1 Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
2 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
3 Department of Microbiology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
4 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
5 The Croucher Laboratory for Human Genomics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
6 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
7 Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China
Correspondence
Stephen K. W. Tsui
kwtsui{at}cuhk.edu.hk
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ABSTRACT |
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INTRODUCTION |
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The 3a locus (also known as X1 or ORF3; nt 2526826092 in the Tor2 strain of SARS-CoV) encodes one of the ORFs of unknown function and is located between two structural genes encoding the spike and the envelope proteins of SARS-CoV (Marra et al., 2003). Interestingly, the 3a ORF is not found in two human coronaviruses (OC43 and 229E) or other coronavirus species identified to date. This suggests that the 3a protein is a newly emerged protein in coronaviruses. In SARS-CoV-infected monkey kidney Vero E6 cells, 3a mRNA (Zeng et al., 2003
) and 3a protein (Yu et al., 2004
) have been detected. In addition, the 3a protein has been detected in the lung tissue of a SARS patient (Yu et al., 2004
).
When the 3a sequence was searched against the SMART server (Letunic et al., 2004), a predicted signal sequence was found at aa 116. In addition, three transmembrane domains were predicted at aa 3456, 7799 and 103125. Furthermore, the C-terminal region of the 3a protein shares 53 % (aa 209264) and 40 % (aa 152254) similarity, respectively, with the Plasmodium calcium pump and the Shewanella outer-membrane porin. Notably, the outer-membrane porins are a family of bacterial proteins that may oligomerize to form transmembrane channels for the passive diffusion of small molecules across membranes.
Previous studies have shown that many coronaviruses, including murine hepatitis virus, avian infectious bronchitis virus and transmissible gastroenteritis coronavirus, are able to induce apoptosis of host cells (An et al., 1999; Eléouët et al., 2000
; Liu et al., 2001
, 2003
; Chen & Makino, 2002
), but little is known about this ability in SARS-CoV. Apoptosis was observed in liver specimens from patients with SARS-associated viral hepatitis (Chau et al., 2004
) and lymphopenia is commonly observed in SARS patients and has been postulated to be caused by apoptosis induced by SARS-CoV infection (O'Donnell et al., 2003
). Although lymphopenia may be a result of glucocorticoid treatment (Panesar et al., 2004
), it may also be due to the upregulation of apoptotic genes in SARS-CoV-infected human peripheral blood mononuclear cells, as shown by oligonucleotide array analysis (Ng et al., 2004
). Furthermore, SARS-CoV can induce a cytopathic effect and apoptosis (Yan et al., 2004
) in some cell-culture models, such as Vero E6 cells, and the nucleocapsid protein is able to induce apoptosis in COS-1 monkey kidney cells in the absence of growth factors (Surjit et al., 2004
). Recently, the ORF7a protein has been shown to induce apoptosis when overexpressed in Vero E6 cells (Tan et al., 2004b
). In the present study, we demonstrated that overexpression of the non-structural 3a protein triggers apoptosis in Vero E6 cells.
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METHODS |
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Immunohistochemistry.
Sections of 4 µm were prepared from 10 % formalin-fixed, routinely processed paraffin blocks of autopsy specimens. A standard avidinbiotin staining method was used for immunohistochemical studies, using antibodies specific for peptides of the nucleocapsid (N terminus, aa 117) and 3a (C terminus, aa 250274) proteins (diluted 1 : 100) and pre-immune serum. Antibodies were generated from rabbits immunized with a keyhole limpet haemocyanin-conjugated synthetic peptide. Antigen retrieval was performed by microwave pre-treatment in 10 mM citrate buffer, pH 6·0, with preliminary heating at 780 W for 4 min, followed by 480 W twice for 5 min each.
Plasmid construction.
To clone the 3a gene into pcDNA4/HisMaxTOPO (Invitrogen Life Technologies), the 3a ORF was amplified by using primers 3a-pcDNAF (5'-ATGGATTTGTTTATGAGATTTTTTACTCTTGG-3') and 3a-pcDNAR (5'-TTACAAAGGCACGCTAGTAGTCGTCG-3'). cDNA prepared from the Su-10 coronavirus (Tsui et al., 2003) was used as template. The PCR product was then ligated to linearized pcDNA4/HisMaxTOPO vector according to the manufacturer's protocol (Invitrogen Life Technologies) to produce the recombinant construct pcDNA4-3a. To prepare the recombinant construct pEGFP-3a, the 3a ORF was PCR-amplified by using the primers 3aC1F (5'-GCAGATCTATGGATTTGTTTATGAGATTTTTTACTCTTGGATC-3') and 3aC1R (5'-GCGGTACCTTACAAAGGCACGCTAGTAGTCGTCGTC-3'). The PCR product was digested with restriction enzymes (BglII and KpnI) and ligated to the pEGFP-C1 vector [encoding a variant of wild-type green fluorescent protein (GFP); BD Biosciences Clontech].
Subcellular localization of the 3a protein.
Vero E6 cells seeded on coverslips were co-transfected with 1 µg pcDNA4-3a and pDsRed2-ER (BD Biosciences Clontech) constructs. At 1 day post-transfection, cells were washed and fixed in 100 % chilled methanol for 3 min. After blocking with 2 % BSA in PBS for 30 min, cells were incubated with anti-3a antibody diluted 1 : 100 for 30 min. Cells were then washed three times with PBS. For fluorescent visualization, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody diluted in 1 : 500 in 2 % BSA in PBS for 30 min. After washing five times with PBS, cell nuclei were stained with Hoechst 33342 (2·5 µg ml1 in PBS; Molecular Probes) for 30 min. Fluorescent signals were detected by using a Nikon TE2000U microscope and images were captured with the SPOT RTKE imaging system (Diagnostic Instruments).
Overexpression of the 3a protein and Western blotting analysis.
Approximately 1·7x105 Vero E6 cells were transfected with 1 µg pcDNA4-3a or control plasmids in a six-well plate by using Lipofectamine PLUS reagent (Invitrogen Life Technologies). Cells were collected daily for 5 days, washed with 1x PBS and trypsinized. The cell pellet was then resuspended in 100 µl lysis buffer [2 % SDS, 10 % glycerol, 0·0625 M Tris/HCl (pH 8·0)] and incubated on ice for 30 min. After boiling for 10 min and centrifugation at 12 000 g for 20 min, the supernatant was saved for protein quantification, polyacrylamide-gel electrophoresis and transfer to PVDF membrane (Millipore). The blot was incubated with primary antibody (diluted 1 : 1000) overnight and horseradish peroxidase-conjugated secondary antibody (diluted 1 : 1000) for 1 h. Antibodies used in this study for the detection of apoptotic markers were purchased from Santa Cruz Biotechnology, Sigma or Invitrogen Life Technologies. Signals were detected by using an Enhanced Chemiluminescence Western Blotting kit (Amersham Biosciences).
DNA-ladder analysis.
Approximately 1·7x105 Vero E6 cells were transfected with 1 µg pcDNA4-3a or control plasmids in a six-well plate by using Lipofectamine PLUS reagent (Invitrogen Life Technologies). Cells were collected daily for 5 days, washed with 1x PBS and lysed in 400 µl lysis buffer [200 mM Tris (pH 8·3), 100 mM EDTA and 1 % SDS] supplemented with 10 µl proteinase K (20 mg ml1). After incubation at 37 °C for 2 h, 150 µl saturated NaCl was added to the lysate and the mixture was shaken vigorously for 1 min. After centrifugation at 6500 g for 15 min, the supernatant was mixed with 1 ml ice-cold absolute ethanol and the mixture was subjected to centrifugation at 15 000 g for 20 min. The pellet was then washed with 1 ml ice-cold 75 % ethanol and centrifuged at 7000 g for 5 min. The supernatant was discarded and the DNA pellet was allowed to dry in air. Finally, the pellet was resuspended in 20 µl RNase A solution (0·2 mg ml1) and incubated at 37 °C for 90 min. DNA fragmentation was then analysed in a 2 % agarose gel.
Chromatin-condensation analysis and immunostaining.
The recombinant constructs pEGFP-3a, pEGFP-C1 and pcDNA4-3a and the pcDNA4 empty vector were transfected independently into Vero E6 cells by using Lipofectamine PLUS reagent according to the manufacturer's protocol (Invitrogen Life Technologies). For chromatin-condensation analysis, cells were fixed in 4 % paraformaldehyde and counterstained with either propidium iodide (1 mg ml1; Sigma) or Hoechst 33342 (2·5 µg ml1; Molecular Probes) in PBS for 30 min. For immunostaining, cells were treated as described above.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay.
Approximately 1·7x105 Vero E6 cells seeded on coverslips were transfected with pcDNA4-3a or pcDNA4 empty vector by using Lipofectamine PLUS reagent according to the manufacturer's protocol (Invitrogen Life Technologies). Cells were analysed for DNA fragmentation at 35 days post-transfection. Cells treated with DNase I (3000 U ml1) served as the positive control. Cells were fixed in 4 % paraformaldehyde for 1 h and then treated with permeabilization solution (0·1 % Triton X-100, 0·1 % sodium citrate in PBS) for 5 min on ice. Cells were then incubated with a mixture of 45 µl labelling solution and 2 µl TUNEL reaction mix (Roche) in a humidified CO2 incubator at 37 °C for 1 h. Cell nuclei were counterstained with 300 nM 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes). Fluorescent signals were detected by using a Nikon TE2000U microscope and images were captured with the SPOT RTKE imaging system (Diagnostic Instruments).
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RESULTS |
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DISCUSSION |
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Sequence analysis has suggested that the 3a protein contains an N-terminal signal sequence and three transmembrane domains. Although the 3a protein was reported to be localized in the perinuclear region and plasma membrane (Tan et al., 2004a), others have suggested that the 3a protein is distributed in the cytoplasm and Golgi apparatus (Yu et al., 2004
). In contrast to these findings, our results indicated that the 3a protein localized to the ER. The discrepancy in subcellular localization may be due to differences in the specificities of antibodies being used. Another possible explanation is the involvement of the 3a protein in different subcellular compartments, as it probably is a multifunctional protein. Most notably, the 3a protein is unique to SARS-CoV and there is no homologue in other coronaviruses, which may explain the unexpectedly high virulence of SARS-CoV. The 3a protein has a cysteine-rich region located at the junction of the transmembrane and cytoplasmic regions, which can facilitate the formation of inter-chain disulfide bonds with the spike protein and may play an important role in the virulence of SARS-CoV (Zeng et al., 2004
). In addition, Tan et al. (2004a)
suggested that the 3a protein can interact with structural proteins and may participate in virus assembly and propagation. Thus, the 3a protein may play different roles in virus virulence and assembly, as well as in the induction of apoptosis.
Apoptosis is an important host-defence mechanism that controls viral infection (O'Brien, 1998; Roulston et al., 1999
). However, virus-induced apoptosis can limit the inflammatory response and somehow facilitate the dissemination of progeny undetected by the host immune system (O'Brien, 1998
). Many RNA viruses are known to induce apoptosis and this can play an important role in viral pathogenesis (Mori et al., 2004
). For instance, human immunodeficiency virus induces apoptotic cell death in T cells (Plymale et al., 1999a
, b
) and influenza virus induces apoptosis in cultured MDCK and U937 cells (Price et al., 1997
), whilst Dengue virus causes apoptotic cell death in hepatoma cell lines (Marianneau et al., 1997
, 1998
) and rubella virus provokes programmed cell death in Vero E6 cells (Hofmann et al., 1999
). Apoptotic cell death has been reported in SARS-CoV-infected Vero E6 cells (Yan et al., 2004
) and upregulation of apoptotic genes is observed in SARS-CoV-infected human peripheral blood mononuclear cells (Ng et al., 2004
). However, the precise mechanism and the viral protein(s) involved remain largely unknown. Recently, the nucleocapsid protein and the non-structural protein ORF7a have been shown to induce apoptosis when overexpressed in COS-1 and Vero E6 cells, respectively (Surjit et al., 2004
; Tan et al., 2004b
). Here, we have demonstrated for the first time that the non-structural protein 3a alone can induce apoptosis in SARS-CoV-susceptible Vero E6 cells.
Although expression of the 3a protein in mammalian cells was reported to be unsuccessful without the use of the vaccinia virus-infected transfection method (Yu et al., 2004), we detected 3a protein expression in various cell lines, including Vero E6 cells, by using a simple transient transfection, and the recombinant 3a protein migrated as a single-band protein with a size of approximately 35 kDa. The time lag between peak expression of the 3a protein (Fig. 4b
) and the appearance of DNA fragmentation (Figs 4a and 5
) is possibly due to the fact that DNA fragmentation is a late apoptotic event. Our study has shown that overexpression of the 3a protein can induce chromatin condensation and low-molecular-mass apoptotic DNA fragmentation from 3 days post-transfection. These data were consistent with the results of the TUNEL assay, showing a significant amount of internucleosomal DNA cleavage from 3 days post-transfection. As the receptor-mediated apoptotic pathway is Bcl-2-insensitive (Chen & Makino, 2002
) and we found that caspase-8 (an apical caspase in the death-receptor apoptotic pathway) was activated, we postulate that overexpression of the 3a protein induces apoptosis, mediated through a caspase-8-dependent pathway, which may be similar to the death-receptor signalling cascades.
Analyses of the 3a protein has shown that it is homologous to the Shewanella outer-membrane porin and the Plasmodium calcium pump; therefore, we speculate that the 3a protein may alter membrane calcium-ion permeability. Calcium ions released from the ER perturb the ER/cytosolic calcium gradient and increase the cytosolic calcium concentration, which subsequently leads to ER stress-induced apoptosis (Breckenridge et al., 2003). This may provide another possible mechanism of 3a-induced apoptosis.
In summary, the 3a protein was detected in lung and intestinal specimens of SARS patients. We demonstrated that the 3a protein is an ER membrane-bound protein that can induce apoptosis thorough a caspase-8-dependent pathway in Vero E6 cells. The present study provides a molecular link between SARS and apoptosis, and further investigations are needed to define in more detail the functional role of the 3a protein in SARS infection.
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ACKNOWLEDGEMENTS |
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Received 8 December 2004;
accepted 2 April 2005.