MG-132 reduces virus release in Bovine herpesvirus-1 infection

Abstract

Bovine herpesvirus 1 (BoHV-1) can provoke conjunctivitis, abortions and shipping fever. BoHV-1 infection can also cause immunosuppression and increased susceptibility to secondary bacterial infections, leading to pneumonia and occasionally to death. Herein, we investigated the influence of MG-132, a proteasome inhibitor, on BoHV-1 infection in bovine kidney (MDBK) cells. Infection of MDBK cells with BoHV-1 induces apoptotic cell death that enhances virus release. Whereas, MG-132 inhibited virus-induced apoptosis and stimulated autophagy. Protein expression of viral infected cell protein 0 (bICP0), which is constitutively expressed during infection and is able to stimulate Nuclear factor kappa B (NF-κB), was completely inhibited by MG-132. These results were accompanied by a significant delay in the NF-κB activation. Interestingly, the efficient virus release provoked by BoHV-1-induced apoptosis was significantly reduced by MG-132. Overall, this study suggests that MG-132, through the activation of autophagy, may limit BoHV-1 replication during productive infection, by providing an antiviral defense mechanism.

Figure 1

MG-132 decreases cytotoxicity of MDBK during BoHV-1 infection. (A) MDBK cells were treated with MG-132 for 4, 24 or 48 h. (B) MDBK cells were infected with BoHV-1 alone or in association with MG-132 for 4, 24 or 48 h. At different times of treatment cells were stained with Trypan-blue and scored with a Burker chamber at a light microscope. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Significant differences between infected untreated and infected MG-132-treated groups are indicated by probability P. *P < 0.05 and **P < 0.01.

Figure 2

MG-132 inhibits BoHV-1-induced apoptosis in MDBK cells. (A) Cell lysate was prepared from mock-infected cells (lanes C), or infected cells, exposed or not to MG-132 and, for the indicated times, Western blot analysis was performed with an antibody which specifically recognized pro-caspase 9, cleaved caspase 9, pro-caspase 3, cleaved caspase 3, Bax, Bcl-2, Bcl-X_L, PARP, cleaved PARP, p53, p-p53 or β-actin. β-actin was used as an internal loading control. Blots are representative of three separate experiments. (B,C) Bax/Bcl-2 and p-p53/p53 ratio were obtained by densitometry analysis of the relative blots shown in (A). Results are the mean ± S.E.M. of three separate experiments.

Figure 3

MG-132 stimulates autophagy in MDBK cells during BoHV-1 infection. (A) Cell lysate was prepared from mock-infected cells (lanes C), or infected cells, exposed or not to MG-132 and, for the indicated times. Western blot analysis was performed with an antibody which specifically recognized LC3-I, LC3-II, beclin 1, ATG5 or β-actin. Blots are representative of three separate experiments. (B) Densitometry analysis of the LC3-II blots shown in (A). (C) Cell lysate was prepared from infected cells, exposed or not to 3-MA and LiCl for 24 h. Western blot analysis was for LC3-I, LC3-II or β-actin. Blots are representative of three separate experiments. (D) Photomicrographs showing morphology of MDBK cells stained with Giemsa. After 48 of infection, compared to the control groups, in MG-132 exposed groups a large number of cells exhibited an increase of characteristic signs of autophagy, such as an elevated degree of vacuolization in the cytoplasm (arrow) which often indicates an increased autophagic flux (magnification × 150); microphotographs of cells staining with acridine orange that revealed the induction of acidic vesicular organelles. Moreover, in infected cells were detected both typical necrotic morphology (star) and apoptotic features (arrow). Whereas, we distinguished only necrotic features in MG-132 infected cells (star). Detection of green and red fluorescence in acridine orange-stained cells was performed using a fluorescence microscope in MDBK control cells, infected and treated or not with MG-132 at 48 h p.i., (magnification × 1000); immunofluorescence for LC3 in infected cells treated or not with MG132 for 48 h, studied as described in Methods Section (magnification × 400).

Figure 4

MG-132 inhibits protein expression of bICP0 during infection in MDBK cells. Cell lysate was prepared from mock-infected cells (lanes C), or infected cells, exposed or not to MG-132 and, for the indicated times, Western blot analysis was performed with an antibody which specifically recognized bICP0 or β-actin. β-actin was used as an internal loading control. The molecular weight (in kDa) of protein size standards is shown on the left hand side. Blots are representative of three separate experiments.

Figure 5

MG-132 delayed the translocation of NF-κB from cytoplasm to nuclei during infection in MDBK cells. Cell lysate was prepared from mock-infected cells (lanes C), or infected cells, exposed or not to MG-132 and, for the indicated times, Western blot analysis was performed with an antibody which specifically recognized NF-κB, β-tubulin or Lamin A/C. β-tubulin and Lamin A/C were used as internal loading controls. The molecular weight (in kDa) of protein size standards is shown on the left hand side. Blots are representative of three separate experiments.

Figure 6

MG-132 reduces virus production during productive infection in MDBK cells. (A) Representative microphotographs by phase-contrast light microscopy of MDBK control cells, untreated or treated with MG-132, infected with BoHV-1 and untreated or treated with MG-132 and LiCl at 48 h p.i., showing the cytopathic effects and the morphological changes on cellular monolayers. (B) Virus titers, assayed by TCID₅₀ method and reported as Log TCID₅₀/ml.

Figure 7

Schematic diagram illustrating the hypothesized mechanisms as to how MG-132 exerts its effects on the BoHV-1 replication, resulting in stimulated autophagy and decreased virus replication in MDBK cells.