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Abstract
The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-beta), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-beta, IRF-3. N protein dramatically inhibited expression from an NF-kappaB-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.
Figures
Identification of SARS-CoV ORF 3b, ORF 6, and N proteins as potential interferon antagonists. A. A549 cells were transfected with either control plasmids or plasmids expressing HA-tagged SARS-CoV proteins. At 24 h posttransfection, cells were infected with NDV-GFP, which grows in the presence of interferon antagonists. Images were obtained at 24 h postinfection using a 10× objective and are representative of three experiments. B. A549 cells were transfected with plasmids expressing HA-tagged SARS-CoV proteins for 24 h, fixed, and analyzed for expression of the SARS-CoV proteins using an antibody to the HA tag.
Localization and apoptosis analysis of SARS-CoV ORF 3b, ORF 6, and N proteins. 293T cells were transfected with plasmids expressing GFP-tagged ORF 3b (A), ORF 6 (B), or N (C) proteins for 24 h. Cells were fixed, permeabilized, and stained for either the Golgi using BODIPY TR, the ER using an antibody to PDI, the mitochondria using an antibody to cytochrome c, or chromatin using a DAPI stain. Green represents GFP-tagged protein, red represents the indicated organelle, and blue represents the chromatin. Cells were analyzed by confocal microscopy using a 63× objective, and representative images are shown. D. 293T cells were transfected with GFP or plasmids expressing GFP-tagged ORF 7a, ORF 3b, ORF 6, or N proteins for 24 h. Cells were analyzed by fluorescence and phase-contrast microscopy for morphological changes associated with apoptosis. E. 293T cells were transfected for 24 h, harvested, lysed, and analyzed for caspase-3 activity.
SARS-CoV ORF 3b, ORF 6, and N proteins inhibit production of interferon. A. 293T cells were cotransfected with a plasmid expressing red fluorescence protein under the control of the IFN-β promoter and empty vector plasmid, a plasmid expressing influenza virus NS1, or plasmids expressing SARS-CoV proteins. At 24 h posttransfection, cells were infected with Sendai virus to stimulate the production of interferon. Images were captured 24 h postinfection using a 10× objective. B. Images from three experiments described for panel A were quantitated using Image J software. Data are averages plus standard deviations for three experiments. C. Cells were transfected with the indicated plasmids for 24 h and then infected with Sendai virus for 24 h. Cells were fixed, permeabilized, and analyzed for Sendai virus proteins using monoclonal antibodies to Sendai virus.
SARS-CoV ORF 3b, ORF 6, and N proteins inhibit activation of IRF-3. A. 293T cells were cotransfected with the p55-CIB promoter, which contains three IRF-3 binding sites, a plasmid that constitutively expresses Renilla luciferase, and plasmids expressing SARS-CoV proteins or the indicated control plasmids. Cells were infected with Sendai virus 24 h posttransfection. Cells were harvested at 24 h postinfection and analyzed for firefly and Renilla luciferase. Data were normalized using the Renilla luciferase values. Data are averages plus standard deviations for three experiments. A value of 100% represents approximately 10,000,000 firefly luciferase units(p55-CIB) and 6,000,000 Renilla luciferase units. B. 293T cells were transfected with the indicated plasmid and infected with Sendai virus for 6 h. Cells were harvested, and lysates were analyzed by Western blotting using a phospho-IRF-3 antibody. Quantitations are given below each band and are percentages of the value for the empty, infected control. C. 293T cells were transfected with the indicated plasmids for 24 h and then infected with Sendai virus. Cells were fixed 8 h postinfection and analyzed by microscopy using an antibody that recognizes the HA tag.
N protein inhibits NF-κB activation. 293T cells were cotransfected with a plasmid expressing firefly luciferase under the control of an NF-κB-responsive promoter containing two NF-κB binding sites, the Renilla luciferase plasmid, and the indicated plasmids. At 24 h posttransfection, cells were infected with Sendai virus. At 24 h postinfection, cells were harvested and analyzed for firefly and Renilla luciferase activities. Cells were normalized to the Renilla luciferase control. Data are averages plus standard deviations for three experiments. A value of 100% represents approximately 8,000,000 firefly luciferase units and 6,000,000 Renilla luciferase units. The P values for N and ORF 6 proteins were less than 0.05.
Inhibition of a promoter containing an ISRE by SARS-CoV proteins. 293T cells were cotransfected with ISRE-GFP and the indicated plasmid for 24 h. Cells were then infected with Sendai virus (A and B) or treated with IFN-β (C and D) for 24 h and then analyzed by microscopy using a 10× objective. Images were quantitated with ImageJ software (B and D). Data are averages plus standard deviations for three experiments.
ORF 6 protein inhibits translocation of STAT1 to the nucleus. A and B. 293T cells were cotransfected with STAT1-GFP and the indicated plasmids. At 24 h posttransfection, cells were treated with IFN-β for 1 h and then fixed and stained. Viral proteins were visualized with an antibody to the HA tag, and DNA was visualized using Hoechst 33342 stain. Green represents STAT1-GFP, red represents the viral protein, and blue represents DNA. Cells were analyzed by confocal microscopy, and representative images are shown. All images were obtained using a 63× objective; the images in panel B were magnified using Zeiss confocal software.
Analysis of STAT1 phosphorylation by SARS-CoV proteins. A. 293T cells were transfected with the indicated plasmid for 24 h and then treated with IFN-β for 1 h. Cells were harvested, and lysates were analyzed by Western blot analysis using antibodies recognizing the phospho- and total forms of STAT1. B. Cells were transfected with the indicated plasmid and STAT-1 GFP for 24 h and then treated with IFN-β for 1 h. Cells were fixed, permeabilized, and analyzed for phospho-STAT1 by confocal microscopy using a 10× objective. Representative images are shown.
References
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- Chan, W. S., C. Wu, S. C. Chow, T. Cheung, K. F. To, W. K. Leung, P. K. Chan, K. C. Lee, H. K. Ng, D. M. Au, and A. W. Lo. 2005. Coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (SARS). Mod. Pathol. 18:1432-1439. - PMC - PubMed
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