Interferon-induced ISG15 conjugation inhibits influenza A virus gene expression and replication in human cells

Abstract

The ubiquitin-like ISG15 protein, as well as its conjugating enzymes, is induced by type I interferons (IFNs). Experiments using ISG15 knockout (ISG15(-/-)) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus. However, in contrast to the virus inhibition results for mice, the rates of virus replication in ISG15(+/+) and ISG15(-/-) mouse embryo fibroblasts in tissue culture were similar. Here we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on influenza A virus gene expression and replication in such cells. We demonstrate that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs directed against ISG15-conjugating enzymes. IFN-induced antiviral activity against influenza A virus protein synthesis was reduced 5- to 20-fold by suppressing ISG15 conjugation. The amounts of the viral proteins that were restored by these siRNA treatments were approximately 40 to 50% of the amounts produced in cells that were not pretreated with IFN. Further, we show that ISG15 conjugation inhibits influenza A virus replication 10- to 20-fold at early times after infection in human cells. These results show that ISG15 conjugation plays a substantial role in the antiviral state induced by IFN in human cells. In contrast, we show that in mouse embryo fibroblasts ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression.

FIG. 1.

siRNA knockdown of UbE1L in A549 cells inhibits ISG15 conjugation and causes a 5- to 20-fold increase in the synthesis of several viral proteins during influenza A virus infection. The experimental design is shown at the top. Extracts collected 8 h after Ud virus infection of cells subjected to the indicated treatments were analyzed by immunoblotting (WB) using antibodies against the indicated proteins. Quantitation of the immunoblots was carried out as described in Materials and Methods. In addition, the RNA extracted from these extracts was analyzed by a Northern blotting (NB) for the amount of NS1A mRNA as described in Materials and Methods.

FIG. 2.

siRNA knockdown of UbcH8 in A549 cells inhibits ISG15 conjugation and causes a 12-fold reduction in the IFN-induced antiviral activity against the synthesis of the NS1A protein during influenza A virus infection. (A) Extracts of cells transfected with a siRNA against UbcH8 or a control siRNA, followed by IFN-β treatment for 24 h, were analyzed by immunoblotting (WB) using antibody against UbcH8, ISG15, or tubulin. (B) Extracts collected 8 h after Ud virus infection of cells subjected to the indicated treatments were analyzed by quantitative immunoblotting using antibody against the viral NS1A protein or against tubulin.

FIG. 3.

Inhibition of IFN-induced ISG15 conjugation in Calu3 cells by siRNA knockdown of UbE1L leads to a significant increase in the rate of influenza A virus replication at early times after infection. (A) Single-cycle growth curve for Ud virus in Calu3 cells. (B) Extracts of cells collected 8 h after Ud virus infection of Calu3 cells subjected to the indicated treatments were analyzed by immunoblotting (WB) using antibodies against the indicated proteins. Quantitation of the immunoblots was carried out as described in Materials and Methods. (C) Calu3 cells were transfected with either ISG15 and UbE1L siRNAs or a control siRNA and 24 h later were treated with 1,000 units/ml IFN-β. After an additional 24 h at 37°C, the two sets of cells were infected with 5 PFU/cell of the Ud virus, and the amounts of virus produced at 2, 4, 6, and 8 h postinfection were determined by plaque assays of MDCK cells.

FIG. 4.

ISG15 conjugation in MEFs does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression. ISG15^+/+ and ISG15^−/− MEFs were treated with 1,000 units/ml of mouse IFN-β (Sigma) or were untreated. After 24 h of incubation, the MEFs were infected with Ud virus, and cells were collected 8 h later. Extracts of the cells were analyzed by immunoblotting (WB) using the indicated antibodies. Quantitation of the immunoblots was carried out as described in Materials and Methods.