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. 2005 Feb;79(4):2079-86.
doi: 10.1128/JVI.79.4.2079-2086.2005.

Inhibition of Beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3

Martin Spiegel  1 , Andreas Pichlmair, Luis Martínez-Sobrido, Jerome Cros, Adolfo García-Sastre, Otto Haller, Friedemann Weber
Affiliations

Inhibition of Beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3

Martin Spiegel et al. J Virol. 2005 Feb.

Abstract

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. We and others have previously shown that the replication of SARS-CoV can be suppressed by exogenously added interferon (IFN), a cytokine which is normally synthesized by cells as a reaction to virus infection. Here, we demonstrate that SARS-CoV escapes IFN-mediated growth inhibition by preventing the induction of IFN-beta. In SARS-CoV-infected cells, no endogenous IFN-beta transcripts and no IFN-beta promoter activity were detected. Nevertheless, the transcription factor interferon regulatory factor 3 (IRF-3), which is essential for IFN-beta promoter activity, was transported from the cytoplasm to the nucleus early after infection with SARS-CoV. However, at a later time point in infection, IRF-3 was again localized in the cytoplasm. By contrast, IRF-3 remained in the nucleus of cells infected with the IFN-inducing control virus Bunyamwera delNSs. Other signs of IRF-3 activation such as hyperphosphorylation, homodimer formation, and recruitment of the coactivator CREB-binding protein (CBP) were found late after infection with the control virus but not with SARS-CoV. Our data suggest that nuclear transport of IRF-3 is an immediate-early reaction to virus infection and may precede its hyperphosphorylation, homodimer formation, and binding to CBP. In order to escape activation of the IFN system, SARS-CoV appears to block a step after the early nuclear transport of IRF-3.

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Figures

FIG. 1.
FIG. 1.
Absence of IFN-β induction in SARS-CoV-infected cells. (a) IFN-β promoter activation. Human 293 cells were transfected with plasmid p-125Luc containing the firefly luciferase gene under control of the IFN-β promoter, along with a control plasmid encoding Renilla luciferase under control of the constitutively active simian virus 40 promoter. At 6 h posttransfection, cells were infected with SARS-CoV or the IFN-inducing control virus Bunyamwera delNSs (CTRL) or left uninfected (mock). At 18 h postinfection, luciferase activities were measured. Firefly luciferase values (reflecting IFN-β promoter activation) were normalized to the Renilla luciferase activities. (b) Reverse transcription-PCR analysis. Total RNA from 293 cells which were infected for 18 h was assayed by reverse transcription-PCR for the presence of IFN-β mRNA (panel 1), mRNA of the N gene of SARS-CoV (panel 2) or the control virus (panel 3), or cellular γ-actin mRNA (panel 4).
FIG. 2.
FIG. 2.
Subcellular localization of IRF-3 early in infection. Confocal double immunofluorescence pictures of cells which were either mock infected (panels 1 to 3) or infected for 8 h with SARS-CoV (panels 4 to 6) or the control virus (panels 7 to 9). Viral N proteins (panels 4 and 7) and IRF-3 (panels 2, 5, and 8) were detected with specific rabbit and mouse antisera, respectively.
FIG. 3.
FIG. 3.
Subcellular localization of IRF-3 late in infection. Cells were infected for 16 h and immunostained as indicated for Fig. 2.
FIG. 4.
FIG. 4.
Hyperphosphorylation of IRF-3. Extracts from cells infected for 8 h (a) or 16 h (b) were analyzed by sodium dodecyl sulfate gel electrophoresis, followed by immunoblot analyses to detect IRF-3 (panels 1), the N protein of SARS-CoV (panels 2), or the N protein of the control virus (panels 3). The two forms of unphosphorylated IRF-3 are indicated by arrowheads, and the hyperphosphorylated form of IRF-3 is indicated by an asterisk (*).
FIG. 5.
FIG. 5.
Homodimerization of IRF-3. Extracts from cells infected with SARS-CoV or the control virus for 8 h (a) or 16 h (b) were analyzed by nondenaturing gel electrophoresis followed by an immunoblot to detect IRF-3. The positions of IRF-3 monomers and dimers are indicated by arrowheads.
FIG. 6.
FIG. 6.
CBP recruitment by IRF-3. Cells were infected for 8 h (a) or 16 h (b), and anti-CBP immunocomplexes (IP) were analyzed for the presence of IRF-3 (panels 1) and CBP (panels 2). Infections were monitored by probing the postprecipitation supernatants (SN) for the viral N proteins (panels 3 and 4).
FIG. 7.
FIG. 7.
Nuclear import and export. (a) Nuclear localization of IRF-2. Cells were transfected with plasmid pEGFP-C1-hIRF-2 for 24 h and then superinfected with SARS-CoV for 16 h (right panel) or left uninfected (left panel). Viral N protein was detected with a specific rabbit antiserum and a Cy3-conjugated secondary antibody. (b) Nuclear export assays. Cells were transfected with the Rev expression construct pcRev, the Rev export-dependent CAT reporter plasmid pDM128 (19), and the Renilla luciferase control plasmid pRL-SV40. After 4 h at 37°C, cells were either infected with SARS-CoV or left uninfected (black columns). As a control, cells were treated in parallel with the CRM-1 inhibitor leptomycin B (LMB) at a concentration of 2 ng/ml (grey columns). At 16 h postinfection, reporter activities were measured. CAT activities were normalized to the corresponding Renilla luciferase activities to determine induction. Data from a representative experiment are shown.
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References

    1. Aurisicchio, L., P. Delmastro, V. Salucci, O. G. Paz, P. Rovere, G. Ciliberto, N. La Monica, and F. Palombo. 2000. Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis. J. Virol. 74:4816-4823. - PMC - PubMed
    1. Baigent, S. J., G. Zhang, M. D. Fray, H. Flick-Smith, S. Goodbourn, and J. W. McCauley. 2002. Inhibition of β interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J. Virol. 76:8979-8988. - PMC - PubMed
    1. Basler, C. F., and A. Garcia-Sastre. 2002. Viruses and the type I interferon antiviral system: induction and evasion. Int. Rev. Immunol. 21:305-337. - PubMed
    1. Basler, C. F., A. Mikulasova, L. Martinez-Sobrido, J. Paragas, E. Muhlberger, M. Bray, H. D. Klenk, P. Palese, and A. Garcia-Sastre. 2003. The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J. Virol. 77:7945-7956. - PMC - PubMed
    1. Baudoux, P., L. Besnardeau, C. Carrat, P. Rottier, B. Charley, and H. Laude. 1998. Interferon alpha inducing property of coronavirus particles and pseudoparticles. Adv. Exp. Med. Biol. 440:377-386. - PubMed

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