Semliki forest virus-induced endoplasmic reticulum stress accelerates apoptotic death of mammalian cells

Abstract

The alphavirus Semliki Forest virus (SFV) and its derived vectors induce apoptosis in mammalian cells. Here, we show that apoptosis is associated with the loss of mitochondrial membrane potential followed by the activation of caspase-3, caspase-8, and caspase-9. Cell death can be partially suppressed by treatment with the pan-caspase inhibitor zVAD-fmk. To determine the role of SFV structural proteins in cell death, the temporal course of cell death was compared in cells infected with SFV and cells infected with SFV virus replicon particles (VRPs) lacking some or all of the virus structural genes. In the absence of virus structural proteins, cell death was delayed. The endoplasmic reticulum (ER) stress response, as determined by the splicing of X-box binding protein 1 (XBP1) transcripts and the activation of caspase-12, was activated in virus-infected cells but not in VRP (SFV lacking structural genes)-infected cells. The C/EBP-homologous protein (CHOP) was upregulated by both virus and VRP infections. The virus envelope proteins but not the virus capsid protein triggered ER stress. These results demonstrate that in NIH 3T3 cells, SFV envelope glycoproteins trigger the unfolded protein response of the ER and accelerate apoptotic cell death initiated by virus replicase activity.

FIG. 1.

(A) Loss of MMP in SFV4-infected NIH 3T3 cells as visualized by JC-1 staining. (a) Cells with (orange) and without (green) an intact MMP are easily distinguished using JC-1 staining (SFV4-infected NIH-3T3 cells; MOI = 1.0 at 24 h). Green cells are rounded up and condensed. The inset shows a couple of cells at higher-power magnification (×100). (b) Time course of the loss of MMP following SFV4 infection (MOI, 30); the percentage of green cells (green line) increased, and the percentage of orange cells (red lines) decreased. Each point is the mean count from three replicate cultures in 15 fields selected at random. The experiment was repeated independently twice with similar results. (c) JC-1 staining of uninfected NIH 3T3 cells. (d) JC-1 staining of formaldehyde-treated NIH 3T3 cells. (B) Viability (Wst-1 assay) of NIH 3T3 cells 30 h after infection with SFV4 (MOI, 30) in the presence or absence of zVAD (200 μM), a pan-caspase inhibitor. As controls, cells were treated with staurosporine (STS; 1 μM), a known inducer of apoptosis, for 30 h in the presence or absence of zVAD or were left untreated. The bars represent the means of quadruplicate cultures at each time point. Error bars are standard deviations from the means. This experiment was repeated three times with similar results.

FIG. 2.

(A) Representation of the genomes of SFV (upper) and SFV1-d1eGFP VRPs (lower). The replicase ORF comprised of nsP1 to nsP4 is present in both. In addition, the viral genome has the structural ORF comprised of the capsid (C), p62 (precursor of the E2 and E3 envelope glycoproteins), and the E1 envelope glycoprotein. The structural ORF of the VRP genome is replaced with d1eGFP. (B) Viability (Wst-1 assay) of uninfected NIH 3T3 cells (□) and NIH 3T3 cells infected (MOI, 30) with SFV4 (▴) or VRPs (▪). Each point is the mean of quadruplicate cultures. Error bars are standard deviations from the means. The experiment was repeated three times with similar results. (C) Replicate cultures of NIH 3T3 cells were infected (MOI, 30) with SFV4 (▪) or VRPs (□). Levels of activated caspase-3/7, -8, and -9 were determined at 12, 14, 15, 16, and 18 h postinfection. The bars are means from quadruplicate cultures at each time point. Error bars are standard deviations from the means. This experiment was repeated three times with similar results.

FIG. 3.

Levels of CHOP, activated caspase-12, and β-actin proteins as determined by immunoblotting in NIH 3T3 cells infected (MOI, 30) with SFV4 or VRPs. The negative control (-ve) was mock-infected cells. The positive control (+ve) was tunicamycin (5 μg/ml)-treated cells. This experiment was repeated three times with the same result.

FIG. 4.

(A) XBP1 transcript splicing in NIH 3T3 cells infected (MOI, 30) with SFV4 or VRPs. RNA from infected cells was reverse transcribed into cDNA, and XBP1 amplicons were generated by PCR. These were digested with PstI, which only cleaves amplicons derived from unspliced XBP1 transcripts. The digests were run on an agarose gel (shown). The upper (undigested) band represents spliced transcript and the lower (digested) band unspliced transcript. The negative (-ve) control was RNA from mock-infected cells. The positive (+ve) control was RNA from tunicamycin (5 μg/ml)-treated cells. The ladder (L) is a 1-kb DNA ladder (Promega) showing 250-bp (lower), 500-bp (middle), and 750-bp (upper) bands. (B) Genome structure of SFV4 (V), VRPs expressing the nonstructural and envelope proteins only (RE), VRPs expressing the nonstructural and the capsid protein only (RC), or VRPs lacking all structural genes (R) used to infect (MOI, 30) NIH 3T3 cells. (C) Following infection with the virus or VRPs from panel B, XBP1 transcript splicing was assessed (as described above) at 13 h postinfection. The negative control (-ve) was RNA from mock-infected cells. The positive control (+ve) was RNA from tunicamycin (5 μg/ml)-treated cells. The ladder (L) was a 100-bp DNA ladder (New England Biolabs). The experiment was repeated three times with similar results.

FIG. 5.

Rate of cell death was unaffected by a knockdown of caspase-12. (A) NIH 3T3 cells were treated (+ve) with siRNA against caspase-12 (C-12) or with a scrambled control siRNA (-ve). The effects on caspase-12 and, as a control, β-actin transcripts were determined by PCR. (B) Caspase-12 and β-actin protein levels after siRNA treatment. (C) NIH 3T3 cells treated with siRNA against caspase-12 (▪) or control siRNA (▴) were infected (MOI, 30) with SFV4, and cell viability (Wst-1 assay) was measured over time. Uninfected cells (□) were included as a control. This was repeated three times with similar results.