Viroporins from RNA viruses induce caspase-dependent apoptosis

Abstract

The virus-encoded viroporins are known to modify membrane permeability and play an essential role in virus budding. Here, a comparative analysis of the membrane permeabilization capacity of a number of viroporins was performed in baby hamster kidney cells. Synthesis of 6K protein from Sindbis virus, E from mouse hepatitis virus, M2 from influenza A virus, and 2B and 3A from poliovirus enhanced membrane permeability to different extents. We show that two proteins from hepatitis C virus, p7 and NS4A, also display viroporin activity to a level comparable to 6K protein. In addition to their capacity to disrupt ionic cellular homeostasis and promote bacterial cell lysis, the expressed viroporins were able to induce cell death. Degradation of internucleosomal DNA and generation of apoptotic bodies were observed upon viroporin expression. Consistently, cleavage of translation initiation factor 4GI and poly-(ADP-ribose) polymerase indicated activation of effector caspase-3. We found that poliovirus 2B localizes partially in mitochondria and induces an anomalous perinuclear distribution of these organelles. Mitochondria morphology was also altered after expression of other viroporins. Finally, detection of cytochrome c release from mitochondria suggests involvement of the mitochondrial pathway in viroporin-induced apoptosis. These findings suggest that viroporins induce caspase-dependent programmed cell death.

Figure 1

Schematic representation of the SV genome and SV‐derived replicons (RNA) with or without additional viroporin sequences. s.p., subgenomic promoter.

Figure 2

Membrane permeabilization induced by different viroporins in BHK cells. BHK cells were electroporated with in vitro‐synthesized RNA from the different constructs shown in Fig. 1. At 4, 7 or 15 hpe, proteins were labelled with [³⁵S]Met/Cys in the absence (–) or presence (+) of 1 mM HB for 40 min. Samples were processed by SDS‐PAGE (17.5%), followed by fluorography and autoradiography.
A. Membrane permeabilization assayed by the inhibition of translation as a result of HB entry induced by viroporins at 8 hpe. As negative controls, the cells were electroporated with transcription buffer alone (BHK) or with rep C. The numbers below each lane indicate the percentages of protein synthesis obtained by dividing the values obtained by densitometry in HB‐treated cells by the values obtained in untreated cells. Expression of NS4A and the non‐proteolysed product C‐NS4A at 8 hpe were analysed by Western blot using a monoclonal anti‐NS4A antibody and a rabbit polyclonal anti‐C antibody respectively (lower panels).
B. Statistical analysis of membrane permeabilization caused by the indicated viroporins at different time points. Each bar represents the percentage of protein synthesis in HB‐treated cells compared with untreated cells. The SV C protein or cellular proteins in the case of control BHK cells were quantified by densitometry. All data are shown as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.005. Mr, molecular weight markers.

Figure 3

Nuclear fragmentation in BHK cells upon expression of different viroporins.
A. Detection of apoptotic cells by DAPI staining and TUNEL assay. BHK cells were electroporated with the indicated viroporin replicons. At 16 hpe, the cells were fixed and permeabilized, and TUNEL assay was performed (see Experimental procedures). Cells were stained with 0.5 μg ml⁻¹ DAPI. Cells expressing C protein or electroporated with transcription buffer alone (BHK) served as negative controls. Cells electroporated with rep C + 3C^pro, which express PV 3C^pro, and cells treated with 1 μM staurosporine (STS) for 23 h were used as positive controls.
B. Percentage of TUNEL‐positive cells and cells showing nuclear fragmentation (mean ± SD). *P < 0.005.

Figure 4

Activation of caspase‐3 induced by several viroporins. Whole‐cell lysates from BHK cells expressing viroporins at 5, 8 or 16 hpe were processed by SDS‐PAGE. Cells expressing C protein or electroporated with transcription buffer alone (BHK) served as negative controls.
A. Cleavage of eIF4GI mediated by caspase‐3 activation. Two eIF4GI apoptotic cleavage products (c.p) were detected by Western blotting using a polyclonal anti‐eIF4GI antibody. In the lower left panel, the cleavage products of eIF4GI generated by viral proteases (PV 2 A and HIV‐1 PR) are shown. c‐t, eIF4GI carboxyl‐terminal fragment. As positive controls, cells were incubated with 5 μg ml⁻¹ ActD and 25 μg ml⁻¹ CHX for 16 h to induce apoptotic cleavage of eIF4GI by caspase‐3. The 150 kDa protein related to eIF4GI is indicated with an asterisk.
B. Cleavage of PARP in BHK cells expressing viroporins. Proteolysis of PARP was analysed by Western blotting using a monoclonal anti‐PARP antibody. A representative experiment shows early PARP cleavage induced by NS4A expression (left panel). c.p, apoptotic PARP cleavage product. The percentage of intact PARP at 5, 8 and 16 hpe was calculated by densitometry of the PARP band (right diagram). Cells treated with 2 μM staurosporine (STS) for 8 h were used as positive controls (mean ± SD of three independent experiments). *P < 0.05.
C. Inhibition of eIF4GI cleavage. Cells expressing the indicated viroporins were treated with or without DEVD‐fmk (40 μM) at 4 hpe and incubated for 12 h at 37°C. Cell lysates were analysed by Western blotting using anti‐eIF4GI antibodies. Inhibition of early eIF4GI cleavage in cells expressing NS4A at 8 hpe (left panel). To measure protein loading, eIF4A or α‐tubulin (not shown) were detected. Percentage of eIF4GI intact in untreated (solid bars) and DEVD‐fmk‐treated cells (grey bars) at 16 hpe was calculated by densitometry of the eIF4GI bands. Each bar shows the percentage of intact eIF4GI compared with control BHK cells (mean ± SD of three independent experiments). *P < 0.05.

Figure 5

Colocalization of PV 2B and HCV NS4A proteins with mitochondrial and organelle markers. BHK cells expressing PV 2B (A–C) or HCV NS4A (D–F) were fixed at 8 hpe, permeabilized and double‐stained with anti‐2B and anti‐NS4A antibodies respectively, and different organelle markers. Antibodies against GRP94 and calnexin were used as ER markers (A, D). Antibody 25H8 and anti‐giantin antibodies were used as Golgi markers (B, E). Cells were incubated prior to fixation for 45 min with Mitotracker Red to stain mitochondria (C, F). Overlaid images are shown on the right.
G. Mitochondria staining in cells transfected with viroporin replicons at 16 hpe.

Figure 6

Cytochrome c release in cells expressing viroporins.
A. BHK cells expressing the different viroporins as well as PV 3C^pro (upper middle panel) were fixed at 16 hpe, permeabilized and incubated with mouse anti‐cytochrome c antibodies and secondary Alexa Fluor 488‐conjugated anti‐mouse IgG. Cells treated with 5 μg ml⁻¹ ActD and 100 μg ml⁻¹ CHX, or 3C‐expressing cells were used as positive controls (upper right and middle panels). Cells expressing C protein or electroporated with transcription buffer alone (BHK) served as negative controls. Arrows indicate cells in which efflux of cytochrome c is clearly observed.
B. Percentage of cells exhibiting cytochrome c release (mean ± SD). *P < 0.05.