The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor

Abstract

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (3) ATF4-dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN) signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.

Figure 1. Induction of ER stress by the 3a protein.

(A) The grp78-luc reporter plasmid (0.25 µg) was co-transfected in Huh7 with plasmid pSGI, pEGFP or pEYFP (vector controls) or pSGI-3a-HA, pEGFP-Vpu, pEGFP-Orf3 or pEYFP-Nef (0.75 µg each). COS or Vero cells were also co-transfected with grp78-luc (0.25 µg) and either plasmid pSGI-HA (vector control) or pSGI-3a-HA (0.75 µg each). (B) The grp94-luc reporter plasmid (0.25 µg) was similarly co-transfected in Huh7 or COS cells along with plasmid pSGI-HA (vector control) or pSGI-3a-HA (0.75 µg each). The transfections were carried out in 12-well plates. Cell lysates were prepared 48 hr post-transfection, the protein content estimated and the normalized lysates were analyzed for luciferase activity, expressed as Relative Light Units. Each bar is representative of three separate experiments, each with three independent transfections.

Figure 2. The 3a protein mediates ER stress through eIF2α.

(A) Huh7 cells were transfected with 1 µg of either pSGI or pSGI-3a-HA, or untransfected cells were treated with 500 nM thapsigargin (Tg) for 16 hr prior to harvest. The cells were harvested at the indicated times, RNA prepared with Trizol and amplified by RT-PCR for the XBP-1 unspliced (XBP-1^u) and spliced (XBP-1^s) RNA forms. RT-PCR for histone mRNA was used as a loading control. (B) Huh7 cells were transfected with 1 µg of either pSGI or pSGI-3a-HA; untransfected cells were treated with 1.5 mM dithiothreitol for 45 min prior to harvest, as a positive control for ER stress. Cell lysates were prepared 48 hr post-transfection, equal amounts of proteins were separated by SDS-PAGE and western blotted for the indicated proteins. (C) Huh7 cells were co-transfected with the grp78-luc reporter plasmid (0.2 µg), pSGI or pSGI-3a-HA (0.4 µg), and an expression plasmid for either wild type, dominant-active (DA) or dominant-negative (DN) eIF2α (0.4 µg). After 48 hr, cell lysates were prepared, normalized and analyzed for luciferase activity. Each bar is representative of two separate experiments, each with three independent transfections. All the transfections were carried out in 12-well plates.

Figure 3. The 3a protein activates the PERK pathway.

The (A) ATF4-UTR-luc or (B) CHOP-luc plasmids (0.25 µg each), which express luciferase under control of the ATF4 untranslated region or the CHOP gene promoter, respectively, were co-transfected in Huh7 cells together with 0.75 µg of plasmid pSGI-HA, pEGFP, pEYFP or pIRES-EGFP (as controls) or pSGI-3a-HA, pEGFP-Vpu, pEGFP-Orf3, pEYFP-Nef, pIRES-GFP-XO (as test). (C) The grp78-luc reporter plasmids (0.25 µg each) were co-transfected in Huh7 with either plasmid pSGI-HA (vector control) or pSGI-3a-HA (0.75 µg each), except that one set of cells were treated with 20 µM SB203580 for 12 hr before harvest, while the controls were treated with the same volume of DMSO. Cell lysates were prepared 48 hr post-transfection, and normalized lysates were analyzed for luciferase activity. Each bar is representative of two separate experiments, each with three independent transfections, which were carried out in 12-well plates. In (C), the p value for comparisons between untreated and SB203580-treated pSGI-3a-HA transfected cells was calculated to be 0.345.

Figure 4. The 3a protein increases phosphorylation of IFNAR1 and its subsequent ubiquitination.

(A) Huh7 cells were transfected with plasmid pSGI-HA or pSGI-3a-HA (2 µg each); untransfected cells were also treated with 1 µM tunicamycin for 16 hr before harvest as a positive control for ER stress. Cell lysates were prepared 48 hr post-transfection and equal amounts of proteins were subjected to immunoprecipitation (IP) using IFNAR1 antibody followed by western blotting with a phosphoserine antibody. Cell lysates were also subjected to western blotting with indicated antibodies as controls. (B) Transfections were as in (A) above. Normalized cell lysates were subjected to immunoprecipitation (IP) with an anti-IFNAR1 antibody followed by western blotting (WB) with an anti-Ubiquitin antibody. Cell lysates were also directly subjected to western blotting with indicated antibodies as controls. Arrows indicate the partially (lower band) and heavily glycosylated (higher band) forms of the IFNAR1 protein. The transfections were carried out in 60 mm dishes.

Figure 5. The 3a protein increases lysosomal accumulation of IFNAR1.

Huh7 cells were grown on coverslips placed in the wells of 6-well dishes and transfected with 2.5 µg of plasmid pECFP or p3a-ECFP. After 48 hr, the cells were washed and stained with Lysotracker as well as an anti-IFNAR1 antibody, as described in Materials and Methods. The cells were then imaged with a confocal microscope, the images quantitated and colocalization coefficients calculated as described. Separate images for Lysotracker (red), IFNAR1 (green) and ECFP/3a-ECFP (cyan) are shown, together with merged images for Lysotracker and IFNAR1. Scale bar, 10 µm. The bar graph shows Pearson's colocalization coefficients; p = 1.8×10⁻⁷.

Figure 6. The 3a protein reduces endogenous levels of IFNAR1.

Huh7 cells in 60 mm dishes were transfected with 2.5 µg of either pECFP or p3a-ECFP. In one set, the p3a-ECFP transfected cells were also treated with 20 mM 3-MA for 1 hr prior to harvest. The cells were harvested 48 hr post-transfection and endogenous levels of IFNAR1 were analyzed by flow cytometry as described in Materials and Methods. The mean fluorescence intensity (MFI) of IFNAR1 signal was estimated and p values shown were calculated with the Student's T test. The flow cytometry plot is representative of three separate experiments, while the MFI values shown are an average (±SD) of these experiments.