It is believed that human SARS-CoV may originate via zoonosis, given that similar viruses have been identified in the civet cat and bats [6, 7]. A feature of the human SARS-CoV that distinguishes it from its animal counterparts PROTAC ERRα ligand 2 is a 29-nt deletion from (previously known as ) resulting in which has the potential to encode a protein with only 39 aa [7]. The VLs in the stable clones expressing ORF8a were significantly higher than those in control subjects 5 days after infection. siRNA against significantly reduced VLs and interrupted the CPE. ORF8a was found to be localized in mitochondria, and overexpression resulted in increases in mitochondrial transmembrane potential, reactive oxygen species production, caspase 3 activity, and cellular apoptosis. ORF8a not only enhances viral replication but also induces apoptosis through a mitochondria-dependent pathway. The severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the causal agent of SARS [1-3]. The 29.7-kb RNA genome of SARS-CoV contains open reading frames (ORFs) for 5 structural proteins-replicase, spike, envelope, membrane, and nucleocapsid-as well as a number of accessory proteins that may participate in the viral life cycle [4, 5]. It is believed that human SARS-CoV may originate via zoonosis, given that comparable viruses have been identified in the civet cat and bats [6, 7]. A feature of the human SARS-CoV that distinguishes it from its animal counterparts is usually a 29-nt deletion from (previously known as ) resulting in which has the potential to encode a protein with only 39 aa [7]. The 29-nt sequence in the animal versions of the virus results in the merging of and into a new that encodes a putative protein 122 aa in length [7]. It is interesting to note that, among the 80 human SARS-CoVs whose full lengths have been sequenced, all of them contained with 1 exception: GD01, a strain identified early in the SARS endemic in Guangdong Province that contains [8]. We hypothesized that may have important functions in human SARS-CoV and its pathogenesis in cells that enabled SARS-CoV containing to become the predominant strain in the 2003 pandemic. In the present study, we used Western-blot (WB) assays with a recombinant glutathione S-transferase (GST)-ORF8a fusion protein to demonstrate that 2 patients with SARS had antiORF8a antibody reactivity. Subsequently, we used 2 stable clones expressing ORF8a and cells transfected with small interfering RNA (siRNA) to demonstrate that ORF8a enhances viral replication and the cytopathic effect (CPE). Finally, we showed that ORF8a was localized in mitochondria, where it could perturb the mitochondrial membrane potential and induce apoptosis via a caspase 3-dependent pathway. Patients, Materials, and Methods Serum samples from 37 patients infected with SARS-CoV were collected from Taipei Veterans General Hospital and Taipei Ho-Ping PROTAC ERRα ligand 2 Hospital. Infection in all patients had been confirmed serologically using a WB assay with recombinant spike and nucleocapsid proteins [9]. In addition, serum samples collected from 31 healthy subjects were used as normal controls. Total RNA was extracted from SARS-CoV-infected cells using Trizol (Invitrogen). A cDNA fragment of made up of RI and I restriction enzyme sites at the 5 and 3 ends, respectively, was generated by use of reverse-transcription polymerase chain reaction (RT-PCR). The RT-PCR product was digested by RI and I and inserted between the RI and I sites of a pcDNA3 vector made up of a hemagglutinin (HA) tag sequence and pGEX-5X-1 made up PROTAC ERRα ligand 2 of a GST gene to generate pHA-ORF8a and pGEXORF8a plasmids. The plasmid called pORF8a-EGFP (expressing green fluorescent protein [GFP]-tagged ORF8a) was constructed by inserting the cDNA of that contained I and dIII flanking sequences at the 5 and 3 ends into the upstream region of the EGFP gene in pEGFP-1 vector. All plasmids were confirmed by DNA sequencing using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). A synthetic peptide (SP) made up of amino acid residues 25- 37 of ORF8a was generated using the solid-phase method (Kelowna). The SP was coupled with keyhole limpet hemocyanin in accordance with procedures published elsewhere [10] and was used to immunize rabbits. ORF8a recombinant protein (RP) expressed in (BL21) as a GST fusion protein was purified by glutathione-sepharose affinity chromatography in accordance with the manufacturers recommendation. The GST-ORF8a RP (0.5 g for each strip) was used to evaluate the Rabbit Polyclonal to PNPLA8 antibody reactivity of serum from SARS-CoV-infected patients. In addition to patients serum, an anti-GST monoclonal antibody (MAb; Santa Cruz Biotechnology), rabbit preimmunized serum, and rabbit anti-ORF8a-SP antiserum (R26) were also used to examine recombinant GST-ORF8a protein. Patients serum samples were diluted at 1:100 and incubated with WB strips for 1 h at 37C. Details of the WB have been described elsewhere [11]. Three cell lines-VeroE6, HEK 293T, and HuH-7-were used in this study [12-14]. They PROTAC ERRα ligand 2 were cultured in Dulbeccos altered Eagle medium (DMEM; GIBCO) with 10% fetal calf serum (HyClone), penicillin, streptomycin, and l-glutamine. For transfection experiments, either VeroE6 or HEK 293T cells were seeded in plates and cultured overnight at 37C with 5% CO2. HuH-7 cells were transfected with.