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 (eIF2α) and inhibitory effects of a dominant-negative form of eIF2α 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.

The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis. INTRODUCTIONThe aetiological agent for severe acute respiratory syndrome (SARS) was identified as a novel coronavirus (SARS-CoV) (Peiris et al., 2003). SARS-CoV has a polyadenylated, positive-sense RNA genome of approximately 30 kb (Marra et al., 2003). In addition to the prototypic coronavirus genes, the SARS-CoV genome also contains nine unique open reading frames (ORFs) (Marra et al., 2003). Of these, orf3a is the largest and encodes a protein of 274 aa, variously called ORF3A (Ito et al., 2005), X1 (Rota et al., 2003) or U274 (Tan et al., 2004b). The 3a protein has been predicted to contain an N-terminal signal peptide followed by three transmembrane domains and a C-terminal cytoplasmic domain of approximately 150 aa (Zeng et al., 2004).The 3a protein is associated with virus particles produced following infection of Vero E6 or Caco-2 cells (Ito et al., 2005;Shen et al., 2005) and can assemble into virus-like particles when co-expressed with the membrane and envelope proteins in insect cells (Shen et al., 2005). In vitro studies have also shown the 3a protein to interact with the viral envelope, membrane and spike proteins (Tan et al., 2004b;Tan, 2005) and the cellular protein caveolin-1 (Padhan et al., 2007). A deletion of orf3a was shown to reduce virus titres, but not to eliminate virus replication (Yount et al., 2005), and convalescent sera from SARS patients have antibodies to the 3a protein (Tan et al., 2004a), suggesting that it is expressed during virus infection of the host. The 3a protein was shown to upregulate the expression of fibrinogen in A549 lung epithelial cells and to possess an ionchannel activity selective for monovalent cations (Lu et al., 2006). Ectopic expression of the 3a protein has been shown to induce apoptosis in Vero E6 cells through activation of caspase-8, chromatin c...

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BackgroundThe SARS coronavirus (SARS-CoV) 3a protein functions as an ion channel, induces apoptosis and is important for viral pathogenesis. It is expressed on the cell surface and contains a tyrosine-based sorting motif and a di-acidic motif, which may be crucial for its intracellular trafficking. However the role of these motifs is not fully understood in the case of 3a protein.MethodsThe subcellular distribution of the 3a protein was studied by immunofluorescence staining of cells transfected with wild type and mutant constructs along with markers for different intracellular compartments. Semi-quantitative RT-PCR was performed to estimate the mRNA where as western blotting was carried out to detect protein levels of wild type and mutant 3a proteins. In vitro transcription- translation was performed to estimate cell free protein synthesis.ResultsWhile the wild type 3a protein is efficiently transported to the plasma membrane, the protein with mutations in the tyrosine and valine residues within the YXXV motif (ΔYXXΦ) accumulated in the Golgi compartment. However the 3a protein with mutations within the EXD di-acidic motif (ΔEXD) showed an intracellular distribution similar to the wild type protein. Increased retention of the ΔYXXΦ protein in the Golgi compartment also increased its association with lipid droplets. The ΔYXXΦ protein also expressed at significantly lower levels compared to the wild type 3a protein, which was reversed with Brefeldin A and Aprotinin.ConclusionsThe data suggest that the YXXΦ motif of the SARS-CoV 3a protein is necessary for Golgi to plasma membrane transport, in the absence of which the protein is targeted to lysosomal degradation compartment via lipid droplets.