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. 2007 Aug 1;196(3):405-15.
doi: 10.1086/519166. Epub 2007 Jun 19.

Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis

Chia-Yen Chen  1 , Yueh-Hsin Ping, Hsin-Chen Lee, Kuan-Hsuan Chen, Yuan-Ming Lee, Yu-Juin Chan, Te-Cheng Lien, Tjin-Shing Jap, Chi-Hung Lin, Lung-Sen Kao, Yi-Ming Arthur Chen
Affiliations

Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis

Chia-Yen Chen et al. J Infect Dis. .

Abstract

Background: A unique genomic difference between human and civet severe acute respiratory syndrome coronaviruses (SARS-CoVs) is that the former has a deletion of 29 nucleotides from open reading frame (orf) 8a' that results in the generation of orf8a and orf8b. The objectives of the present study were to analyze antibody reactivity to ORF8a in patients with SARS and to elucidate the function of ORF8a.

Methods: Western-blot and immunofluorescent antibody assays were used to detect anti-ORF8a antibody. SARS-CoV HKU39849 was used to infect stable clones expressing ORF8a and cells transfected with small interfering RNA (siRNA). The virus loads (VLs) and cytopathic effects (CPEs) were recorded. Confocal microscopy and several mitochondria-related tests were used to study the function of ORF8a.

Results: Two (5.4%) of 37 patients with SARS had anti-ORF8a antibodies. The VLs in the stable clones expressing ORF8a were significantly higher than those in control subjects 5 days after infection. siRNA against orf8a 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.

Conclusions: ORF8a not only enhances viral replication but also induces apoptosis through a mitochondria-dependent pathway.

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Figures

Figure 1.
Figure 1.
Detection of anti-open reading frame (ORF)-8a antibody reactivity in patients infected with severe acute respiratory syndrome coronavirus. A, Induction and purification of the recombinant glutathione S-transferase (GST)-ORF8a protein. Lanes 1-3, Coomassie blue staining of an SDS-PAGE gel containing bacterial lysates (lanes 1 and 2) or GST-ORF8a fusion protein purified from glutathione-sepharose 4B bead column (lane 3). Lane 1, Before isopropyl-β-D-thiogalactopyranoside (IPTG) induction; lane 2, after IPTG induction. B, Western-blot assay with a recombinant GST-ORF8a protein. Lane 1, Preimmunized rabbit serum; lane 2, rabbit anti-ORF8a antiserum R26; lane 3, anti-GST monoclonal antibody (MAb); lane 4, normal human serum; lane 5, serum sample from patient HP631; and lane 6, serum sample from patient C596. Nos. along the left margin of panels A and B are molecular-weight markers in kilodaltons. C, Immunofluorescent antibody assay test using VeroE6 cells transfected with pHA-ORF8a. a, Anti-hemagglutinin antibody; b, preimmunized rabbit serum; c, rabbit anti-ORF8a antiserum R26 at 1:500 dilution; d, normal human serum; e, serum from patient HP631; f, serum from patient C596. The nucleus was stained with Hoechst H33258.
Figure 2.
Figure 2.
Enhancement viral replication and cytopathic effects (CPEs) of severe acute respiratory syndrome coronavirus (SARS-CoV) HKU39849 in 2 stable clones by open reading frame (ORF)-8a protein. A, Reverse-transcription polymerase chain reaction (PCR) analysis of orf8a mRNA levels in 3 stable clones from the HuH-7 cell line. Lane 1, control clone transfected with vector plasmid pcDNA3 DNA; lanes 2 and 3, stable clones expressing ORF8a (lane 2, clone 18; lane 3, clone 24). B, Measurements of viral loads in different stable clones infected with 100 TCID50 of SARS-CoV HKU39849. After infection, the cultural supernatants were collected at various time points for 5 days. The no. of copies of nucleocapsid mRNA was determined using real-time PCR. Plus symbols (+) refer to the percentage of cells showing a CPE in cell cultures: +, <25%; ++, 25%-50%; +++, 50%-75%; ++++, >75%. C, CPEs of SARS-CoV HKU39849 in 3 stable clones for 5 days after the viral infection.
Figure 3.
Figure 3.
The effect of orf8a small interfering RNA (siRNA) on viral replication and cytopathic effects of severe acute respiratory syndrome coronavirus (SARS-CoV) HKU39849. A, Verification of the effects of 2 sets of orf8a siRNAs. Top, Results of reverse-transcription polymerase chain reaction (RTPCR) of orf8a mRNA using total RNA extracted from VeroE6 cells cotransfected with pHA-ORF8a and different siRNAs. Lane 1, pcDNA3-HA; lanes 2-9, pHA-ORF8a. The siRNAs used in different assays were as follows: lanes 2 and 3, siRNA-GFP; lanes 4-6, orf8a siRNA set 1; and lanes 7-9, orf8a siRNA set 2. The cells were harvested at different time points after transfection (18 h for lanes 2, 4, and 7; 36 h for lanes 5 and 8; and 56 h for lanes 1, 3, 6, and 9). The relative intensity (ratio) of the orf8a, compared with b-actin, in the top panel was calculated and normalized using the ratio of lane 3 (56 h of siRNA-GFP) as the 100% value. B, Measurements of the viral loads in the cultural supernatants from VeroE6 cells that had been transfected with orf8a siRNA for 36 h before they were inoculated with SARS-CoV HKU39849. After infection, the cultural supernatants were collected at various time points for 5 days. Nos. of replicase open reading frame (ORF)-1b mRNA copies were determined using real-time PCR. Error bars indicate SDs. * P < .05 (Student's t test). Plus symbols (+) refer to percentage of cells showing CPE in cell cultures: +, <25%; ++, 25%-50%; +++, 50%-75%; and ++++, 175%. C, Cytopathic effects of SARS-CoV HKU39849 in VeroE6 cells infected 120 h after transfection with different siRNAs for36h.
Figure 4.
Figure 4.
The subcellular localization of open reading frame (ORF)-8a. A, HEK 293T cells transfected with plasmid pORF8a-EGFP. B, Severe acute respiratory syndrome coronavirus (SARS-CoV)-infected VeroE6 cells immunostained with preimmunized rabbit serum. C, SARS-CoV-infected VeroE6 cells immunostained with rabbit anti-ORF8a antiserum R26 (1:100 dilution). D-I, VeroE6 cells cotransfected with a mitochondrial expression plasmid (pEF-CKMt2-green fluorescent protein [GFP]) and pHA-ORF8a and immunostained with rabbit anti-ORF8a antiserum R26 (1:500 dilution). Rhodamine-conjugated goat anti-rabbit antibodies were used as a secondary antibody in the immunofluorescent antibody assay test. Green represents GFP (D and G); red represents ORF8a protein (E and H). Yellowish areas in merged panels (F and I) indicate ORF8a localized in mitochondria. D-F, Low magnification; G-I, high magnification.
Figure 5.
Figure 5.
The effects of open reading frame (ORF)-8a on mitochondria. A, Mitochondrial membrane potential. B, H2O2 production. C, Oxygen content. D, Oxygen consumption. Two stable ORF8a-expressing stable clones (18 and 24), a vector control clone, and the parental cell line HuH-7 were used. Data are averages of experiments performed in triplicate. Error bars indicate SDs. * P < .05. JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenz-imidazolylcarbocyanine iodide; KCN, potassium cyanide.
Figure 6.
Figure 6.
Open reading frame (ORF)-8a triggering apoptosis via a caspase 3-dependent pathway. A, Flow cytometry (FACS) with annexin V staining of HuH-7 cells that had been transfected with the following plasmid DNAs for 24 h: a vector control (pcDNA3-HA, 7.5 μg), pHAORF8a (3.75 μg), pHA-ORF8a (7.5 μg), and a positive control (pcDNA3Tat, 7.5 μg). B, FACS with annexin V staining of 2 stable clones expressing ORF8a (18 and 24), a control clone, and its parental cell line, HuH-7. C, Caspase 3 activity of the clones and cells mentioned above. Data are averages of experiments performed in triplicate. Error bars indicate SDs. *P < .05.
Table 1.
Table 1.
Putative mitochondrial targeting signal found in the open reading frame (ORF)-8a protein of severe acute respiratory syndrome coronavirus (SARS-CoV) and gene products of other viruses.
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References

    1. Fouchier RAM, Kuiken T, Schutten M, et al. Aetiology: Koch's postulates fulfilled for SARS virus. Nature. 2003;423:240. - PMC - PubMed
    1. Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. 2003;361:1319–25. - PMC - PubMed
    1. Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348:1953–66. - PubMed
    1. Rota PA, Oberste MS, Monroe SS, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300:1394–9. - PubMed
    1. Marra MA, Jones SJM, Astell CR, et al. The genome sequence of the SARS-associated coronavirus. Science. 2003;300:1399–404. - PubMed

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