Characterization of the antiviral effects of interferon-alpha against a SARS-like coronoavirus infection in vitro - PubMed (original) (raw)
Characterization of the antiviral effects of interferon-alpha against a SARS-like coronoavirus infection in vitro
Joanna Zorzitto et al. Cell Res. 2006 Feb.
Abstract
Interferon (IFN)-alphas bind to and activate their cognate cell surface receptor to invoke an antiviral response in target cells. Well-described receptor-mediated signaling events result in transcriptional regulation of IFN sensitive genes, effectors of this antiviral response. Results from a pilot study to evaluate the clinical efficacy of IFN-alpha treatment of SARS patients provided evidence for IFN-inducible resolution of disease. In this report we examined the contribution of IFN-inducible phosphorylation-activation of specific signaling effectors to protection from infection by a SARS-related murine coronavirus, MHV-1. As anticipated, the earliest receptor-activation event, Jak1 phosphorylation, is critical for IFN-inducible protection from MHV-1 infection. Additionally, we provide evidence for the contribution of two kinases, the MAP kinase p38MAPK, and protein kinase C (PKC) delta to antiviral protection from MHV-1 infection. Notably, our data suggest that MHV-1 infection, as for the Urbani SARS coronoavirus, inhibits an IFN response, inferred from the lack of activation of pkr and 2'5'-oas, genes associated with mediating the antiviral activities of IFN-alphas. To identify potential target genes that are activated downstream of the IFN-inducible signaling effectors we identified, and that mediate protection from coronavirus infection, we examined the gene expression profiles in the peripheral blood mononuclear cells of SARS patients who received IFN treatment. A subset of differentially regulated genes were distinguished with functional properties associated with antimicrobial activities.
Figures
Figure 1
The pharmacological inhibitors: Jak inhibitor 1, Rottlerin and SB203580 inhibit IFN-inducible anti-MHV-1 activity. (A) 106 L2 cells were either left untreated, or treated with the indicated doses of IFN-α4 for 14 h, then challenged with MHV-1 for 36 h. Cells were lysed, RNA extracted and resolved by electrophoresis in agarose and Northern analyses of 28S RNA and cleavage products performed. Data are representative of 2 independent experiments. (B) 104 L2 cells were either left untreated (▴) or treated with 10μM Jak inhibitor 1(▪), 1μM Rottlerin (□), 10μM SB203580 (▵), or 100μM AG490 (O) for 1 h. Cells were then incubated, in triplicate, with the indicated dose of IFN-α4 for 16 h, then challenged with MHV-1. After 24 h CPE were quantitated using a colorimetric assay (Materials and Methods). Data are expressed as percent protection from CPE. Mean values ± SE of three independent experiments are shown. (C) 107 L2 cells were either left untreated, or treated with 10μM Jak Inhibitor 1, 10 μM SB203580 or 1μM Rottlerin for 1 h prior to a 30 min IFN-α4 (104 U/ml) treatment. Cells were lysed and equal amounts of whole cell lysates were resolved by SDS-PAGE and examined for Jak1, p38 MAPK or PKCδ phosphorylation by immunoblotting with anti-phospho-Jak1,-p38 MAPK or -PKCδ antibodies. Membranes were stripped and re-probed with anti-Jak1, anti-p38 MAPK, or anti-PKCδ antibodies to control for loading. Blots shown are representative of 3 independent experiments. (D) 107 L2 cells were either left untreated, or treated with 10 μM Jak Inhibitor 1 or 1 μM Rottlerin for 1 h prior to a 30 min IFN-α4 (104 U/ml) treatment. Cells were lysed and equal amounts of whole cell lysates were resolved by SDS-PAGE and examined for STAT tyrosine phosphorylation by immunoblotting with anti-phospho-STAT antibodies. Membranes were stripped and re-probed with anti-STAT antibodies to control for loading. Blots shown are representative of 3 independent experiments.
Figure 2
MHV-1 infection leads to negligible transcriptional activation of 2′5′-oas and pkr. 106 L2 cells were either left untreated (UT), infected with MHV-1 (A), treated with 104U/ml IFN-α4 for the times indicated (B), or treated with 10 μM SB203580, 1 μM Rottlerin or 10μM Jak 1 inhibitor for 1h then 104U/ml IFN-α4 for 12h (C). RNA was extracted, cDNA synthesized and quantitative real-time PCR analyses performed for reference GAPDH and target genes 2′5′-oas and pkr expression. Data are presented as the fold-change in expression compared to untreated L2 cells. Values ± SE were calculated using Relative Quantification software (Roche) and are the mean of three separate reactions, each performed in triplicate.
Figure 3
Heat map of genes regulated by IFN alfacon-1 in PBMC of SARS patients. The relative expression levels of the genes identified in Table 3 are described. Upregulated genes (A) are shown in red and downregulated genes (B) are shown in green. Maximum intensity values +/− 9 are shown. Patient 1, SARS patient treated with corticosteroids alone; Patients 2 and 3, SARS patients treated with IFN alfacon-1 (as per Tables 1 and 2).
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