The Gamma Interferon (IFN-γ) Mimetic Peptide IFN-γ(95-133) Prevents Encephalomyocarditis Virus Infection both in Tissue Culture and in Mice

Cell culture, virus, B8R protein, and interferons.Murine L929 fibroblasts were from the American Type Culture Collection (Manassas, VA) and maintained in Eagle's minimal essential medium (JHR Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (complete medium) at 37°C in a 5% CO₂ atmosphere incubator. EMC virus was obtained from the American Type Culture Collection and stored at −70°C until use. The B8R protein was a kind gift from Tilahun Yilma (University of California, Davis). Rat and mouse IFN-γ (both at 10⁷ U/mg) were purchased from PBL Biomedical Laboratories (Piscataway, NJ) and kept at −70°C until use.

Peptide synthesis.All peptides [IFN-γ(95-133), IFN-γ(95-125), IFNGR(253-287) (the IFN-γ receptor peptide consisting of residues 253 to 287), IFN-γ(126-133), and the SV40 T antigen NLS] were synthesized with an Applied Biosystems 9050 automated peptide synthesizer (Foster City, CA) using conventional fluorenylmethyloxycarbonyl chemistry as described previously (17, 18, 21). IFN-γ(95-133) and IFN-γ(126-133) peptides were synthesized without the cysteine residue at the C-terminal end (residue no. 133). The SV40 and the IFN-γ(95-133)SV40 peptides were synthesized with the addition of a cysteine at the N terminus of the sequence. The addition of a lipophilic group (palmitoyl-lysine) to the N termini of the synthetic peptides was performed as the last step by using a semiautomated protocol. Peptides were characterized by mass spectrometry and purified by high-performance liquid chromatography. Peptides were dissolved in deionized water and used for experimentation. All peptide solutions were negative for endotoxins as determined by a Limulus amebocyte lysate test (E-toxate kit; Sigma, St. Louis, MO).

Antiviral assays.Antiviral assays were performed by using a cytopathic effect reduction assay with EMC virus. Murine L929 cells (6 × 10⁴ cell/well) were plated in a 96-well plate and grown overnight to confluence for optimal growth. Various concentrations of IFN-γ, IFN-γ(95-133) and derivatives, and control peptides were incubated for various times (7 to 24 h) at 37°C. In other experiments, B8R protein (33 μg/ml) was preincubated for 2 h with rat IFN-γ and IFN-γ mimetic and control peptides. EMC virus (200 PFU/well) was added to the plate and incubated for 1 h, after which plates were washed and media added. Cells were incubated for at least 24 h and then stained with 0.1% crystal violet. Unbound crystal violet was aspirated, and the plates were thoroughly rinsed with deionized water, blotted, and allowed to air dry. Plates were then scanned using an Astra 2100U flatbed computer scanner (UMAX Technologies, Dallas, TX) and analyzed using Image J 1.29 software (National Institutes of Health, Bethesda, MD) to assess cell survival. Percentages of cell survival were determined by comparing survival for the experimental treatment groups with that for the virus-only control group.

Virus yield reduction and plaque assay.Murine L929 cells seeded to confluence in a 25-cm² flask were pretreated with 2,500 U/ml of mouse IFN-γ, 20 μM of IFN-γ(95-133), or 20 μM of the IFNGR(253-287) control peptide for 24 h, after which the cells were challenged with EMC virus at 100 PFU/ml for 1 h. The flasks were washed and incubated with Eagle's minimal essential medium containing 2% fetal bovine serum and incubated for 24 h (37°C, 5% CO₂). EMC virus produced was harvested via freezing and thawing three times in liquid nitrogen, and supernatants were titrated by a standard viral plaque assay using L929 cells, as previously described (21). Plaques for IFN-γ and IFN-γ(95-133) mimetic and IFNGR(253-287) control peptides were counted at a 1/200,000 dilution, whereas medium-only treatment was counted at a 1/2,000,000 dilution.

In vivo studies of mice.One-year-old mice (C57BL/6) were purchased from Jackson Laboratories and cared for at the Animal Resource Center of the University of Florida (Gainesville, FL). The Institutional Animal Care and Use Committee (IACUC) of the University of Florida approved all protocols prior to any study initiation. C57BL/6 mice were pretreated by intraperitoneal injection using a tuberculin syringe for 3 or 6 days with rat or mouse IFN-γ and IFN-γ(95-133) and control peptides at various concentrations (100 to 200 μg/mouse) every day. In some studies, B8R protein (250 μg/ml, diluted from crude stock preparation) was preincubated with the rat IFN-γ (200 U/mouse) and peptide (100 μg/mouse) injection cocktails prior to intraperitoneal administration. On the last day of treatment, mice were challenged with 50 PFU of EMC virus. In other studies, mice were injected with supernatants taken from murine L929 cells pretreated with IFN-γ and IFN-γ(95-133) mimetic and control peptides and infected with EMC virus. The numbers of surviving mice were recorded starting on the day of EMC virus challenge (day 0) and are presented as percent survival. Ten mice per treatment group were used in all mouse studies.

Detection of EMC virus in mouse tissues.For detection of virus in organs of infected C57BL/6 mice, heart, spleen, and liver tissues were extracted 6 days post-EMC virus injection from variously treated groups and homogenized into single-cell suspensions of 1-ml volumes. Cells were then lysed by freeze-thawing three times in liquid nitrogen. Samples were centrifuged at 2,000 rpm and supernatants collected. Supernatants were then diluted and incubated with murine L929 cells that were plated to confluence in a 96-well plate. After 24 to 48 h of incubation, plates were stained with 0.1% crystal violet and cell survival was assessed using Image J 1.29 software from NIH (Bethesda, MD) as described above in “Antiviral assays.”

The IFN-γ mimetic peptide IFN-γ(95-133) possesses antiviral activity against EMC virus infection in tissue culture.The antiviral activity of IFN-γ(95-133) was determined on murine L929 cells by use of EMC virus. Table 1 lists the sequences of IFN-γ(95-133) and control peptides, including their derivatives, used in this study. Mouse IFN-γ (300 U/ml to 11 U/ml) and various lipophilic (palmitoyl-lysine) IFN-γ mimetic and control peptides (100 μM to 3.7 μM) were incubated with murine L929 cells grown to confluence. Addition of a lipophilic group (palmitoyl-lysine) to these peptides enabled cytoplasmic delivery into cells (23, 24). After 7 h of incubation, EMC virus (200 PFU/well) was added for 1 h of incubation, after which plates were washed with media and reincubated in media for at least 28 h for establishment of EMC virus cytopathic effect in EMC virus control wells. As shown in Fig. 1, mouse IFN-γ had antiviral activity at all concentrations. The IFN-γ(95-133) mimetic peptide had antiviral activity at the 100 μM and 33 μM concentrations. Deletion of the NLS region, as per peptide IFN-γ(95-125), resulted in a loss of antiviral activity, even at the highest concentration used (100 μM). Furthermore, the NLS region of IFN-γ, IFN-γ(126-133), did not have any antiviral activity. Substitution of the IFN-γ NLS with the SV40 large T antigen NLS, IFN-γ(95-133)SV40, resulted in antiviral activity at 100 μM and 33 μM concentrations similar to that of the IFN-γ(95-133) mimetic peptide. Thus, protection against EMC virus required an NLS as well as the IFN-γ C-terminal amino acids 95 to 125, whereas individually these peptides lacked antiviral activity.

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FIG. 1.

IFN-γ mimetics possess antiviral activity against EMC virus in tissue culture. Mouse L929 cells were plated and grown to confluence on a 96-well plate. Various concentrations of mouse IFN-γ (300 U/ml to 11 U/ml) and various concentrations of IFN-γ(95-133), IFN-γ(95-133)SV40, SV40, IFN-γ(126-133), and IFN-γ(95-125) peptides, ranging from 100 μM to 3.7 μM, were incubated with L929 cells for 7 h, after which EMC virus (EMCV) (2,000 PFU/ml) was added. After 1 h, virus was removed and media added to all wells, followed by incubation for at least 28 h. Cells were stained with crystal violet solution, and plates were scanned and analyzed to assess cell survival. (A) Digital image of the plate. (B) Image J 1.29 software was used to analyze the image to obtain gray value to assess cell survival, presented here as percentages of the medium control value (100% cell survival). Representative data from one of three experiments are presented. Error bars indicate standard errors of the means.

In vitro toxicity studies using L929 cells grown to confluence on a 96-well plate and incubated with the mimetic and control peptides for 7 h demonstrated that all peptides, with the exception of SV40 large T antigen (at 100 μg), were nontoxic to cells at the concentrations used (100 μM to 3.7 μM), as shown in Fig. 2. Thus, the cytopathic effect shown in Fig. 1 was due to EMC virus infection rather than cellular toxicity caused by the IFN-γ mimetic and control peptides.

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FIG. 2.

Cellular toxicity of IFN-γ(95-133), derivatives, and control peptides. Mouse L929 cells were plated and grown to confluence on a 96-well plate. Various concentrations of IFN-γ(95-133), IFN-γ(95-133)SV40, SV40, IFN-γ(126-133), and IFN-γ(95-125) peptides, ranging from 100 μM to 3.7 μM, were incubated with L929 cells for 35 h. Cells were stained with crystal violet solution, and plates were scanned and analyzed to assess cell survival. (A) Digital image of the plate. (B) Image J 1.29 software was used to analyze the image to obtain gray value to assess cell survival, presented here as percentages of the medium control value (100% cell survival). Representative data from one of three experiments are presented. Error bars indicate standard errors of the means.

IFN-γ and IFN-γ(95-133) have reduced EMC virus yield compared to control groups.To determine if the mimetic peptide reduces virus yield after EMC virus infection in tissue culture, murine L929 cells seeded to confluence in a 25-cm² flask were pretreated with IFN-γ, IFN-γ(95-133), media, or the control peptide IFNGR(253-287) for 24 h, after which cells were incubated with EMC virus (100 PFU/ml) for 1 h. As shown in Table 2, IFN-γ and IFN-γ(95-133) reduced virus yield by 37.3- and 24.1-fold, respectively, compared to 0.7-fold for the control peptide IFNGR(253-287). Thus, similarly to IFN-γ, the IFN-γ(95-133) peptide reduced virus yield from EMC virus-infected cells.

Supernatants from EMC virus-infected cells treated with IFN-γ and IFN-γ(95-133) protect mice against lethal EMC virus infection.Supernatants taken from cells from experiments shown in Table 2 were injected into mice for determination of infectivity as measured in a lethal model of EMC virus infection. In the lethal EMC virus infection, mice become lethargic and eventually die, starting 4 days after the EMC virus injection (50 PFU or greater/mouse) (14, 25). As shown in Table 3, supernatants taken from cells treated with media and injected intraperitoneally into mice resulted in 75% death (25% survival), while supernatants taken from IFN-γ-treated cells resulted in 50% death. In contrast, mice injected with supernatants taken from cells treated with IFN-γ(95-133) did not succumb to EMC virus lethality (100% survival). No change was seen in any group after the eighth day after the supernatant injection. Thus, IFN-γ(95-133)-treated cells produced EMC virus amounts that were not lethal, as reflected by survival of mice.

Protection of mice from EMC virus challenge by the IFN-γ mimetic peptide IFN-γ(95-133) is dose dependent.Based on the tissue culture results showing antiviral activity of IFN-γ(95-133) and the in vivo results presented in Table 3, the IFN-γ mimetic peptide IFN-γ(95-133), along with a control peptide, IFN-γ(95-125), which does not have any antiviral activity in tissue culture due to the loss of the NLS region, were tested directly in vivo for their therapeutic effects in the mouse model of lethal EMC virus infection used as described above. C57BL/6 mice were prophylactically treated with phosphate-buffered saline (PBS), IFN-γ (3,000 U/day), IFN-γ(95-133) (100 μg/day), and the control peptide IFN-γ(95-125) for 6 days. On the last day of intraperitoneal injection, EMC virus (200 PFU/mouse) was also included in the injection cocktail. Mice were monitored every day and the numbers of surviving mice recorded. As shown in Fig. 3A, all PBS- and control peptide IFN-γ(95-125)-treated mice died by the seventh and eighth days after the EMC virus challenge, respectively. In contrast, mice injected with IFN-γ at 3,000 U/mouse per day showed 100% survival, while administration of the IFN-γ mimetic peptide at 100 μg/mouse per day resulted in approximately 30% of mice surviving EMC virus challenge (P < 0.05). Thus, administration of IFN-γ mimetic peptide at 100 μg/day for 6 days protected mice significantly compared to administration of PBS and control peptide.

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FIG. 3.

Protection of mice from EMC virus infection by IFN-γ and the IFN-γ mimetic peptide IFN-γ(95-133) is dose dependent. (A) C57BL/6 mice (10 mice per treatment group) were pretreated by intraperitoneal injection for 6 days with PBS, IFN-γ(95-125) and IFN-γ(95-133) peptides (100 μg/day), or IFN-γ (3,000 U/day). (B) Mice (five mice per treatment group) were pretreated with 200 μg/day of either IFN-γ(95-133) or IFN-γ(95-125) peptide for 3 days. On the last day of treatment, mice were challenged by intraperitoneal injection with 50 PFU of EMC virus. The numbers of surviving mice were recorded starting on the day of EMC virus challenge (day 0) and are presented as percent survival. Representative data from one of two experiments are shown.

To determine if an increased dosage of the IFN-γ mimetic peptide results in enhanced therapeutic activity against EMC virus challenge using the above-described mouse model of lethal EMC virus infection, IFN-γ(95-133) was administered at 200 μg/day intraperitoneally for 3 days. This treatment regimen differs from that described above for Fig. 3A in that a higher dose (200 μg/day) was administered in a shorter pretreatment time (3 days). On the third day of treatment, mice were challenged with EMC virus and the numbers of surviving mice recorded. As shown in Fig. 3B, PBS and control peptide IFN-γ(95-125) did not have any antiviral protection in mice (0% survival) whereas 100% of mice were protected from EMC virus challenge by IFN-γ(95-133) treatment. Thus, a higher dose of IFN-γ(95-133) in a reduced treatment time period enhances antiviral activity in mice infected with the EMC virus.

Analysis of sera and tissues of EMC virus-infected mice treated with the IFN-γ mimetic peptide.Infection with lethal EMC virus dosages results in certain organ failures and thus mortality (11, 14, 25). In cases where levels of EMC virus are below lethal doses, viral particles may still be found in the internal tissues of infected mice. Thus, viral titers of EMC virus in the blood and liver, spleen, and heart tissues of mice treated with IFN-γ, IFN-γ(95-133), PBS, and the control peptide IFN-γ(95-125) from the experiments performed as described above for Fig. 3B were determined. Treatment with the control peptide IFN-γ(95-125) showed detectable levels of EMC virus at dilutions of 1/270, 1/21,870, and 1/90 in the spleen, heart, and liver tissue samples, respectively, as shown in Fig. 4A to D. Spleen and liver tissue samples from mice treated with IFN-γ(95-133) did not have any quantifiable levels of EMC virus. Although the heart tissue did have detectable levels of EMC virus up to a dilution of 1/810 for IFN-γ(95-133), none of these mice died as indicated previously for Fig. 3B. The viral levels in the heart tissues of IFN-γ(95-133)-treated mice were 27 times lower than those for the control peptide [IFN-γ(95-125)] group. This is consistent with results seen in virus reduction in tissue culture experiments. Furthermore, 7 days later (13 days after the EMC virus challenge), EMC virus was not present in any tissue from the IFN-γ(95-133)-treated mice. Tissue samples from mice that were treated with IFN-γ had no detectable levels of EMC virus. In addition, as shown in Fig. 4E, EMC virus was not present in any serum samples from mice, including the control groups. Thus, IFN-γ(95-133)-treated mice have no or low EMC virus levels, consistent with mimetic protection.

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FIG. 4.

Mice treated with IFN-γ(95-133) peptide have reduced or no EMC virus particles in sera and various tissues. C57BL/6 mice were pretreated for 3 days with IFN-γ (3,000 U/day), IFN-γ(95-133) (200 μg/day), or the control peptide IFN-γ(95-125) (200 μg/day). On the last day of treatment, mice were challenged with 50 PFU of EMC virus. On day 6 after EMC virus infection, mice were bled and sacrificed, after which equal amounts of heart, spleen, and liver tissues were extracted from each group, homogenized, and lysed by thawing/freezing. (A to D) Samples were centrifuged, and supernatants were diluted and incubated on murine L929 cells plated to confluence in a 96-well plate for detection of EMC virus cytopathic effect after being stained with crystal violet solution. (E) Sera from various treatment groups were diluted and incubated with L929 cells as described above for determination of cytopathic effect. Tissues and sera from three mice per treatment group were analyzed, and representative data from one of two experiments are shown.

Antiviral activity of IFN-γ(95-133) peptide in the presence of the B8R protein in tissue culture and in vivo.Since we have shown previously that IFN-γ(95-133) binds to the cytoplasmic domain of the IFN-γ receptor and thus triggers signal transduction events associated with IFN-γ, this mimetic peptide was evaluated in the presence of the B8R protein, which is produced by poxviruses for neutralization of IFN-γ activity. The B8R protein is a homolog of the IFN-γ receptor extracellular domain; therefore, IFN-γ(95-133) should retain its antiviral activity in the presence of this virulence factor of poxviruses. To demonstrate this in tissue culture, murine L929 cells were plated to confluence on a 96-well plate, after which rat IFN-γ, IFN-γ(95-133), and IFN-γ(95-125), which were all preincubated for 2 h with B8R protein, were added to the plate. Rat IFN-γ was used here instead of mouse IFN-γ due to the fact that B8R does not bind to mouse IFN-γ but does bind to rat IFN-γ (3). Furthermore, rat IFN-γ has activity on mouse cells, as shown previously (3). After overnight incubation, EMC virus was added, and the cells were washed and reincubated in media for 24 h, followed by determination of EMC virus cytopathic effects. As shown in Fig. 5A, IFN-γ had antiviral activity against EMC virus at all concentrations (100 U/ml, 33 U/ml, and 11 U/ml) used, but in the presence of B8R, IFN-γ antiviral activity was lost at 33 U/ml and 11 U/ml. In contrast, the antiviral activities of IFN-γ(95-133) were similar in the presence or absence of B8R protein, as denoted by almost 100% cell viability at the 11 μM peptide concentration used (Fig. 5A and B). The control peptide IFN-γ(95-125) did not have any antiviral activity in either case. Thus, B8R protein neutralized IFN-γ antiviral activity but not IFN-γ(95-133) antiviral activity against EMC virus in tissue culture.

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FIG. 5.

B8R neutralizes IFN-γ but not IFN-γ(95-133) antiviral activity. Murine L929 cells were plated to confluence, after which media and various concentrations of IFN-γ, IFN-γ(95-133), and IFN-γ(95-125) that were preincubated for 2 h with or without B8R (33 μg/ml) were added to the plate. After 24 h of incubation, EMC virus (EMCV) (200 PFU/ml) was added for 1 h of incubation and washed with media. Cells were then incubated with media for 24 h, after which wells were stained with crystal violet and washed. (A) Digital image of the plate. (B) The plate was scanned for cell viability assessment using Image J software (NIH). Percent cell viability is presented for 33 U/ml of IFN-γ and 11 μM of IFN-γ(95-133). Error bars indicate standard errors of the means.

Based on the above-described tissue culture study, the antiviral activity of IFN-γ(95-133) was assessed in the presence of B8R protein in the mouse model of lethal EMC virus infection. C57BL/6 mice were pretreated for 3 days with PBS, IFN-γ(95-133) (100 μg/day), or rat IFN-γ (200 U/day). The 200-U/day dose of rat IFN-γ, which still had significant antiviral activity, was administered in order to detect the neutralizing effect of the B8R protein. On the last day of treatment, mice were challenged with 50 PFU of EMC virus. The numbers of surviving mice were recorded over time. As shown in Fig. 6, injection of rat IFN-γ resulted in 20% survival of mice in response to EMC virus challenge. In contrast, administration of B8R with rat IFN-γ resulted in 0% of mice surviving (100% death), which was similar to results for PBS controls. Administration of IFN-γ(95-133) in the presence or absence of B8R resulted in 40% and 30% survival, respectively, 9 days after the EMC virus challenge. Furthermore, there was a delay in the onset of lethality in IFN-γ(95-133)- and rat IFN-γ-treated groups compared to results for PBS- and IFN-γ/B8R-treated groups. These percentages of survival are similar to those seen for Fig. 3A, where 100 μg/day of IFN-γ(95-133) peptide was used. Thus, the IFN-γ mimetic peptide IFN-γ(95-133) has antiviral activity in tissue culture as well as in vivo in the presence of B8R protein, which neutralizes IFN-γ and which is encoded by poxviruses.

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FIG. 6.

Protection of mice from EMC virus challenge by the IFN-γ(95-133) peptide in the presence of B8R protein. C57BL/6 mice were pretreated for 3 days with PBS, IFN-γ(95-133) (100 μg/day), or rat IFN-γ (200 U/day) in the presence or absence of the B8R protein (25 μg). On the last day of treatment, mice were challenged with 50 PFU of EMC virus. The numbers of surviving mice were recorded starting on the day of EMC virus challenge (day 0) and are presented as percent survival. Ten mice per treatment group were used, and representative data from one of two experiments are shown.