Identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections

Abstract

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-alpha for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-alpha is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

FIG. 1.

Characterization of ISG expression following tetracycline (Tet) induction in FLP-IN/ISG cell lines. (A) Demonstration of the desired ISG expression in the seven newly established cell lines. FLP-IN T Rex-derived cell lines that inducibly express individual ISGs were established as described previously (21). The cells were cultured in the absence or presence of tetracycline for 48 h and then harvested. The levels of N-terminally FLAG-tagged ISG protein expression in cell lysates were determined by Western blot analysis with a monoclonal antibody against the FLAG tag as previously described (21). M. W., molecular mass. (B) Comparison of the kinetics and levels of ISG expression upon IFN-α treatment and tetracycline induction. Huh7, HeLa, and parental FLP-IN T Rex cells were left untreated or treated with 1,000 IU/ml IFN-α, and cells were harvested at the indicated times after IFN-α treatment. Three FLP-IN/ISG cell lines that express MxA, PKR, and ISG15, respectively, were cultured in the absence or presence of 1 μg/ml tetracycline, and cells were harvested at the indicated times after the addition of the antibiotic. The levels of the three IFN-induced proteins in cell lysates were determined by Western blot analysis. Briefly, cell monolayers were washed once with phosphate-buffered saline and lysed with 1× Laemmli buffer. A fraction of the cell lysate was separated on SDS-12% polyacrylamide gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with phosphate-buffered saline containing 5% nonfat dry milk and probed with antibodies against MxA (Proteintech Group), PKR (a gift from Pat Romano, Institute for Hepatitis Virus Research, Hepatitis B Foundation, Doylestown, PA), ISG15 (Cell Signaling), and β-actin (Chemicon International). Bound antibody was revealed by IRDye secondary antibodies and visualized by the Li-COR Odyssey system.

FIG. 2.

Effects of ISG expression on WNV and DENV-1 VLP infection. (A) Schematic representation of the structures of subgenomic replicons packaged within WNV and DENV VLPs. Compared with the full-length WNV or DENV genome, the VLP-containing reporter replicon Rluc2A-Rep contains an in-frame replacement of the structural genes with the Renilla luciferase gene fused with the 2A fragment from food-and-mouth disease virus (39). (B and C) FLP-IN/ISG and FLP-IN/CAT cells were seeded into the wells of a 96-well plate at a density of 5 × 10⁴ cells per well. At 24 h postseeding, the cells were cultured in the absence or presence of 1 μg/ml tetracycline for 24 h, infected with WNV (panel B) or DENV-1 (panel C) VLPs at a multiplicity of infection of 1, refed with medium with or without 1 μg/ml tetracycline for an additional 24 h, and assayed for luciferase activity with a Renilla luciferase assay kit (Promega). As controls, parental FLP-IN T Rex cells were left untreated or pretreated with the indicated concentrations of IFN-α for 24 h, infected with WNV VLPs, cultured further with or without the indicated concentrations of IFN-α for an additional 24 h, and assayed for luciferase activity as described above. The RLA represents the mean ± the standard deviation (n = 4) of the ratio of light units obtained from wells treated with IFN or cultured in the presence of tetracycline to the light units obtained from wells that were left untreated or cultured in the absence of tetracycline.

FIG. 3.

Requirements of the enzymatic activities of viperin, ISG20, and PKR for their antiviral effects against WNV and/or DENV. FLP-IN T Rex-derived cell lines inducibly expressing viperin, ISG20, PKR, the respective enzymatically inactive mutant proteins, and the control protein CAT were seeded into the wells of a 96-well plate at a density of 5 × 10⁴ per well. Tetracycline pretreatment and WNV or DENV VLP infection were as described in the legend to Fig. 2. After infection, cells were cultured with medium with or without tetracycline for 24 h and assayed for luciferase activity. RLA is as defined in the legend to Fig. 2.

FIG. 4.

Effects of ISGs on DENV infection. (A) The stable cell lines that express CAT protein or ISGs and the respective mutant proteins were seeded into the wells of a 96-well plate at a density of 5 × 10⁴ per well. At 24 h postseeding, the cells were cultured in the absence or presence of 1 μg/ml tetracycline for 24 h, infected with serotype 2 DENV at a multiplicity of infection of 0.1, and refed with medium with or without tetracycline. As controls, parental FLP-IN T Rex cells were left untreated or pretreated with the indicated concentrations of IFN-α for 24 h, infected with DENV-2, and cultured further in medium with or without the indicated concentrations of IFN-α. At 48 h after infection, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline, incubated with anti-E antibody 4G2 (1:200; Millipore) for 1 h, and incubated with an anti-mouse IRDye 800CW-labeled secondary antibody together with two reagents for cell staining (DRAQ5 from Biostatus and Sapphire700 from LI-COR). The DENV E protein was visualized in LI-COR Odyssey in the 800-nm channel as green. The cell viability was determined by Sapphire 700 staining (Red color). (B) E protein levels were determined with LI-COR Odyssey and normalized to cell viability. The relative infectivity represents the mean ± the standard deviation (n = 3) of the ratio of the density obtained from wells treated with IFN-α or cultured in the presence of tetracycline to that obtained from wells that were left untreated or cultured in the absence of tetracycline. (C) Effects of ISGs on DENV production. The indicated cell lines were cultured in the absence or presence of 1 μg/ml tetracycline (Tet) for 24 h, infected with DENV-2 at a multiplicity of infection of 0.1, refed with medium with or without tetracycline, and cultured for 3 days. The culture medium was changed every 24 h, and virus yields between 48 and 72 h postinfection were determined by plaque assay and expressed as PFU per milliliter of culture medium (n = 3).

FIG. 5.

Effects of ISG expression on WNV replicon-conferred cell colony formation. (A) WNV subgenomic replicon WNVneoRep contains an in-frame deletion of the structural genes and insertion of the neomycin phosphotransferase gene (neo) driven by an internal ribosome entry site derived from encephalomyocarditis virus in the 3′ untranslated region. (B) CAT- and ISG-expressing FLP-IN T Rex stable cell lines were electroporated with WNVrep3′neo derived from HEK293/WNVrep cells and selected with G418 in the absence or presence of 1 μg/ml tetracycline for 2 weeks as described previously (4). Cell foci were stained with crystal violet and photographed. A pair of representative plates of each of the five ISGs and their mutant protein-expressing and control protein CAT-expressing cell lines that were cultured in the absence (top) or presence (bottom) of tetracycline (Tet) is presented. (C) The foci of each of the ISG- and CAT-expressing cell lines cultured in three plates in either the absence or the presence of tetracycline were counted. Relative colony formation efficiency (RCFE) was expressed and plotted as the mean ± the standard deviation (n = 3) of the ratios of the number of foci of cells that were selected in the presence of tetracycline to the number obtained from cells that were cultured in the absence of the antibiotic.

FIG. 6.

Effects of antiviral ISGs on DENV-1 replicon replication. FLP-IN/ISG and FLP-IN/CAT cells were cultured in the absence or presence of 1 μg/ml tetracycline (Tet) for 24 h. The cells were electroporated with 10 μg of DENV-1 luciferase replicon RNA (Rluc2A-Rep) per 4 × 10⁶ cells. The transfected cells were seeded into the wells of a 96-well plate at a density of 4 × 10⁵ per well and cultured in the absence or presence of tetracycline. At the indicated times after seeding, cells were washed once with cold phosphate-buffered saline and 250 μl of 1× lysis buffer (Promega) was added. The luciferase activity in the cell lysates were measured in TopCount NXT. Average results and standard deviations (n = 4) are presented. Luciferase activities at the two earlier time points are presented in the inserted graphs on a smaller scale.

FIG. 7.

IFITM restricts DENV infection. (A) HeLa cells were transfected with SMARTpool siRNA targeting IFITM2 and IFITM3 or a nontargeting siRNA (control) by following the directions of the manufacturer (Dharmacon). At 72 h posttransfection, cells were retransfected with the same siRNA and left untreated or treated with 100 or 10 IU/ml IFN-α. Cells were harvested at 30 h posttreatment. Levels of IFITM in the cell lysates were detected by Western blot assay with an antibody recognizing IFITM2 and IFITM3 (Proteintech Group). β-Actin served as a loading control. (B) HeLa cells were transfected with SMARTpool siRNA targeting IFITM2 and IFITM3 or a nontargeting siRNA (control) and infected at 72 h posttransfection with DENV-2 at a multiplicity of infection of 1 for 1 h. The infected cells were either left untreated or treated immediately after infection with the indicated concentrations of IFN-α. Virus yields in culture medium harvested at 30 h postinfection were determined with a plaque assay and are expressed as PFU per milliliter of culture medium (n = 3).