BX-795 inhibits HSV-1 and HSV-2 replication by blocking the JNK/p38 pathways without interfering with PDK1 activity in host cells

Abstract

BX-795 is an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1), but also a potent inhibitor of the IKK-related kinase, TANKbinding kinase 1 (TBK1) and IKKɛ. In this study we attempted to elucidate the molecular mechanism(s) underlying the inhibition of BX-795 on Herpes simplex virus (HSV) replication. HEC-1-A or Vero cells were treated with BX-795 and infected with HSV-1 or HSV-2 for different periods. BX-795 (3.125-25 μmol/L) dose-dependently suppressed HSV-2 replication, and displayed a low cytotoxicity to the host cells. BX-795 treatment dose-dependently suppressed the expression of two HSV immediate-early (IE) genes (ICP0 and ICP27) and the late gene (gD) at 12 h postinfection. HSV-2 infection resulted in the activation of PI3K and Akt in the host cells, and BX-795 treatment inhibited HSV-2-induced Akt phosphorylation and activation. However, the blockage of PI3K/Akt/mTOR with LY294002 and rapamycin did not affect HSV-2 replication. HSV-2 infection increased the phosphorylation of JNK and p38, and reduced ERK phosphorylation at 8 h postinfection in the host cells; BX-795 treatment inhibited HSV-2-induced activation of JNK and p38 MAP kinase as well as the phosphorylation of c-Jun and ATF-2, the downstream targets of JNK and p38 MAP kinase. Furthermore, SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) dose-dependently inhibited the viral replication in the host cells, whereas PD98059 (an ERK inhibitor) was not effective. Moreover, BX-795 blocked PMA-stimulated c-Jun activation as well as HSV-2-mediated c-Jun nuclear translocation. BX-795 dose-dependently inhibited HSV-2, PMA, TNF-α-stimulated AP-1 activation, but not HSV-induced NF-κB activation. Overexpression of p38/JNK attenuated the inhibitory effect of BX-795 on HSV replication. BX-795 completely blocked HSV-2-induced MKK4 phosphorylation, suggesting that BX-795 acting upstream of JNK and p38 MAP kinase. In conclusion, this study identifies the anti-HSV activity of BX-795 and its targeting of the JNK/p38 MAP kinase pathways in host cells.

Figure 1

BX-795 inhibition of HSV replication and gD expression. (A) The molecular structure of BX-795. (B–E) BX-795 inhibition of HSV gD expression of HSV-1 (HF)/HEC-1-A (B), HSV-2 (G)/HEC-1-A (C), HSV-1 (HF)/Vero (D) or HSV-2 (G)/Vero (E); HSV was used at moi=1 and the gD-1/2 expression was determined by In-cell Western and normalized by β-catenin level 24 h postinfection. (F, G) BX-795 inhibition of HSV-2 replication in HEC-1-A and Vero cells (F). The cells were treated with BX-795 prior to HSV-2 infection (moi=1). The virus was released by three freeze-thaw cycles of the infected cells and the viral titers were determined on Vero-ICP10-promoter cells. The cytotoxicity of BX-795 on two cell lines used in the inhibitory activity analyses (G) All experiments were performed three times and the representative results are shown.

Figure 2

Effects of BX-795 on HSV IE gene expression at early stage of viral infection, and on the viral infection. (A–C) BX-795 inhibition of HSV IE expression. Confluent HEC-1-A cells were exposed to serial concentrations of BX-795 prior to infection with HSV-1 (moi=1). ICP0, ICP4 and ICP27 expression was determined by In-cell Western and normalized by β-catenin level 24 h postinfection. All experiments were performed three times and the representative results were shown. BX-795 did not block the expressions of HSV-1 (D) and HSV-2 (E) ICP0 and ICP27 6 h postinfection, but blocked their expressions 12 h postinfection. HEC-1-A cells were treated with serial concentrations of BX-795 and infected with HSV-1 and HSV-2 (moi=1), respectively. The levels of ICP0-1/2, ICP27-1/2 or gD-1/2 mRNA were quantified by qPCR analysis as described. All experiments were performed three times and the representative results are shown.

Figure 3

BX-795 inhibition of HSV replication not mediated by its PDK1 inhibitory activity. (A) HSV-2 infection resulted in the activation of PI3K and Akt. HEC-1-A cells infected with HSV-2 (moi=1) were collected at the indicated time points and lysed. Akt, p-Akt, p-PI3K and gD were determined by Western blot. (B) BX-795 inhibited HSV-2-induced Akt phosphorylation and activation. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence or absence of BX-795. Cells were lysed 12 h postinfection. Akt and its phosphorylated form were determined by Western blot. (C) The blockage of PI3K/Akt/mTOR by their respective inhibitors did not affect HSV-2 replication. Confluent cells were treated with LY294002, rapamycin or BX-795 (25 μmol/L), and infected with HSV-2 (moi=1). Viral gD expression was determined by In-cell Western 24 h postinfection and normalized by β-catenin level. The experiment was carried out three times and the representative results are shown.

Figure 4A–4H

BX-795 inhibition of HSV replication by blocking JNK/p38 pathway. (A) HSV-2 infection stimulated JNK and p38 MAP kinase pathway, but not ERK pathway. (B) HSV-2 infection also activated the phosphorylation of c-Jun and ATF-2. Cells infected with HSV-2 (moi=1) were collected at the indicated time points. ERK, JNK, p38 MAP kinase, c-Jun and their phosphorylated forms and p-ATF-2 were determined by Western blot. (C) JNK and p38 MAP kinase inhibitors blocked HSV-2 replication. Confluent HEC-1-A cells or Vero cells were infected with HSV-2 (moi=1) in the presence of serial concentrations of SB203580, SP600125 or PD98059. gD expression was determined 24 h postinfection. (D) BX-795 inhibited HSV-2-induced activation of JNK and p38 MAP kinase pathways. (E) BX-795 inhibited further activation of c-Jun and ATF-2 caused by HSV-2 infection. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence or absence of BX-795. JNK, p38 MAP kinase, c-Jun and their phosphorylated forms and p-ATF-2 were determined 12 h postinfection. (F) BX-795 blocked PMA-stimulated c-Jun activation. HEC-1-A cells were either mock-treated or treated with PMA (5 μg/mL) in the presence or absence of BX-795. The cells were collected after 2 h treatment, and c-Jun and its phosphorylated form were determined. (G) BX-795 inhibited HSV-2-mediated c-Jun nuclear translocation. HEC-1-A cells were either mocked-treated or treated with PMA (5 μg/mL) or infected with HSV-2 (moi=1) in the presence or absence of BX-795. c-Jun nuclear translocation was determined by immunofluorescence staining 2 h post-treatment with PMA or 12 h post-infected with HSV-2. (H) BX-795 inhibited HSV-2-induced AP-1 activation in a dose-dependent manner. HEC-1-A cells were transfected with AP-1 luc reporter plasmid. After 24 h, the cells were either mock-infected or infected with HSV-2 (moi=1) in the presence of serial concentrations of BX-795. AP-1 activity was determined by luciferase assay.

Figure 4I, 4J

BX-795 inhibition of HSV replication by blocking JNK/p38 pathway. (I) BX-795 also inhibited PMA and TNF-α stimulated AP-1 activation. HEC-1-A cells were transfected with AP-1 luc reporter plasmid. After 24 h, cells were mock-treated, treated with PMA (5 μg/mL) or rhTNF-α (100 ng/mL) or infected with HSV-2 (moi=1) in the presence of BX-795 (25 μmol/L). AP-1 activity was determined. (J) BX-795 inhibition on HSV replication attenuated by overexpression of p38/JNK. HeLa cells with or without JNK1 or p38 overexpression were infected with HSV-2 and total cellular protein was prepared for WB analysis. gD-2, the phosphorylation level of p38 MAP kinase, JNK and their substrates were determined via Western blot.

Figure 5

BX-795 acted at the upstream of JNK and p38 MAP kinase. (A) HSV-2 infection activated MKK4 in HEC-1-A cells. Cells infected with HSV-2 (moi=1) were collected at each time point. Cell lysates were prepared as described and p-MKK4 level were determined by Western blot. (B) BX-795 inhibited activation of MKK4 caused by HSV-2 infection. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence or absence of BX-795. p-MKK4 was analyzed 12 h postinfection.

Figure 6

BX-795 inhibited ICP0-mediated AP-1 activation, but not NF-κB activation. (A) BX-795 inhibited ICP0-mediated AP-1 activation. HEC-1-A, Vero and 293T cells were co-transfected with pcDNA3-ICP0-1/2-GFP or pcDNA3 and AP-1 luc reporter plasmid. After 24 h, cells were either mock-treated or treated with BX-795 (25 μmol/L). The luciferase activities were measured 24 h post-treatment. (B) BX-795 did not impede ICP0-mediated NF-κB activation. HEC-1-A cells were co-transfected with pcDNA3-ICP0-1/2-GFP or pcDNA3 and NF-κB luc reporter plasmid. After 24 h culture, cells were either mock-treated or treated with BX-795 (25 μmol/L), BAY11-7082 (20 μmol/L), PDTC (10 μg/mL), PGA1 (10 μg/mL) or MG132 (20 μmol/L). The luciferase activities were measured 24 h post-treatment.

Figure 7

BX-795 did not block HSV-induced NF-κB activation. (A) HSV-2 infection activated NF-κB in HEC-1-A cells. Cells infected with HSV-2 (moi=1) were collected at the indicated time points. Cell lysates were prepared as described in the context and p65 and IκB-α were determined by Western blot. (B) BX-795 did not inhibit HSV-2-induced NF-κB activation. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence or absence of BX-795 or PGA1. Cells were lysed and cytoplasmic proteins were extracted 12 or 24 h postinfection. Cytoplasmic IκB-α and β-actin were determined by Western blot. (C) BX-795 did not inhibit HSV-2-induced p65 nuclear translocation. HEC-1-A cells were either mock-infected or infected with HSV-2 (moi=1) in the presence of DMSO or BX-795 or PGA1, and p65 translocation was determined by immunofluorescence assay 24 h postinfection.