Identification of alpha interferon-induced genes associated with antiviral activity in Daudi cells and characterization of IFIT3 as a novel antiviral gene

Abstract

A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.

FIG. 1.

AP and AV effects of IFN-α2c on Daudi cells. Daudi cells were treated with IFN-α2c in the presence or absence of VSV. Plates were developed 48 h after IFN treatment.

FIG. 2.

Kinetics of IFN AV activity on Daudi cells using anti-IFNAR1 MAb 64.10. Daudi cells were treated with IFN-α2c, HY-2, or IFN-α2a standard for 6, 15, or 24 h prior to the addition of neutralizing anti-IFNAR1 MAb 64.10 (1 μg/ml). At 24 h, cells were infected with VSV. Plates were developed 24 h postinfection.

FIG. 3.

(A) Detection of IFN-α induced AV proteins after 6 h of treatment and the influence of anti-IFNAR1 MAb 64.10 on the expression levels of these proteins at 24 h. To study early IFN-α-induced proteins, Daudi cells were treated for 6 h with concentrations of IFN-α2c that cause an AP effect, or a concentration of IFN-α2a standard, causing AV protection only (2.5 IU/ml). To compare the levels of IFN-induced proteins under conditions of AP activity versus conditions causing AV protection only and to confirm the association of upregulated proteins with IFN treatment, the same samples were incubated with or without neutralizing anti-IFNAR1 MAb 64.10 for 24 h. Lanes: M, molecular weight marker; 1, IFN-α2c (0.36 ng/ml) AP; 2, IFN-α2a standard (2.5 IU/ml) AV; 3, negative control (NC), untreated cells; 4, IFN-α2c (0.36 ng/ml) AP; 5, IFN-α2c (0.36 ng/ml) + MAb 64.10 AV; 6, IFN-α2a standard (2.5 IU/ml) AV; 7, IFN-α2a standard (2.5 IU/ml) + MAb 64.10, no activity; 8, negative control (NC), untreated cells. AP, concentrations of IFN causing antiproliferative effect on Daudi cells. AV, concentrations of IFN causing antiviral effect on Daudi cells. No effect, concentrations of IFN not having any effect on Daudi cells. (B) Detection of IFN-α-induced AV proteins after 24 h of treatment. Daudi cells were treated for 24 h with different concentrations of IFNs. Lanes: M, molecular weight marker; 1, IFN-α2c (3.6 ng/ml) AP; 2, IFN-α2c (0.36 ng/ml) AP; 3, IFN-α2c (0.036 ng/ml) AP; 4, IFN-α2c (0.0036 ng/ml) AV; 5, IFN-α2c (0.00036 ng/ml) no activity; 6, negative control (NC), untreated cells.

FIG. 4.

Detection of IFIT3 by Western blotting (24 h). (A) Adherent cell lines. Lanes: M, molecular weight marker; 1, A549, negative control, untreated cells (NC); 2, A549 IFN-α2c (0.036 ng/ml); 3, Huh7 (NC); 4, Huh7 IFN-α2c (3.6 ng/ml); 5, OVCAR-3 (NC); 6, OVCAR-3 (0.36 ng/ml); 7, 2fTGH (NC); 8, 2fTGH (0.36 ng/ml); 9, HeLa (NC); 10, HeLa (0.36 ng/ml). (B) Suspension cell lines. Lanes: M, molecular weight marker; 1, Daudi IFN-α2c (3.6 ng/ml), positive control; 2, monocyte (NC); 3, monocyte IFN-α2c (0.36 ng/ml); 4, B-JAB (NC); 5, B-JAB (0.36 ng/ml); 6, U-937 (NC); 7, U-937 (0.36 ng/ml); 8, Jurkat (NC); 9, Jurkat (0.36 ng/ml).

FIG. 5.

Detection of AV proteins at 6 and 24 h after EMCV infection. Lanes: M, molecular weight marker (MWM); 1, negative control (NC), no virus present; 2, cells 6 h after EMCV treatment; 3, cells 24 h after EMCV infection.

FIG. 6.

IFIT3 RNA interference decreases efficiency of IFN AV effect. (A) Influence of IFIT3 siRNA on IFN AV protection against EMCV (expressed as percentage of cells protected by IFN from EMCV) and analysis of the efficiency of IFIT3 RNA interference by WB (inset). Lanes: M, molecular weight marker; 1, untreated cells; 2, nonspecific siRNA (20 nM); 3, nonspecific siRNA + IFN-α2a (2.5 IU/ml); 4, IFIT3 siRNA (20 nM); 5, IFIT3 siRNA + IFN-α2a (2.5 IU/ml). (B) Analysis of efficiency of IFIT3 RNA interference by plaque assay. To analyze effect of IFIT3-siRNA transfection on virus titers, cell supernatants were harvested after treatment with 5 IU of IFN-α2a/ml 24 h postinfection. The virus titer was examined by plaque assay on Vero cells.

FIG. 7.

Overexpression of IFIT3 decreases virus titers. Influence of IFIT3 on VSV and EMCV virus titers in Vero cells, and analysis of IFIT3 overexpression by WB (inset). Lanes: M, molecular weight marker; 1, cells transfected with plasmid pIRESpuro3; 2, cells transfected with plasmid pIRESpuro3-IFIT3.