The gamma interferon (IFN-gamma) mimetic peptide IFN-gamma (95-133) prevents encephalomyocarditis virus infection both in tissue culture and in mice

Abstract

We have demonstrated previously that the C-terminal gamma interferon (IFN-gamma) mimetic peptide consisting of residues 95 to 133 [IFN-gamma(95-133)], which contains the crucial IFN-gamma nuclear localization sequence (NLS), has antiviral activity in tissue culture. Here we evaluate the efficacy of this peptide and its derivatives first in vitro and then in an animal model of lethal viral infection with the encephalomyocarditis (EMC) virus. Deletion of the NLS region from the IFN-gamma mimetic peptide IFN-gamma(95-133) resulted in loss of antiviral activity. However, the NLS region does not have antiviral activity in itself. Replacing the NLS region of IFN-gamma(95-133) with the NLS region of the simian virus 40 large T antigen retains the antiviral activity in tissue culture. IFN-gamma(95-133) prevented EMC virus-induced lethality in mice in a dose-dependent manner compared to controls. Mice treated with IFN-gamma(95-133) had no or low EMC virus titers in their internal organs, whereas control mice had consistently high viral titers, especially in the heart tissues. Injection of B8R protein, which is encoded by poxviruses as a defense mechanism to neutralize host IFN-gamma, did not inhibit IFN-gamma(95-133) protection against a lethal dose of EMC virus, whereas mice treated with rat IFN-gamma were not protected. The data presented here show that the IFN-gamma mimetic peptide IFN-gamma(95-133) prevents EMC virus infection in vivo and in vitro and may have potential against other lethal viruses, such as the smallpox virus, which encodes the B8R protein.

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.

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.

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.

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.

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.

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.