The SARS coronavirus nucleocapsid protein induces actin reorganization and apoptosis in COS-1 cells in the absence of growth factors

Abstract

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia, and was subsequently proven to be the causative agent of the disease now referred to as SARS (severe acute respiratory syndrome). The complete genome of the SARS-CoV (SARS coronavirus) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) protein shares little homology with other members of the coronavirus family. In the present paper, we show that SARS-CoV N is capable of inducing apoptosis of COS-1 monkey kidney cells in the absence of growth factors by down-regulating ERK (extracellular-signal-regulated kinase), up-regulating JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and affecting their downstream effectors. SARS-CoV N expression also down-regulated phospho-Akt and Bcl-2 levels, and activated caspases 3 and 7. However, apoptosis was independent of the p53 and Fas signalling pathways. Furthermore, activation of the p38 MAPK pathway was found to induce actin reorganization in cells devoid of growth factors. At the cytoskeletal level, SARS-CoV N down-regulated FAK (focal adhesion kinase) activity and also down-regulated fibronectin expression. This is the first report showing the ability of the N protein of SARS-CoV to induce apoptosis and actin reorganization in mammalian cells under stressed conditions.

Figure 1. SARS Co-V N induces cell death

(A) Expression of N protein was analysed by immunoprecipitating mock-transfected (C) or pcDNA3.1 myc-tagged N- (myc-N) or pSGI HA-tagged N- (HA-N) transfected cell lysates with corresponding antibodies. Lanes 2 and 4 show N expression, while lanes 1 and 3 are respective controls. (B) Results of TUNEL assay. Lanes 1 and 2 respectively show mock- (C) or N-transfected (N) cells in presence of serum (Ser), while lanes 3 and 4 show the same cells in the absence of serum. TUNEL-positive cells were scored from a total of 500 cells viewed from each of the three sets of experiments. The histogram shows the means±S.E.M. for three independent sets of experiments. The P values are 0.17 for lanes 1 and 2, and 0.02 for lanes 3 and 4. TUNEL-positive cells in the absence of serum constituted approx. 30% of the total cell count. Pr., protein. (C) A representative field of TUNEL-positive cells as observed under the microscope.

Figure 2. N down-regulates fibronectin expression

(A) Mock-transfected (C) or N-transfected (N) cells were maintained in the presence (Ser+) or absence (Ser−) of serum. For extracellular fibronectin level (upper panel), growth medium was immunoprecipitated using the respective antibody. For intracellular fibronectin level, cells were treated for 2 h with brefeldin A (10 μg/ml), lysed in RIPA buffer, immunoprecipitated using anti-fibronectin antibody, resolved by SDS/6% PAGE and immunoblotted. Values were normalized with reference to a non-specific band (calnexin) in the same gel (for extracellular fibronectin or to the level of calnexin for intracellular fibronectin). (B) Cells grown under similar conditions as above were lysed in SDS buffer, resolved by SDS/7% PAGE and immunoblotted using anti-(integrin α_V) or anti-calnexin antibody (loading control). (C) Level of p-FAK (Tyr³⁹⁷). The lower gel shows calnexin level as a loading control. N(1.5) and N(3) represents samples transfected with 1.5 μg and 3 μg DNA respectively. Mock samples were transfected with 3 μg of empty vector DNA. The histogram shows the means±S.E.M. for three independent sets of experiments. (D) N expression as assessed by immunoprecipitation with anti-Myc antibody in the presence (lanes 1, 2 and 3) or absence of (lanes 4 and 5) serum respectively, with different concentrations of DNA. The histogram shows the means±S.E.M. for three independent sets of experiments.

Figure 3. N protein expression modulates cellular MAPK activity

(A) Mock-transfected or Myc–N clone-transfected cells were maintained in serum (Ser+) for 48 h (lanes 1–3) or serum-starved for 24 h (Ser−, lanes 4–6); harvested with SDS lysis buffer, resolved by SDS/12% PAGE, and immunoblotted using corresponding antibodies. The Figure shows the levels of p-ERK and total ERK. The histogram shows means±S.E.M. for three independent sets of experiments. (B) Levels of p-JNK, p-Jun and total c-Jun level. Calnexin was used to ensure equal loading. The histogram shows the means±S.E.M. for three independent sets of experiments. Black bars represent p-JNK, dark grey bars represent p-c-Jun and light grey bars represent c-Jun. (C) The level of p-p38 MAPK, p-MAPKAPK2 (MAPK-activated protein kinase 2) and p-HSP27. Total p38 MAPK level served as the loading control for p-p38 MAPK. The histogram shows the means±S.E.M. for three independent sets of experiments. Black bars represent p-p38 MAPK, dark grey bars represent p-MAPKAPK2 and light grey bars represent p-HSP27.

Figure 4. Actin reorganization induced by the SARS Co-V N protein

Immunofluorescence study using rhodamine-conjugated phalloidin and HA-tagged N. (A) Actin distribution in the presence of serum. The yellow arrow shows an N-transfected cell, and the green arrow shows a non-transfected cell. (B) The same field for N expression stained with FITC-conjugated anti-mouse antibody (merged image). (C) Actin distribution in the absence of serum. Arrows as in (A). (E) Actin redistribution in the presence of cytochalasin D (5 nM). (G) Effect of SB203580 (10 μg/ml). (D, F and H) Superimposition of FITC-stained images on rhodamine-stained images showing N expression in the absence of serum, cytochalasin D or SB203580 respectively.

Figure 5. N expression down-regulates pro-survival factors and induces apoptosis in the absence of growth factors

(A) The level of p-Akt (Thr³⁰⁸) in the presence (Ser+) and absence (Ser−) of serum. The lower gel shows the level of total Akt under the same conditions. The histogram shows the means±S.E.M. for three independent sets of experiments. (B) Bcl-2 was immunoprecipitated from mock (C) or N-transfected (N) cell lysates in the presence (Ser+) or absence (Ser−) of serum respectively, and was detected by Western blotting. The calnexin level is shown as loading control in the bottom gel. The histogram shows the means±S.E.M. for three independent sets of experiments. (C) The activation of caspases 3 and 7 in N-expressing cells (lane 4). Anti-(caspase 3) antibody detects both the precursor and cleavage product. (D) Anti-(caspase 7) antibody is specific for cleaved product only. (E) Western blot of caspase 3 in serum-deprived N-expressing cells in the presence of overexpressed p110α subunit of PI3K (110, lane 1), 10 μg/ml SB203580 (SB, lane 2) or 100 μM Z-VAD-FMK (Z, lane 3).