Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Sequences of murine IFN-γ(95-133), derivatives, and control peptidesa
Peptide Sequence IFN-γ(95-133) AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC IFN-γ(95-125) AKFEVNNPQVQRQAFNELIRVVHQLLPESSL IFN-γ(95-133)SV40 AKFEVNNPQVQRQAFNELIRVVHQLLPESSLPKKKRKV SV40 T antigen PKKKRKV IFN-γ(126-133) RKRKRSRC IFNGR(253-287) TKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYS ↵a Peptides were synthesized as described in Materials and Methods. The NLS (RKRKRSR) of IFN-γ(95-133) and the NLS (PKKKRKV) of IFN-γ(95-133)SV40 are highlighted in boldface type.
- TABLE 2.
IFN-γ and IFN-γ(95-133) reduction of EMC virus yielda
Treatment Concn Virus yield (PFU/ml) Fold reduction Medium only 4.1 × 108 IFN-γ 2,500 U/ml 1.1 × 107 37.3 IFN-γ(95-133) 20 μM 1.7 × 107 24.1 IFNGR(253-287) 20 μM 6.3 × 108 0.7 ↵a Mouse L929 fibroblasts seeded to confluence in a 25-cm2 flask were pretreated with 2,500 U/ml of IFN-γ, 20 μM of IFN-γ(95-133), or 20 μM of IFNGR(253-287) control peptide for 24 h, after which cells were challenged with EMC virus at 100 PFU/ml for 1 h. Flasks were washed and incubated with media for another 24 h. Virus produced was harvested and titrated by a standard viral plaque assay (see Materials and Methods). The numbers of PFU/ml of the original samples were reported and reduction (n-fold) calculated via dividing the number of PFU/ml of the medium-only control by the number of PFU/ml of each experimental sample.
- TABLE 3.
Induction of EMC virus infection in C57BL/6 micea
Treatment % Survival at the indicated day after EMC virus infection 0 4 5 6 7 8 Medium 100 100 50 50 25 25 IFN-γ 100 100 100 75 50 50 IFN-γ(95-133) 100 100 100 100 100 100 ↵a Mouse L929 fibroblasts were treated with media, 2.5 × 103 U/ml of IFN-γ, or 20 μM of IFN-γ(95-133) for 24 h, after which cells were incubated with 100 PFU/ml of EMC virus for 1 h. Cells were then washed three times with media to wash away residual IFN-γ and peptides from the cells. Cells were resuspended with media for 24 h and supernatants collected. All supernatants were diluted with media (1:200,000) prior to intraperitoneal injection of mice on day 0. Mortality from EMC virus infection was recorded, and the percentage of surviving mice for each treatment group is presented. No change was seen in the mice after the eighth day postinjection with supernatants. A total of eight mice per group were used.
