Proteasome inhibition reduces avian reovirus replication and apoptosis induction in cultured cells

Abstract

The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins sigmaC, sigmaA, and sigmaNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV sigmaC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.

Fig. 1

Proteasome inhibitor MG132 reduces ARV replication. (A) BHK-21 cells were infected with ARV (MOI = 5) and treated with various concentrations of MG132. Virus titers at 18 h.p.i. were determined by a plaque assay. (B) BHK-21 cells were infected with ARV at various MOIs and then treated with 25 μM of MG132 for 18 h. Virus titers at 18 h.p.i. were determined by a plaque assay. (C) Representative morphological changes of BHK-21 cells treated with MG132 or DMSO at 18 h.p.i. by a phase-contrast microscopy.

Fig. 2

Effects of proteasome inactivation at increasing periods of time after infection and effect of MG132 on virus internalization. (A) Experimental design for pulse treatment with MG132 is shown. (B) BHK-21 cells were treated with MG132 during different time windows. The virus titer at each time point was examined by a plaque assay. (C–D) Effect of MG132 on virus internalization was evaluated. BHK-21 cells were either pre-incubated 30 min before infection or 2 h.p.i. with MG132. Whole cell extracts prepared from infected cells 18 h after treatment were assayed by examination of virus titer (C) and Western blot assay for the presence of σA, σNS, and σC expression (D) in ARV-infected BHK-21 cells. Values are means ± standard errors (SE) of three independent experiments.

Fig. 3

Proteasome inhibition decreases viral transcription and protein translation in ARV-infected cells. (A) The effects of proteasome inhibitor on ARV RNA level were examined. Total RNAs isolated from ARV-infected BHK-21 cells 18 h.p.i. after treatment with MG132 were subject to a RT-PCR analysis of ARV RNA. GAPDH was used as a control. M: mock infection; NC: water. (B) Representative IFA staining of ARV-infected BHK-21 cells used to detect σC expression. (C) BHK-21 cells were pre-incubated with various concentration of proteasome inhibitor MG132. Cell lysates were collected 18 h.p.i. and Western blot for σC was performed. This same blot was also stained with an antibody against actin to illustrate equal protein loading. Mock: without ARV. (D) To examine whether the protease inhibitor MG132 inhibits host cell translation, the pcDNA-GFP construct was transfected into ARV-infected BHK-21cells treated with various concentrations of MG132. Cell lysates were collected from infected cells 18 h.p.i. were assayed by Western blot for the presence of GFP and σC protein expression in ARV-infected BHK-21 cells.

Fig. 4

Inhibition of ARV σC-induced apoptosis by a protease inhibitor MG132. (A) BHK-21 cells were either pre-incubated 30 min before infection or 2 h.p.i. with MG132. Cell lysates collected from infected cells 18 h after treatment were assayed by Western blot assay for the presence of ser⁴⁶-phosphorylated p53 protein expression in ARV-infected BHK-21 cells. (B) Activated caspase 3 staining of ARV-infected BHK-21 cells was carried out to examine inhibition of ARV-induced apoptosis by MG132. Mock and ARV-infected BHK-21 cells treated with or without MG132 were stained18 h.p.i. (C) BHK-21 cells were pre-incubated 30 min before infection with MG132. Cell lysates were collected from infected cells 18 h after treatment with various concentrations of MG132 and assayed by DNA fragmentation analysis. (D) BHK-21 cells were transfected with construct pcDNA-σC and then cells were treated with various concentrations of MG132. Cell lysates were collected 18 h after transfection and assayed by Western blot assay for the presence of ser⁴⁶-phosphorylated p53 and σC protein expression (upper panel). DNA fragmentation assay was performed to check whether apoptosis was also induced in mock-treated, ARV-infected, and MG132-treated cells (lower panel). Actin, mock-transfection with pcDNA3.1 (−) and ARV-infected cells were used controls, respectively.