Antiviral activity of limitin against encephalomyocarditis virus, herpes simplex virus, and mouse hepatitis virus: diverse requirements by limitin and alpha interferon for interferon regulatory factor 1

Abstract

Limitin has sequence homology with alpha interferon (IFN-alpha) and IFN-beta and utilizes the IFN-alpha/beta receptor. However, it has no influence on the proliferation of normal myeloid and erythroid progenitors. In this study, we show that limitin has antiviral activity in vitro as well as in vivo. Limitin inhibited not only cytopathic effects in encephalomyocarditis virus- or herpes simplex virus (HSV) type 1-infected L929 cells, but also plaque formation in mouse hepatitis virus (MHV) type 2-infected DBT cells. In addition, administration of limitin to mice suppressed MHV-induced hepatitis and HSV-induced death. The antiviral activity may be mediated in part by 2',5'-oligoadenylate synthetase, RNA-dependent protein kinase, and Mx protein, which inhibit viral replication or degrade viral components, because limitin induced their mRNA expression and enzyme activity. While limitin has antiviral activity as strong as that of IFN-alpha in vitro (the concentration that provided 50% inhibition of cytopathic effect is approximately 30 pg/ml), IFN regulatory factor 1 (IRF-1) dependencies for induction of an antiviral state were different for limitin and IFN-alpha. In IRF-1-deficient fibroblasts, a higher concentration of limitin than of IFN-alpha was required for the induction of antiviral activity and the transcription of proteins from IFN-stimulated response element. The unique signals and the fewer properties of myelosuppression suggest that a human homolog of limitin may be used as a new antiviral drug.

FIG. 1.

Characterization of recombinant limitin. (A) Gel filtration profile of limitin on a high-performance liquid chromatography Superdex 75 PC 3.2/30 column precalibrated with the standard protein markers. Recombinant limitin (1.5 μg) was applied, and the flow rate was 75 μl/min. The arrows indicate the elution peaks of the marker proteins (albumin, 67.0 kDa [1]; ovalbumin, 43.0 kDa [2]; chymotrypsinogen A, 25.0 kDa [3]; and RNase A, 13.7 kDa [4]). (B) WEHI3 cells (2.5 × 10³) were seeded in each well and stimulated with the indicated dose of recombinant limitin (solid circles), limitin-Ig (open circles), and CD44-Ig (open squares) for 48 h. The results represent the means ± standard deviations of triplicate cultures. Similar results were obtained in three independent experiments.

FIG. 2.

Reduction of susceptibility of L929 cells to EMCV by limitin and IFN-α. (A) L929 cell monolayers on 96-well plates were pretreated with the indicated concentrations of limitin (solid circles) or IFN-α (open circles) overnight and challenged with EMCV (5 × 10² PFU/well). After 24 h of incubation, the cells protected from EMCV virulence were evaluated by the CPE dye uptake assay. The percent inhibition of CPE was calculated as 100 × [OD₆₅₀ (each sample) − OD₆₅₀ (virus control)/OD₆₅₀ (cell control) − OD₆₅₀ (virus control)]. The results represent the means ± standard deviations (SD) of triplicate cultures. Similar results were obtained in three independent experiments. (B) Limitin (1,000 pg/ml) or IFN-α (1,000 pg/ml) was incubated with the indicated antibodies (10 μg/ml) for 30 min on ice. L929 cells were cultured in the presence of the mixtures overnight and then challenged with EMCV (5 × 10² PFU/well). After 24 h of incubation, the cells protected from EMCV virulence were evaluated by the CPE dye uptake assay. The results represent the means ± SD of triplicate cultures. Similar results were obtained in three independent experiments.

FIG. 3.

Antiviral effects of limitin and IFN-α against HSV and MHV. (A) L929 cell monolayers on 96-well plates were pretreated with the indicated concentrations of limitin (solid circles) or IFN-α (open circles) overnight and challenged with HSV (50 PFU/well). After 72 h of incubation, the cells protected from EMCV virulence were evaluated by the CPE dye uptake assay. The results represent the means ± standard deviations (SD) of triplicate cultures. Similar results were obtained in three independent experiments. (B) DBT cells were seeded in a six-well dish, and then the subconfluent monolayer was treated with 30 EU of limitin/ml or 30 EU of IFN-α/ml for 24 h. The pretreated DBT cells were infected with MHV (10³ PFU/well) for 1 h, followed by a plaque assay. The results represent the means ± SD of triplicate cultures. Similar results were obtained in three independent experiments.

FIG. 4.

Repression of MHV infection in vivo by limitin. (A) Ten BALB/c mice were divided into two groups. One group was injected with 3 × 10⁴ EU of limitin/day. The other group was treated with PBS as controls. The mice were treated intraperitoneally with PBS or limitin on days −1, 0, 1, and 2 relative to MHV exposure (10³ PFU/mouse). After 72 h of virus inoculation, the mice were anesthetized with pentobarbital and bled to measure the activity of GPT in serum. Each dot represents the GPT concentration in the indicated mouse. Similar results were obtained in two independent experiments. (B) Two mice from each group were anatomized, and their livers were investigated histologically. Magnification, ×100.

FIG. 5.

Prolongation of survival of HSV-challenged mice by limitin. (A) Twenty-four BALB/c mice were separated into two groups and challenged with HSV (5 × 10⁴ PFU/mouse). The mice were treated with 3 × 10⁴ EU of limitin (solid circle) or PBS (open circles)/mouse on days −1, 0, 1, 2, 3, 4, and 5 relative to HSV exposure. The duration of observation for survival was 14 days. Similar results were obtained in two independent experiments. (B) Eighteen athymic nude mice were separated into two groups and challenged with HSV (10⁴ PFU/mouse). The mice were treated with 3 × 10⁴ EU of limitin (solid circles) or PBS (open circles)/mouse on days −1, 0, 1, 2, 3, 4, and 5 relative to HSV exposure. The duration of observation for survival was 14 days. Similar results were obtained in two independent experiments. Statistical analysis was performed by the Kaplan-Meier method, and the P value was calculated by the generalized Wilcoxon test.

FIG. 6.

Induction of antiviral molecules by limitin. (A) Whole-cell lysates of L929 cells stimulated with limitin (30 EU/ml) for 4 h were prepared and subjected to measurement of 2′,5′-OAS activity using a ¹²⁵I radioimmunoassay kit. The results were represented as 2-5A production by each sample within 1 h. The assay range of this method is 10 to 810 pmol/dl. The results represent the means ± standard deviations of triplicate cultures. Similar results were obtained in three independent experiments. (B) Total RNAs of L929 cells stimulated with the indicated doses of limitin or IFN-α for 4 or 16 h were prepared and subjected to Northern blot analysis and probed with the indicated materials. β-Actin was used as an internal control. Similar results were obtained in three independent experiments.

FIG. 7.

Antiviral activities of limitin and IFN-α in fibroblasts established from wild-type and IRF-1-deficient mice. (A) Fibroblast monolayers on 96-well plates were pretreated with the indicated concentrations of limitin (solid circles) or IFN-α (open circles) overnight and challenged with EMCV (5 × 10² PFU/well). After 24 h of incubation, the cells protected from EMCV virulence were evaluated by the CPE dye uptake assay. The results represent the means ± standard deviations (SD) of triplicate cultures. Similar results were obtained in three independent experiments. (B) Fibroblast monolayers on 96-well plates were pretreated with limitin (10 EU/ml) or IFN-α (10 EU/ml) overnight and challenged with EMCV (5 × 10² PFU/well). After 24 h of incubation, the cells protected from EMCV virulence were evaluated by the CPE dye uptake assay. The results represent the means ± SD of the results obtained from six sets of fibroblasts independently established from wild-type and IRF-1-deficient mice.

FIG. 8.

ISRE-dependent luciferase activity after stimulation of limitin and IFN-α. The pCIS-ISRE plasmid was transfected into fibroblasts derived from wild-type or IRF-1-deficient mice. The transfectants were stimulated with the indicated concentrations of limitin or IFN-α for 6 h. The cells were lysed and subjected to measurement of luciferase activity. The data are expressed as the fold increase over the average for controls and represent the means ± standard deviations of triplicate assays. Similar results were obtained with four sets of fibroblasts independently established from wild-type and IRF-1-deficient mice. N.S., not significant.