Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis

Abstract

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE.

Figure 1. Characterization of infection of Vero E6 and MA-104 cells with rSARS-CoV and rSARS-CoV-ΔE.

Vero E6 and MA-104 cells were infected at an moi of 2 with rSARS-CoV-ΔE or rSARS-CoV. (A) Growth kinetics curves. Virus titers in supernatants of infected cells at different times pi were determined by plaque assay. (B) Levels of intracellular genomic RNA in infected cells at different times pi as determined by qRT-PCR. (C) Levels of intracellular N gene sg mRNA in infected cells at different times pi as determined by qRT-PCR. Standard bars represent standard deviations of the mean of results from three experiments.

Figure 2. Effect of SARS-CoV E protein on host gene expression.

Comparison of gene expression in Vero E6 (at 15 hpi) and MA-104 (at 65 hpi) cells using microarrays: rSARS-CoV versus mock-infected, rSARS-CoV-ΔE versus mock-infected and rSARS-CoV-ΔE versus rSARS-CoV-infected cells. Red spots indicate upregulated gene transcripts while green spots indicate downregulated gene transcripts. Only genes with a fold change higher than two or lower than minus two (FDR<0.01) were considered.

Figure 3. Host cell genes differentially expressed in rSARS-CoV-ΔE versus rSARS-CoV-infected cells using microarrays.

Genes differentially expressed in rSARS-CoV-ΔE versus rSARS-CoV-infected Vero E6 and MA-104 cells, were classified according to their main biological functions. Only genes with a fold change higher than two or lower than minus two (FDR<0.01) were considered. –, indicates that the gene is not detected in the array or is not differentially expressed with a fold change higher than two or lower than minus two. Red color indicates genes upregulated more than 10 fold. Green color indicates genes upregulated between 5 and 10 fold, at least in one cell line. For those genes recognized with more than one probe, the value corresponding to the highest upregulation or downregulation is represented.

Figure 4. Stress response proteins are differentially expressed in rSARS-CoV-ΔE versus rSARS-CoV-infected cells.

Total protein from Vero E6 (A) and MA104 (B) cells infected with rSARS-CoV-ΔE or SARS-CoV was extracted at 22 and 75 hpi, respectively. Levels of hsp90 and hsp60 were normalized to those of GAPDH after Western-blot assay (left panels) and densitometric analysis of the bands (right panels). Columns represent hsp90/GAPDH and hsp60/GAPDH ratios in SARS-CoV-ΔE (red) or SARS-CoV (blue) infected cells. Error bars indicate the standard deviation from three independent experiments.

Figure 5. Upregulation of functionally associated genes in rSARS-CoV-ΔE compared to rSARS-CoV-infected cells using microarrays.

Gene Sets, based on Gene Ontology terms, that correlate with upregulated genes in Vero E6 cells at 15 hpi (A) and in MA-104 cells at 65 hpi (B). X values: −log10 (FDR-q val).

Figure 6. Cellular stress responses induced by rSARS-CoV-ΔE infection.

Vero E6 and MA-104 cells were infected with rSARS-CoV-ΔE or SARS-CoV at an moi of 2. Cellular RNAs were extracted at 22 (Vero E6) and 65 (MA-104) hpi and the expression of cellular mRNAs corresponding to cytosolic, ER and mitochondrial stress genes was measured by qRT-PCR. In the case of cytosolic stress, only genes with fold increases >2.5 measured by microarrays were further evaluated by qRT-PCR. Numbers indicate the levels of gene expression in rSARS-CoV-ΔE compared to SARS-CoV-infected cells. Three independent experiments were analyzed with similar results in all cases.

Figure 7. Effect of SARS-CoV E protein on stress induced by SARS-CoV infection.

Vero E6 cells infected at an moi of 0.5 with rSARS-CoV-ΔE-P1 or rSARS-CoV were transfected with a plasmid expressing E protein (E⁺) or with the empty plasmid (E⁻) as control. (A) Accumulation of SARS-CoV E protein and GAPDH as a loading control, at 15 and 22 hpi were evaluated by Western blot. (B) Virus titers in the presence or absence of E protein provided in trans were evaluated at 15 and 22 hpi. (C) At 22 hpi, cellular RNAs were extracted, and the expression of the stress-induced genes hspA1A, hsp90AA1, hspH1, SERPINH1, and hspE1, and that of polQ and 18S rRNA, as controls, were analyzed by qRT-PCR. In each case, the expression levels of mRNAs encoding representative cell stress proteins were evaluated in rSARS-CoV-ΔE-P1-infected cells in relation to rSARS-CoV-infected cells. Bars represent standard deviations of the mean from three experiments.

Figure 8. Effect of SARS-CoV E protein on the stress induced by RSV infection.

Vero E6 cells transfected with a plasmid expressing SARS-CoV E protein (E⁺) or with the empty plasmid (E⁻) were infected with RSV at a moi 2. (A) Accumulation of SARS-CoV E protein and GAPDH as a loading control, at 2 and 24 hpi in the case of RSV-infected cells and at 15, 22, and 28 hpi in the case of SARS-CoV-infected cells, was evaluated by Western blot. (B) Intracellular RNA was extracted at 2 and 24 hours post-RSV infection and the expression of cellular stress genes, polQ and 18S rRNA, was measured by qRT-PCR. In each case, levels of expression in infected cells were compared to those in mock-infected ones. Bars represent standard deviations of the mean of results from three experiments.

Figure 9. Effect of SARS-CoV E protein on the induction of ER stress caused by drugs.

Vero E6 and MA-104 cells were transfected with a plasmid expressing SARS-CoV E protein (E⁺) or with the empty plasmid (E⁻). At 24 hpt, the cells were treated with 1000 nM thapsigargin (+thap), 2 µg/ml of tunicamycin (+tun) or left untreated (−). (A) Levels of SARS-CoV E and GAPDH (loading control) at 8 and 20 h post treatment in Vero E6 cells were determined by Western blot. The expression of the stress induced genes, GRP78 and GRP94 ER and that of polQ and 18S rRNA was evaluated by qRT-PCR in Vero E6 (B) or MA-104 (C) cells. In each case, levels of expression in treated cells were compared to non-treated cells. Bars represent the standard deviations from the mean in three independent experiments.

Figure 10. Activation of the IRE-1 pathway in rSARS-CoV-ΔE-infected cells.

Vero E6 cells were infected with rSARS-CoV-ΔE or rSARS-CoV at an moi of 2. Splicing of XBP-1 mRNA was analyzed at different times pi using oligonucleotides flanking the splicing region. Numbers below the gel represent the percentage of spliced/unspliced forms of XBP-1.

Figure 11. rSARS-CoV-ΔE-induced apoptosis.

Apoptosis levels in mock, rSARS-CoV-ΔE and rSARS-CoV-infected cells were evaluated at 4 (A), 15 (B) and 24 (C) hpi by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V⁺, PI⁻) from those in late apoptosis (Annexin V⁺, PI⁺).