

BoHV-1 infection in A549 cells stimulated EGFR phosphorylation (A,C) Confluent A549 cells in 60 mm dishes were infected with BoHV-1 at an MOI of 1. After infection for 24, 36, or 48 h, cell lysates were analyzed by Western blotting to detect phosphorylated-EGFR(Y1068) (A) and EGFR (C). Data are representative of three independent experiments. (B,D) The relative band intensity was analyzed with software ImageJ, and each analysis was compared with that of an uninfected control, which was arbitrarily set as 1. Significance was assessed with a Student’s t-test (* p < 0.05); ns: not significant.

Figure 2

BoHV-1 productive infection in MDBK cells stimulated EGFR phosphorylation. (A,C) Confluent MDBK cells in 60 mm dishes were infected with BoHV-1 at an MOI of 1. After infection for 4, 8, 12, or 48 h, cell lysates were analyzed by Western blotting to detect phosphorylated-EGFR(Y1068) (A) and EGFR (C). Data is representative of two or three independent experiments. (B,D) The relative band intensity was analyzed with Image J software, and each analysis was compared with that of the uninfected control, which was arbitrarily set as 1. Significance was assessed with a Student’s t-test (* p < 0.05); ns: not significant.

Figure 3

The EGFR inhibitor Gefitinib affected BoHV-1 replication in A549 cells. (A) A549 cells in 24-well plates were treated with Gefitinib at a concentration of 10 μM for 48 h. The cytotoxicity of Gefitinib was then analyzed by the Trypan-blue exclusion test. (B) A549 cells in 60 mm dishes pretreated with either the DMSO control or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or Gefitinib, respectively. After infection for 36 h, the cell lysates were prepared and p-EGFR(Y1068) was detected by Western blot. (C) A549 cells in 24-well plates pretreated with either DMSO control or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or Gefitinib, respectively. After infection for 24, 36, or 48 h, the cell cultures were collected and virus titers were determined in MDBK cells. (D) A549 cells in 24-well plates were infected with BoHV-1 (MOI = 1) for 24 and 48 h, with or without Gefitinib (10 μM) treatment. The cell morphology was observed under a light microscope. Images shown are representative of two independent experiments (magnification: 200×). (E) Diagram showing five different experimental conditions in the time-of-addition assay: (I) DMSO treatment from −1 to 36 hpi, (II) Gefitinib treatment from −1 to 36 hpi, (III) Gefitinib treatment from 0 to 36 hpi, (IV) Gefitinib treatment from 3 to 36 hpi, and (V) Gefitinib treatment from 8 to 36 hpi. (F) Viral titer for the time-of-addition assay. Results are the mean of three independent experiments, with error bars showing standard deviations. Significance was assessed with student t-test (* p < 0.05); ns: not significant.

Figure 4

The EGFR inhibitor Gefitinib affected BoHV-1 replication in MDBK cells. (A) MDBK cells in 24-well plates were treated with Gefitinib at a concentration of 10 μM for 24 h. The cytotoxicity of Gefitinib was then analyzed by a Trypan-blue exclusion test. (B) MDBK cells in 60 mm dishes pretreated with either DMSO control or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or Gefitinib, respectively. After infection for 24 h, cell lysates were prepared and p-EGFR(Y1068) was detected by Western blot. (C) MDBK cells in 24-well plates pretreated with either DMSO control or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or Gefitinib, respectively. After infection for 24 h, cell cultures were collected and virus titers were determined in MDBK cells. Results are the mean of three independent experiments, with error bars showing standard deviations. Significance was assessed with a Student’s t-test (* p < 0.05); ns: not significant. (D) MDBK cells in 24-well plates, either with or without BoHV-1 infection (MOI = 1), were treated with Gefitinib (10 μM) for 24 h. The cell morphology was observed under a light microscope. Images shown are representative of two independent experiments (magnification: 200×).

Figure 5

BoHV-1 infection in A549 cells stimulated PLC-γ1 signaling. (A) A549 cells in 60 mm dishes were mock-infected or infected with BoHV-1 (MOI = 1) for 24, 36, or 48 h. The cell lysates were then prepared for Western blots to detect p-PLC-γ1(S1248), PLC-γ1, and GAPDH. (B,C) The relative band intensity of both p-PLC-γ1(S1248) (B) and PLC-γ1 (C) was analyzed with ImageJ software, and each analysis was compared with that of the uninfected control which was arbitrarily set as 1. (D) A549 cells in 24-well plates were treated with U73122 at a concentration of 2.5 μM for 48 h. Cytotoxicity was then analyzed by a Trypan-blue exclusion test. (E) A549 cells in 60 mm dishes pretreated with either DMSO control or U73122 (2.5 μM) were infected with BoHV-1 (MOI = 1) in the presence of a DMSO control or U73122, respectively. After infection for 36 h, the cell lysates were prepared and p- PLC-γ1(S1248) was detected by Western blot. (F) A549 cells in 24-well plates pretreated with either DMSO control or U73122 (2.5 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or U73122, respectively. After infection for 36 h, cell cultures were collected and virus titers were determined in MDBK cells. Results are the mean of three independent experiments, with error bars showing standard deviations. Significance was assessed with a Student’s t-test (* p < 0.05); ns: not significant.

Figure 6

The activation of PLC-γ1 signaling in BoHV-1-infected A549 cells is inhibited by EGFR inhibitor Gefitinib. (A) A549 cells in 60 mm dishes pretreated with either DMSO or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of the indicated chemical. After infection for 24, 36, or 48 h, cell lysates were prepared and subjected to Western blot to detect p-PLC-γ1(S1248) (A) and PLC-γ1 (E), respectively. (B–D, F–H) The relative band intensity was analyzed with ImageJ software, and each analysis was compared with that of the uninfected control, which was arbitrarily set as 1. Data shown are representative of three independent experiments. Significance was assessed with a Student’s t-test (* p < 0.05).

Figure 7

BoHV-1 infection stimulated Akt for efficient replication in a cell type-specific manner. (A) A549 cells in 60 mm dishes were mock-infected or infected with BoHV-1 (MOI = 1) for 24, 36, or 48 h. The cell lysates were prepared for Western blot to detect p-Akt(S473) and Akt. (B) The relative band intensity of Akt was analyzed with ImageJ software, and each analysis was compared with that of uninfected control, which was arbitrarily set as 1. (C) A549 cells in 60 mm dishes were mock-infected or infected with BoHV-1 (MOI = 1) for 0.5 and 1 h. The cell lysates were then prepared for Western blot to detect p-Akt(S473). (D,H) Either A549 or MDBK cells in 24-well plates were treated with Ly294002 at a concentration of 5 μM for 48 and 24 h, respectively. The cytotoxicity was then analyzed by a Trypan-blue exclusion test. (E) A549 cells in 60 mm dishes pretreated with either DMSO control or Ly294002 (5 μM) were infected with BoHV-1 (MOI = 1) in the presence of a DMSO control or Ly294002, respectively. After infection for 36 h, cell lysates were prepared and p-Akt(S473) was detected by Western blot. (F) A549 cells in 24-well plates pretreated with either a DMSO control or Ly294002 (5 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO or Ly294002, respectively. At 36 and 48 hpi, cell cultures were collected and virus titers were determined in MDBK cells. (G) MDBK cells in 60 mm dishes pretreated with either the DMSO control or Ly294002 (5 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO control or Ly294002, respectively. After infection for 24 h, the cell lysates were prepared and p-Akt(S473) was detected by Western blot. (I) MDBK cells in 24-well plates pretreated with either the DMSO control or Ly294002 (5 μM) were infected with BoHV-1 (MOI = 1) in the presence of DMSO or Ly294002, respectively. At 24 hpi, cell cultures were collected and virus titers were determined in MDBK cells. Data and error bars denote the variability between three independent experiments. Significance was assessed with a Student’s t-test (* p < 0.05); ns: not significant.

Figure 8

Activated Akt stimulated by BoHV-1 infection in A549 cells was inhibited by the EGFR inhibitor Gefitinib. (A) A549 cells in 60 mm dishes pretreated with either DMSO or Gefitinib (10 μM) were infected with BoHV-1 (MOI = 1) in the presence of the chemical indicated. After infection for 24, 36, or 48 h, cell lysates were prepared and subjected to Western blot to detect p-Akt(S473) (A) and Akt (E). (B–D) and (F–H) The relative band intensity was analyzed with ImageJ software, and each analysis was compared with that of the uninfected control which was arbitrarily set as 1. Data shown are representative of two or three independent experiments. Significance was assessed with a Student’s t-test; ns, not significant.