Peptide mimetics of gamma interferon possess antiviral properties against vaccinia virus and other viruses in the presence of poxvirus B8R protein

Abstract

We have developed peptide mimetics of gamma interferon (IFN-gamma) that play a direct role in the activation and nuclear translocation of STAT1alpha transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-gamma receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-gamma and prevents its interaction with the receptor. Human and murine IFN-gamma mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-gamma mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-gamma, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-gamma receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-gamma was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-gamma mimetics was not affected by B8R protein, while IFN-gamma-induced upregulation was blocked. Finally, the mimetics, but not IFN-gamma, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-gamma can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.

FIG. 1.

Intracellular expression of IFN-γ mimetics with adenoviral vectors. HEK 293 cells were transduced with an empty-vector control or vectors expressing murine IFN-γ(95-133) or human IFN-γ(95-134) peptides. Two days later, cell extracts from empty-vector-transduced cells (column 1), murine peptide vector-transduced cells (column 2), or human peptide vector-transduced cells (column 4) were harvested. Supernatants from murine peptide vector-transduced cells (column 3) and human peptide vector-transduced cells (column 5) were also harvested and quantitated for IFN-γ peptide by using an enzyme-linked immunosorbent assay. Cell extract was taken in a 300-μl volume, while the supernatant volume was 3,000 μl. Wells in a microtiter plate were coated with 10 μl of the cell extract or 100 μl of the supernatant (which would represent equivalent amounts of the transduced cells), which was probed with a rabbit polyclonal antibody raised against the murine IFN-γ(95-133) peptide. The plate was coated with known amounts of chemically synthesized murine IFN-γ(95-133) peptide, which was processed similarly to obtain a standard curve. After a washing to remove nonspecific binding, horseradish peroxidase-conjugated anti-rabbit antibody was allowed to bind. o-Phenylenediamine dihydrochloride (Pierce, Rockford, Ill.) was added as a substrate, and absorption was measured. The results are averages from three independent determinations, plus or minus standard deviations.

FIG. 2.

IFN-γ mimetic peptides induce MHC class I molecules, even in the presence of B8R protein. WISH cells, untreated (column 1) or transduced with an empty-vector control (column 2) or a vector-expressing murine (column 3) or human (column 4) IFN-γ mimetic peptide, B8R protein for 8 h followed by murine IFN-γ peptide (column 5), or B8R protein for 8 h followed by human IFN-γ peptide (column 6) were used. IFN-γ (10 U/ml) was added to untreated cells (column 7) or cells transduced with B8R expression vector 8 h after the transduction (column 8). Lipo-(95-133) peptide (10 μM) was added to untreated cells (column 9) or cells transduced with B8R expression vector 8 h after the transduction (column 11). Cells transduced with B8R expression vector alone are shown in column 10. Cells treated with the control peptide, Lipo-(95-125), are shown in column 12. One day after these treatments, cells were stained with a monoclonal antibody to human MHC I conjugated to PE and analyzed by flow cytometry for its expression. The results are averages from three independent determinations, plus or minus standard deviations. The following abbreviations were used: Untrtd, untreated; Vec Cntrl, vector control; Mu pep, murine IFN-γ(95-133); Hu pep, human IFN-γ(95-134); Lipo-pep and Lipo-cont, chemically synthesized murine IFN-γ(95-133) and murine IFN-γ(95-125), respectively, that have a lipid moiety attached.

FIG. 3.

Binding of ¹²⁵I-IFN-γ to B8R protein and lack of competition by MuIFN-γ(95-133). In columns 1 through 5, wells in a microtiter plate were coated with 5 μg of protein from supernatants of WISH cells transduced with B8R expression vector, while column 6 shows the wells that were coated with 5 μg of protein from supernatant of control-vector-transduced cells. No competitor was included in columns 1 and 6. The following competitors were added: 10 U/ml of unlabeled IFN-γ (column 2), 10 μM of MuIFN-γ(95-133) (column 3), 5 μg of protein from supernatant of B8R-expressing cells (column 4), and 5 μg protein from supernatant of vector control cells (column 5). After the addition of ¹²⁵I-IFN-γ and incubation for 1 h, the wells were washed and the bound radioactivity was measured. The results are averages (counts per minute [CPM]) from three independent determinations, plus or minus standard deviations.

FIG. 4.

Inhibition of EMC virus by IFN-γ mimetic peptides in the presence of B8R protein. WISH cells were transduced with recombinant adenovirus control or vector expressing different proteins at, from left to right, 10⁶, 10 × 10⁶, and 50 × 10⁶ particles/ml in the three columns of sets 1 through 6. An empty-vector control (set 1) or vectors expressing murine IFN-γ(95-133) (set 2), human IFN-γ(95-134) (set 3), vaccinia virus-encoded B8R (set 4), murine IFN-γ(95-133) and B8R protein (set 5), or human IFN-γ(95-133) and B8R protein (set 6) were used to transfect cells. Human IFN-γ, at concentrations of 1, 5 and 10 U/ml, from left to right, was added to untreated cells (set 7). Cells transduced with B8R expression vector at 5 × 10⁷ particles/ml for 8 h were treated with 1, 5, and 10 U/ml of IFN-γ, from left to right (set 8). Lipopeptide MuIFN-γ(95-133) was added to untreated cells at concentrations of 1, 5, and 50 μM, from left to right (set 9), or cells transduced with B8R expression vector for 8 h at 5 × 10⁷ particles/ml (set 10). Cells treated with a control lipopeptide MuIFN-γ(95-125) at 1, 10, and 50 μM are shown in column 11. After incubation for 1 day, EMCV was added for 1 h and replaced with fresh growth medium, and cells were allowed to grow for one more day. Cells were stained with crystal violet, and absorbance was used to measure cytopathicity. The abbreviations that are used for this figure are the same as those used for Fig. 2. The results are averages from three independent determinations, plus or minus standard deviations.

FIG. 5.

Inhibition of VSV replication by IFN-γ mimetic peptides, even in the presence of B8R protein. WISH cells were transduced with recombinant adenovirus control vector or a vector expressing different proteins at, from left to right, 10⁶, 10 × 10⁶, and 50 ×10⁶ particles/ml in the three columns of sets 1 through 5. An empty-vector control (set 1) and vectors expressing murine IFN-γ(95-133) (set 2), human IFN-γ(95-134) (set 3), murine IFN-γ(95-133) and B8R protein (set 4), and human IFN-γ(95-134) and B8R protein (set 5) were used to transfect cells. Human IFN-γ, at concentrations of 1, 5, and 10 U/ml, from left to right, was added to untreated cells (set 6) or cells transduced with B8R expression vector at 5 × 10⁷ particles/ml for 8 h (set 7). Cells transduced with B8R expression vector control alone are shown in set 8. A lipo-IFN-γ(95-125) peptide control at concentrations of 1, 5, and 50 μM is shown in set 9. Lipo-IFN-γ(95-133), at concentrations of 1, 5, and 50 μM, is shown in set 10. Lipo-IFN-γ(95-133) added 8 h after transduction with B8R at 5 × 10⁷ particles/ml is shown in set 11. After incubation for 1 day, VSV was added for 1 h and replaced with fresh growth medium, and cells were allowed to grow for one more day. Cells were stained with crystal violet, and absorbance was used to measure cytopathicity. The abbreviations that are used for this figure are the same as those used for Fig. 2. The results are averages of three independent determinations, plus or minus standard deviations.

FIG. 6.

B8R protein is secreted from cells transduced with the vector expressing B8R. WISH cells (5 × 10⁵) were transfected with 5 × 10⁷ particles/ml of an empty-vector control or a vector expressing B8R protein. After 1 day, the supernatants (3 ml) were harvested. In the next step, 100 μl of the supernatant from the control (column 1) or 100, 50, or 20 μl of supernatant from B8R-expressing cells (columns 2 through 4) was added to WISH cells in the presence of 10 U/ml of IFN-γ. After 1 day of incubation, these cells were challenged with VSV for 1 day, and cytopathicity was determined after staining with crystal violet.

FIG. 7.

Presence of B8R protein dimer in cells transduced with the vector expressing B8R. HEK 293 cells (5 × 10⁵) were transduced with 5 × 10⁷ particles/ml of an empty-vector control or B8R-expressing vector. Eight hours later, cell extracts were harvested. Cell extracts (20, 4, and 1 μl) from the empty vector (lanes 1 through 3) or B8R-expressing vector (lanes 4 through 6) were electrophoresed (a molecular weight marker appears to the side of the gel). After transfer to nitrocellulose membrane, the filter was incubated with human IFN-γ. After washing to remove nonspecific binding, the filter was probed with an antibody to human IFN-γ, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Detection was by chemiluminescence. Inhibition of IFN-γ antiviral activity refers to the observed activity described in Fig. 4 and 5.

FIG. 8.

Inhibition of vaccinia virus replication by IFN-γ mimetic peptides. BSC-40 cells, untreated (column 1) or those treated as follows, were grown for 1 day. Cells transduced with an empty-vector control are shown in column 2, and those treated with 10 U/ml of human IFN-γ are shown in column 3. Cells transduced with a vector expressing murine IFN-γ(95-133) at 5 × 10⁶ and 50 × 10⁶ particles/ml are shown in columns 4 and 5. Human IFN-γ(95-134) vector-transduced cells at 5 × 10⁶ and 50 × 10⁶ particles/ml are shown in columns 6 and 7. Cells treated with 50 μM of a control lipo-IFN-γ(95-125) are shown in column 8, while those treated with 10 and 50 μM of lipo-IFN-γ(95-133) peptide are shown in columns 9 and 10, respectively. These treatments were followed by infection with vaccinia virus at a moiety of infection of 0.01 for 2 days. Cells were stained with neutral red, and the numbers of plaques were counted. The abbreviations that are used for this figure are the same as those used for Fig. 2. The results are averages from three independent determinations, plus or minus standard deviations.