The PDZ-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis

Abstract

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Generation of recombinant SARS-CoVs with E protein PBM truncated or mutated by reverse genetics and growth kinetics in cell culture.

(A) Top, representation of SARS-CoV E protein sequence and its corresponding domains. Below, sequences corresponding to the end of E protein are shown in boxes for the different viruses. SARS-CoV-E-wt represents the wild type sequence. In SARS-CoV-E-ΔPBM and SARS-CoV-E-mutPBM, E protein PBM was eliminated by deletion or point mutations, reducing or maintaining the full-length protein, respectively. In SARS-CoV-E-potPBM, four amino acids of E protein were replaced to alanine, to generate a non-native new potential PBM. PBM+ and PBM− represent the presence or absence of a PBM within E protein sequence, respectively. Red boxes highlight PBMs within E protein. (B) Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, ΔE, ΔPBM, mutPBM and potPBM viruses at an MOI of 0.05. Culture supernatants collected at 4, 24, 48 and 72 hpi were titrated by plaque assay. Error bars represent standard deviations of the mean of results from three experiments. Statistically significant data are indicated with one (P<0.05) or two (P<0.01) asterisks.

Figure 2. Virulence and viral growth of SARS-CoV-E-PBM-infected mice.

16-week-old BALB/c mice were intranasally inoculated with 100,000 pfu of wt, ΔE, ΔPBM, mutPBM and potPBM viruses. Weight loss (A) and survival (B) were monitored for 10 days. Data represent two independent experiments with 5 mice per group. Differences in weight loss between attenuated and virulent viruses were statistically significant (P<0.01). (C) Viral titer in lungs was determined at 2 and 4 days post infection (n = 3, each day). Error bars represent standard deviations. Statistically significant data are indicated with one (P<0.05) asterisk.

Figure 3. Lung pathology of mice infected with recombinant SARS-CoV-E-PBM mutants.

16-week-old BALB/c mice were inoculated intranasally with 100,000 pfu of wt, ΔE, ΔPBM, mutPBM and potPBM viruses. (A) Gross pathology of mouse lungs infected with recombinant viruses at 2 and 4 dpi. (B) Weight of left lungs excised from infected mice sacrificed at the indicated days (n = 3, each day). Error bars represent standard deviations. (C) Lung tissue sections from mice infected with recombinant viruses were prepared at 2 and 4 dpi and stained with hematoxylin and eosin. Three independent mice per group were analyzed. (D and E) Pathology scoring in lung of mice infected with SARS-CoV recombinant mutants. Lungs were harvested at 2 and 4 days and scored in a blinded fashion using 3 mice per condition on a scale of 0 (none) to 3 (severe), estimated according to previously described procedures . Data are presented for edema (D) and cellular infiltrates (E). Mean values are reported and statistically significant data are indicated with two (P<0.01) asterisks. Original magnification was 20x. Representative images are shown.

Figure 4. Effect of SARS-CoV E protein PBM on host gene expression.

(A) Comparison of gene expression in lungs of infected mice using microarrays: wt versus mock-infected, mutPBM versus mock-infected and mutPBM versus wt-infected mice. Red spots indicate upregulated gene transcripts (fold change, >2) and green spots indicate downregulated gene transcripts (fold change, x axis indicate DAVID FDR values. (C) Selection of differentially expressed genes found in at least one functional group using DAVID software. The numbers indicate the fold change for each gene in mutPBM versus wt-infected mice. (D) Expression of inflammatory cytokines evaluated by RT-qPCR. Three independent experiments were analyzed with similar results in all cases. Error bars represent standard deviations of the mean of results from three experiments.

Figure 5. Interaction of SARS-CoV E protein with cellular syntenin.

(A) Sequence of SARS-CoV E protein and the region containing amino acids 36–76 (SARS-CoV ECT) that was used as bait for the yeast two-hybrid screening. (B) Schematic representation of syntenin. Numbers at the bottom indicate the amino acids at the beginning and the end of the different domains. NTD; N-terminal domain; CTD, C-terminal domain; PDZ 1 and 2, PDZ domains. (C) Vero E6 cells transfected with a plasmid encoding an N-terminal HA-tagged syntenin were mock-infected (mock) or infected with recombinant viruses containing (wt and potPBM) or lacking (ΔE, PBM and mutPBM) E protein PBM, respectively. As a control, mock-transfected cells were infected with the wt virus (wt, last lane). Cells were lysed and subjected to immunoprecipitation using a monoclonal anti-HA antibody or polyclonal anti-E antibody to pulldown syntenin or E protein, respectively. The presence of E and syntenin proteins was analyzed in the precipitated fractions.

Figure 6. Colocalization of SARS-CoV E protein and syntenin in transfected and infected cells.

Vero E6 were mock-infected or infected with the wt virus at an MOI of 0.3 (A) or transfected with an empty plasmid (−E) or a plasmid expressing SARS-CoV E protein (+E) (B). At 24 hpi and 24 hours post transfection (hpt) for (A) and (B), respectively, cells were fixed with 4% paraformaldehyde and E or N proteins (red) and syntenin (green) were labeled with specific antibodies, nuclei were stained with DAPI (blue). Areas of colocalization of the two proteins appear yellow in the merged images. Scale bar = 10 µm. (C) Percentage of cells showing a cytoplasmic accumulation of syntenin after mock-infection or infected with wt or mutPBM viruses (n>50). Statistically significant data are indicated with one (P<0.05) or three (P<0.001) asterisks.

Figure 7. Activation of p38 MAPK in SARS-CoV-E-PBM mutants infected mice and cells.

Lung proteins were extracted from infected mice at 2 dpi. (A) The active phosphorylated (p-p38) and total (p38) p38 MAPK in lungs of three infected mice per condition were evaluated by Western blot analysis. (B) The active phosphorylated (p-p38) and total (p38) p38 MAPK in Vero E6 infected cells were evaluated by Western blot analysis. (C and D) Phospho and total p38 MAPK amounts were quantified by densitometric analysis. The graph shows the phosphorylated p38/total p38 MAPK ratio in wt, ΔE, ΔPBM, mutPBM and potPBM infected mice at 2 dpi (C) or Vero E6 cells at 24 hpi (D). Error bars represent the means of three mice analyzed for each condition. Statistically significant data are indicated with one (P<0.05) or two (P<0.01) asterisks.

Figure 8. Role of syntenin in the E protein PBM-dependent p38 MAPK activation.

(A) Vero E6 cells were mock-infected or infected with recombinant viruses containing (wt) or lacking (mutPBM) E protein PBM, and the presence of syntenin in the cytoplasm and nucleus and active p38 MAPK (p-p38) were detected by Western blot analysis at 24 hpi. As controls, histone H3 (H3), total p38 MAPK (Total p38) and actin were analyzed. (B) Vero E6 cells were transfected with an empty plasmid (empty vector) or a plasmid encoding a HA-tagged syntenin (HA-syntenin), and the presence of syntenin in the cytoplasm and nucleus, and active p38 MAPK were detected by Western blot analysis at 24 hpt. As controls, histone H3, total p38 MAPK, and actin were analyzed. (C) Quantification by qRT-PCR of syntenin mRNA in cells transfected with syntenin-specific siRNA (1 and 2) compared to reference levels from cells transfected with a validated-negative control siRNA (NEG). Mean values are reported, and statistically significant data are indicated with two (P<0.01) asterisks. (D) Effect of silencing syntenin expression on Vero E6 cells, mock-infected or infected with the wt virus. The presence of syntenin in the cytoplasm and nucleus and active p38 MAPK were detected by Western blot at 24 hpi. As controls, histone H3, total p38 MAPK and actin were analyzed. (E) Viral titers of wt virus in syntenin silenced Vero E6 cells were determined at 24 hpi. The experiments were performed three times, and the data represent the averages of triplicates. Standard deviations are indicated as error bars. hpi. The experiments were performed three times, and the data represent the averages of triplicates. Standard deviations are indicated as error bars.

Figure 9. Effect of p38 MAPK inhibitor in rSARS-CoV-MA15-infected mice.

16-week-old BALB/c mice were mock-infected or inoculated intranasally with 100,000 pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock hpi and every 12 h from days 1 to 8, mock h from days 1 to 8, mock-infected and wt-infected mice were intraperitoneally injected with SB203580 (6 mg mg/kg of body weight/day). (A) Animals were monitored daily for mortality. Data represent three independent experiments with 5 mice per group. (B) The active phosphorylated (p-HSP27) and total HSP27 in lungs of three infected mice per condition were evaluated by Western blot analysis. (C) Phospho and total HSP27 amounts were quantified by densitometric analysis. The graph shows the phosphorylated HSP27/total HSP27 ratio at 2 dpi in lungs of mock dpi in lungs of mock-infected mice or mice infected with the parental virus, treated or not with SB203580. Error bars represent the means of three mice analyzed for each condition. Statistically significant data are indicated with one (P<0.05) asterisk.