Peptide Mimetics of Gamma Interferon Possess Antiv
http://www.100md.com
病菌学杂志 2005年第9期
Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, Florida 32611-0700
ABSTRACT
We have developed peptide mimetics of gamma interferon (IFN-) that play a direct role in the activation and nuclear translocation of STAT1 transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN- 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- and prevents its interaction with the receptor. Human and murine IFN- mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN- 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-, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN- 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- was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN- mimetics was not affected by B8R protein, while IFN--induced upregulation was blocked. Finally, the mimetics, but not IFN-, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN- can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.
INTRODUCTION
There are several studies using mostly nucleoside analogs to test their efficacy against acute exposure to smallpox and other poxviruses (5, 6, 17). The most promising of these analogs appears to be cidofovir (5). However, undesirable toxic effects, particularly those involving kidneys, have been observed in animal studies (17). Perhaps this is because these drugs do not appear to be virus specific. Thus, there is a timely need for other approaches to the development of poxvirus therapeutic agents. Vaccinia virus, which is used worldwide to vaccinate against smallpox infections, is a prototype of the poxvirus family. These viruses are particularly effective in neutralizing host innate antiviral defense mechanisms, such as the interferon (IFN) system. A major reason for this is the production by these viruses of soluble secreted proteins that bind to and prevent alpha/beta IFN (IFN-/) and IFN- from binding to their respective receptors on the cell membrane (16). An important virulence factor of poxviruses is the B8R protein, which is a homolog of the extracellular domain of the IFN- receptor and can therefore bind to intact IFN- and prevent its interaction with the receptor (24).
We have discovered, characterized, and synthesized small peptide agonists/mimetics of IFN-. These peptides, consisting of IFN- C terminus and C terminus analogs, HuIFN-(95-134) (from amino acid 95 to 134) for humans and MuIFN-(95-133) (from amino acid 95 to 133) for mice, possess potent antiviral activity against vesicular stomatitis virus (VSV) (25-28). VSV is commonly used in the detection and quantitation of IFN activity. The structural motif required for the recognition of the extracellular domain of the IFN- receptor chain IFNGR-1 and that involved in intracellular signaling are contained in two separate domains on the N terminus and C terminus, respectively, of the IFN- molecule (28, 31). The peptide mimetics act intracellularly to activate the Jak/STAT signaling apparatus and, thus, do not recognize the IFN- receptor extracellular domain (28). Therefore, smallpox and vaccinia virus virulence factor B8R should not interact with the IFN- mimetics and, thus, should not block mimetic antiviral activity. The mimetics were delivered into the cell via penetration by attachment of a lipophilic residue to chemically synthesized peptides, as described earlier for the delivery of IFN- peptide mimetics (20, 29, 30). Intracellular expression of mimetic genes was carried out by introducing the coding sequence for the murine (amino acids 95 through 133) or human (amino acids 95 through 134) IFN- sequence driven by the human cytomegalovirus (CMV) promoter in an adenovirus vector that we have previously used for intracellular expression of both IFN-/ and IFN- (1, 2).
We demonstrate in this report that IFN- mimetic peptides possess antiviral activity against vaccinia virus, encephalomyocarditis virus (EMCV), as well as VSV, in both the absence and the presence of coexpressed B8R protein. By contrast, IFN- failed to show antiviral activity against vaccinia virus, because of its endogenous B8R protein. We also show that expression of B8R protein in cells inhibited the antiviral activity of intact IFN- against EMC virus and VSV. The results of this study provide proof of concept of the hypothesis that these IFN- mimetics can exert antiviral activity against viruses, including poxviruses, such as vaccinia, under conditions that inhibit the antiviral activity of intact IFN-.
MATERIALS AND METHODS
Cell lines and recombinant adenoviruses. Human embryonic kidney cell line 293 (American Type Culture Collection [ATCC], Manassas, VA), used for the propagation of recombinant adenoviruses and BSC-40 cells for the propagation of vaccinia virus, was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Human WISH and mouse L929 cells (ATCC) were grown in Eagle's minimal essential medium (EMEM) containing 10% fetal bovine serum. Vaccinia virus Western Reserve strain was a gift from Richard Condit (University of Florida). Vaccinia virus was grown and titrated on BSC-40 cells.
The AdEasy adenoviral vector system from Stratagene (La Jolla, CA) was used. The construction and propagation of adenoviral vectors were carried out according to the manufacturer's protocol. Plasmids containing human or murine IFN- (ATCC) were used to carry out PCR. To obtain the IFN- mimetic peptide sequence, forward primers containing an appropriate restriction site, an initiating methionine, and a coding sequence starting from amino acid 95 through 100 for human or murine IFN- were used. The reverse primer contained an appropriate restriction site, a termination codon, and the coding sequence from amino acid 134 to 128 for human IFN- and from amino acid 133 to 127 for murine IFN-. A plasmid carrying the complete coding sequence for the B8R gene of the vaccinia virus Western Reserve strain was provided by T. Yilma (University of California, Davis). This plasmid was used to obtain a PCR product that contained the entire coding sequence for the B8R protein and the desired restriction sites at their termini. PCR products were digested with appropriate enzymes and cloned in the multiple-cloning site in the plasmid, pShuttleCMV. For the control plasmid, pShuttle MCS, which does not have a transgene, was used. Linearized plasmids, as described above, were cotransformed with pAdeasy plasmid in BJ5183 cells to obtain the recombinant adenovirus sequence. Recombinant plasmids were used to infect human embryonic kidney 293 cells to obtain viruses. The purification of viruses was carried out by using two CsCl gradients. These viruses were characterized by restriction enzyme digestion and DNA sequencing across the coding sequence. Cells that were about 50% confluent were infected with different recombinant adenoviruses at the indicated particles per ml for 1 h, followed by growth in Dulbecco's modified Eagle's medium for the periods indicated.
Peptide synthesis. Peptides corresponding to residues 95 through 133 and 95 through 125 of murine IFN- were synthesized on an Applied Biosystems 9050 automated peptide synthesizer (Foster City, CA) using conventional fluorenylmethyloxycarbonyl chemistry as described previously (26). The addition of a lipophilic group (palmitoyl-lysine) to the N terminus of the synthetic peptide was performed as the last step by using semiautomated protocol. Peptides were characterized by mass spectrometry and purified by high-performance liquid chromatography. Peptides were dissolved in either deionized water or dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO).
Expression of MHC class I. Human WISH cells were transfected with different recombinant adenoviruses for 1 h, followed by growth in EMEM for 48 h. Cells were then washed and incubated with a monoclonal antibody to human major histocompatibility complex (MHC) class I molecules conjugated with R-phycoerythrin (R-PE). Mouse immunoglobulin G2a conjugated with R-PE was used as a control. Both of these R-PE-conjugated antibodies were from Ancell (Bayport, MN). Cells were analyzed for immunofluorescence (FL-2) in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Data were collected in list-mode format and analyzed using CellQuest software (Becton Dickinson Immunocytometry Systems).
Radioligand binding assay for 125I-IFN- and B8R protein. The solid-phase radioimmunoassay for the binding of 125I-IFN- and B8R protein was carried out as described earlier (23). Recombinant human IFN- was purchased from Endogen (Rockford, IL) and was labeled with 125I to a specific activity of approximately 40 μCi/μg. Supernatants from cells transduced with an empty-vector control or a B8R expression vector were harvested, passed through a Centricon-50 column (Millipore, Bedford, MA), and lyophilized for concentration. About 5 μg of the proteins from the supernatant was diluted in 0.1 M carbonate buffer, pH 9.6, and adsorbed to the wells in a microtiter plate overnight at 4°C. The plates were washed once with phosphate-buffered saline (PBS). Blocking buffer (1% bovine serum albumin in PBS) was added to the wells and incubated at room temperature for 1 h. The following competitors diluted in blocking buffer were added: cold IFN- at 10 U/ml, MuIFN-(95-133) at 10 μM, and 5 μg each of proteins from supernatants of B8R expression vector- or control vector-transduced cells. In one of the wells, the plate was coated with 5 μg of protein from supernatant of control vector-transduced cells and no competitor was added. After 30 min of incubation with the competitor, 125I-IFN- was added and incubated for 1 h. After four washings, the counts in each well were counted. All bindings were done in triplicate, and the results represent the means plus or minus standard deviations.
Antiviral assay. Antiviral assays were performed by using a cytopathic-effect reduction assay using VSV and EMCV or by plaque formation for vaccinia virus. For VSV and EMCV, WISH cells (4 x 103) were plated in a microtiter dish and allowed to grow overnight. These cells were then infected with different recombinant adenoviruses for 1 h at the concentration indicated, followed by growth in EMEM medium for 24 h. VSV or EMCV at multiplicity of infection of 0.1 was then added to these cells for 1 h, followed by growth in EMEM medium 24 h. Cells were then stained with crystal violet. The dye retained was extracted in methyl Cellosolve, and absorption at 550 nM was measured (8).
Western blot analysis. WISH cells transduced for 48 h with B8R-expressing vector or empty-vector control were washed with PBS and harvested in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 50 mM NaF, 5 mM EDTA, and protease inhibitor cocktail [Roche Biochemicals, Indianapolis, IN]). Protein concentrations were measured using a bicinchoninic acid kit from Pierce (Rockford, IL). Protein (10 μg or dilutions thereof) was electrophoresed on 12% Tris-glycine gel, transferred to a nylon filter, and incubated with human IFN-, followed by washings to remove unbound IFN-. The filter was next probed with an antibody to human IFN- (PBL Biomedicals, Piscataway, NJ). After washings, a horseradish peroxidase-conjugated secondary antibody was used. Detection was carried out by chemiluminescence.
RESULTS
The adenovirus vector used in these studies, pAdEasy, is a derivative of ad5, which is deleted in early regions I and III. A cassette of CMV promoter-driven transgene replaces early region I. These vectors are replication deficient, owing to the deletion of early region I. The coding sequence of the IFN- mimetic peptide or B8R protein was inserted in the multiple cloning site of pAd vector, driven by the CMV promoter. These vectors were first characterized by restriction enzyme digestion, followed by DNA sequencing across the transgene. In addition to intracellular expression of mimetics, peptide MuIFN-(95-133) was also chemically synthesized with a lipophilic residue, palmitic acid, for penetration across the plasma membrane (20, 29, 30). A peptide encompassing residues 95 through 125 of murine IFN-, [MuIFN-(95-125)], to which the lipophilic moiety was similarly added, was used as a negative control. We have previously shown that MuIFN-(95-125) lacked a required nuclear localization sequence for mimetic activity (26). Data presented below demonstrate that mimetics, whether expressed intracellularly or via delivery of chemically synthesized lipopeptides, possessed similar qualitative IFN- mimetic activity.
To follow the expression of IFN- mimetics, WISH cells were transduced with control or IFN- mimetic expression vectors. Proteins from cell lysate, obtained 2 days after transduction, were quantitated by an enzyme-linked immunosorbent assay using a polyclonal antibody raised in rabbits against the MuIFN-(95-133) peptide (Fig. 1). Vectors expressing nonsecreted mimetics showed the presence of MuIFN-(95-133) and HuIFN-(95-134) at 1.2 and 1.1 μg per 106 cells, respectively, in cell extracts, while the supernatants did not show any detectable IFN--related peptides. Human IFN-(95-134) peptide is recognized by the antibody raised against the murine IFN-(95-133), since the two peptides share sequence homology. No IFN--related peptide was detected in the cells transduced with an empty-vector control, indicating that transduction with recombinant adenovirus in itself does not induce endogenous IFN- expression. Thus, consistent with previously described studies on the mechanism of action of IFN- mimetics, the expressed mimetics reside in the cell (2).
Prior to the determination of IFN- mimetic antiviral activity in the presence and absence of the poxvirus B8R IFN--binding protein, we first established the ability of mimetics to upregulate MHC class I molecules. Accordingly, both MuIFN-(95-133) and HuIFN-(95-134) were expressed in WISH cells in the absence or presence of B8R protein, and MHC class I upregulation was determined. Human IFN- activity was similarly tested, since its biological activity is inhibited by B8R protein (24). As shown in Fig. 2, cells expressing MuIFN-(95-133) or HuIFN-(95-134) and cells treated with human IFN- all upregulated MHC class I molecules about twofold compared with the baseline expression seen for untreated and control-vector-transduced cells. Similarly, chemically synthesized lipo-MuIFN-(95-133) also upregulated MHC class I molecules, while a control peptide, lipo-MuIFN-(95-125), showed only the basal level of MHC class I expression. The expression of B8R protein in mimetic-transfected cells and lipomimetic-treated cells did not inhibit any of the mimetics in the upregulation of MHC class I. IFN--induced upregulation, by contrast, was inhibited by B8R protein. Expression of B8R protein itself did not have any effect on the baseline expression of MHC class I molecules. The data are consistent with mimetic interaction with the cytoplasmic domain of IFN- receptor IFNGR-1 subunit (25). The B8R IFN--binding protein is known to bind to the intact IFN- N terminus and prevent its interaction with the IFNGR-1 extracellular domain (24). Thus, IFN- activity was inhibited by B8R, while IFNGR-1 cytoplasmic-binding mimetic biological activity was not, since B8R should not recognize the mimetic.
To verify that the IFN- mimetics did not bind to the B8R IFN- receptor-binding protein, competition-binding experiments were carried out with 125I-labeled human IFN- and B8R protein in a solid-phase radioligand binding assay. Since B8R is a secreted protein, supernatants from WISH cells transduced with B8R expression vector were concentrated by lyophilization and used for the coating of microtiter plates. Supernatants from control-vector-treated WISH cells were processed similarly. 125I-IFN- was allowed to bind to B8R protein or protein from vector control-treated cells on microtiter plates, and competitions were performed with unlabeled IFN-, IFN- mimetic, and supernatants from B8R expression vector or empty-vector-transduced cells. As shown in Fig. 3, unlabeled intact IFN- reduced 125I-IFN- binding to B8R, while MuIFN-(95-133) at functional concentrations, as per Fig. 2 (10 μM), had no effect on 125I-IFN- binding. Supernatant from B8R-expressing cells inhibited 125I-IFN- binding, while the supernatant from control-vector-treated cells did not significantly affect solid-phase competition for 125I-IFN-. 125I-IFN- did not bind to supernatant from empty-control-vector-treated cells. The competition data are consistent with the MHC class I upregulation data of Fig. 2, where B8R protein competed with the extracellular domain of IFNGR-1 for binding to IFN- at its N terminus but failed to recognize the IFN- mimetics, which specifically bind to the cytoplasmic domain of IFNGR-1 (25). The binding data are also consistent with the secretion of B8R protein with the expression system used and the B8R protein recognition of intact IFN- but not IFN- mimetics.
EMC virus replication is known to be inhibited by IFN-. We therefore determined whether IFN- mimetics could inhibit EMC virus replication in WISH cells as well as the effect of B8R IFN--binding protein on such replication inhibition. Cells were either transfected with mimetic genes as described above for intracellular expression or treated with lipophilic mimetic for penetration of plasma membrane. Human IFN- was also added separately. Mimetic-transfected cells were also simultaneously transfected with the B8R expression vector. In addition, cells were treated with increasing concentrations of human IFN-, lipo-MuIFN-(95-133), or lipo-MuIFN- (95-125) 8 h after transduction with B8R-expressing vector. Cells were allowed to grow for 24 h and then challenged with a 0.1 multiplicity of infection of EMC virus, and the cytopathic effect was quantitated 24 h later by crystal violet staining and absorption measurement, as described previously (8). As can be seen in Fig. 4, cells transfected with a vector expressing MuIFN-(95-133) and HuIFN-(95-134) as well as those treated with lipo-MuIFN-(95-133) and intact IFN- protected WISH cells against EMC virus infection in a dose-dependent manner, while the empty-vector control, the peptide control, or B8R by itself did not have any protective effective against viral infection. Mimetic protection against EMC virus, either via transfection or via lipomimetic addition, was not affected by B8R transfection of the cells. IFN- protection, however, was inhibited by B8R, which is consistent with the anti-IFN- properties of the B8R IFN--binding protein. Thus, the IFN- mimetics possess antiviral activity against EMC virus that was not affected by the poxvirus virulence property of B8R protein.
We next determined the ability of the IFN- mimetics to inhibit replication of the rhabdovirus VSV and the effect of B8R protein on mimetic inhibition. EMC virus is a picornavirus, so the VSV experiment tests the generality of the antiviral properties of the mimetic as well as the effect of B8R protein on the antiviral activity. As can be seen in Fig. 5, WISH cells transduced with the MuIFN-(95-133) and HuIFN-(95-134) genes and those treated with lipo-IFN-(95-133) or intact human IFN- showed antiviral activity in a dose-dependent manner. Further, the mimetic antiviral activity was not blocked by B8R protein, while that of IFN- was blocked. We conclude that mimetics possess antiviral activity against VSV similar to that against EMC virus and that such activity was not affected by the poxvirus virulence factor B8R IFN--binding protein.
To test for the effect of the protein secreted by cells transduced with B8R-expressing construct on IFN- activity, supernatants from B8R expressing or control-vector-treated WISH cells were harvested and, in a second step, added to WISH cells in the presence of IFN- and then challenged with VSV. As shown in Fig. 6, IFN- activity was suppressed in the presence of supernatant from B8R-transduced cells, while supernatant from control-vector-transduced cells did not have any effect on IFN- antiviral activity. This demonstrated that the B8R gene expression resulted in secreted B8R protein that bound to and neutralized added IFN-.
The B8R protein produced in WISH cells was further characterized for its ability to bind to intact IFN- (Fig. 7). Cells were transduced with a B8R-expressing vector or an empty-vector control for 8 h. Cell extracts were electrophoresed on an acrylamide gel. Protein was transferred to a nylon filter and incubated with intact IFN-. Unbound IFN- was removed by washing, and the filter was probed with an antibody to IFN-. A band with an approximate molecular mass of 43 kDa, which corresponds to a dimer of B8R protein, was observed in B8R-expressing cells; its intensity decreased upon dilution. No such band was seen in the extracts of cells transduced with an empty-vector control. Thus, the cells transduced with the B8R expression vector secrete a protein with a molecular mass similar to the size of the B8R dimer (3).
Vaccinia virus is the prototype of the poxvirus family, and it has been used extensively in the study of the pathogenesis of this family of viruses (16). The IFN- mimetics were therefore examined for their antiviral effects against the Western Reserve strain of vaccinia virus, which expresses the intact B8R protein. WISH cells were first transduced with IFN- mimetic expression vectors or treated with lipo-MuIFN-(95-133), lipo-MuIFN-(95-125), or intact human IFN- followed by challenge with vaccinia virus. MuIFN-(95-133) and HuIFN-(95-134) mimetic peptides expressed intracellularly or by the addition of lipo-MuIFN-(95-133) conferred resistance to vaccinia virus infection, while intact human IFN- at 10 U/ml failed to protect WISH cells from vaccinia virus (Fig. 8). Empty vector control or the lipo-MuIFN-(95-125) did not exhibit any protection against vaccinia virus. These observations, combined with the B8R protein studies described above, indicate that human and murine IFN- mimetic peptides can circumvent the B8R IFN--binding protein virulence factor activity, providing proof of concept that IFN- mimetics are a potential approach to development of smallpox antivirals. Thus, the IFN- mimetics regulate virus replication differently from intact IFN-, suggesting unique antiviral properties of these mimetics.
DISCUSSION
Although smallpox was declared eradicated worldwide by the World Health Organization almost 20 years ago, recent events have generated renewed interest in developing new therapeutics for this class of virus. Smallpox or related viruses may be used in biological warfare, primarily through aerosolization (4). Since general vaccinations have been terminated for several decades, there is currently very low immunity in the general population, which leaves many vulnerable to infection. The likelihood of the emergence of new strains through natural recombination within the pox family of viruses, such as that seen in the recent outbreak of monkeypox, does exist (7). That the virulence of this family of viruses can be enhanced further was indicated by the recent demonstration that the introduction of interleukin 4 in the mousepox genome resulted in a virus that was 100% lethal, even in genetically resistant and previously immunized animals (12). Use of the existing vaccine, which is the same as that used 2 centuries ago, carries the risk of side effects, including encephalitis, myocarditis, and myopericarditis (11). Therefore, alternative antiviral treatments are being actively investigated.
Variola is the causative agent of smallpox. Vaccinia virus is a prototype of poxviruses and has been used as a vaccine to eradicate smallpox. It has been studied extensively for its pathogenesis and for its potential use as a gene delivery vehicle (16, 18). DNA sequencing has revealed that the genes common to vaccinia and variola viruses show greater than 90% similarity (10). Therefore, vaccinia virus represents the best system to study the effects of smallpox family of viruses. The genes responsible for viral replication are contained in the central region of the genome, while a large number of immune evasion genes are flanked on either side of this central region.
One of the immune evasion genes of vaccinia virus is the B8R gene, which codes for a soluble protein that resembles the extracellular surface of the IFN- receptor and can thereby neutralize the activity of IFN- from several species, in particular, human IFN- (24). The B8R protein exists as a homodimer naturally, and this apparently provides an advantage to the virus in the efficient binding and inhibition of IFN- in solution (3). It has been shown with rodent models that B8R is a virulence factor for vaccinia virus infection (24). Vaccinia virus vectors with an inactivated IFN- receptor homolog B8R gene are attenuated in vivo (32). An IFN mimetic that bypasses the IFN- receptor extracellular domain should also functionally attenuate the B8R gene. Further, the functional neutralization of B8R and the inhibition of the growth of vaccinia virus (or other poxviruses, such as smallpox) should help reduce the production of other poxvirus proteins that have an inhibitory effect on host defense. The other proteins encoded by vaccinia virus that interact with the IFN system (reviewed in reference 15) are as follows. E3L protein can bind to double-stranded RNA. K3L has structural similarity to the N terminus of eIF-2. VH1 can dephosphorylate STAT1. The B18R gene codes for a soluble IFN-/ receptor. We reason that if virus replication is inhibited by IFN- agonists/mimetics, then the vaccinia virus induction of other virulence factors will be inhibited. A similar situation exists with hepatitis virus C, which produces multiple proteins that can inactivate the IFN system (15), and yet IFN is the only therapy available to date to treat this disease.
We have previously shown that a polycationic sequence in the C terminus of human and murine IFN- is a nuclear localization sequence (NLS) and is responsible for the appearance of IFN- in the nucleus of treated cells (19, 20). Site-directed mutagenesis (2), or chemical synthesis of peptides (23), replacing positively charged amino acids with alanines resulted in loss of biological activity. While the N terminus of IFN- has been shown to bind to the extracellular receptor domains by peptide competition and crystallographic structure determination, no extracellular receptor binding site for the NLS-containing C terminus of IFN- has been demonstrated (reviewed in reference 13).
We have identified a high-affinity, species-nonspecific binding site (Kd, 10–8 M) for the IFN- C terminus on the cytoplasmic domain of both human and murine IFNGR-1 (9). This site, also demonstrable in whole cells, was identified as a membrane proximal region of the cytoplasmic domain within amino acid residues 253 through 287 (13). Consistent with this, we were able to demonstrate that peptides from the C-terminal NLS-containing domains of the human IFN-, HuIFN-(95-134), and the murine IFN-, MuIFN-(95-133), were sufficient to induce IFN--associated activities when internalized by murine macrophages (27). The intracellular peptide mimetics, which contain the NLS of IFN, were found to inhibit virus replication by 106- to 109-fold and upregulated MHC class II expression up to 10-fold in a fashion that depended strictly on the presence of the NLS in these peptide sequences (27). The peptide mimetics interacted exclusively with the cytoplasmic domain of IFNGR-1 within residues 253 through 287 and not with the extracellular region of IFNGR-1. An N-terminal -helix in these peptides within residues 108 through 121 was also found to be important for function by virtue of its requirement in the mimetics for binding to IFNGR-1 (amino acids 253 through 287) (26). Other studies showed that the peptides behaved as IFN- mimetics only when delivered intracellularly in cells and that their mimetic properties required the expression of IFNGR-1 in such cells showing the requirement of the cytoplasmic domain of IFNGR-1 (29, 30). HuIFN-(95-134) and MuIFN-(95-132) share approximately 80% homology and interact with the membrane-proximal IFNGR-1 cytoplasmic region (amino acids 253 through 287) in a non-species-specific manner (27). Therefore, the murine and human peptides were equally potent. The IFN- peptide mimetics, like intracellular IFN-, also were able to induce the activation and nuclear translocation of STAT1, as well as being able to induce the endocytosis and nuclear translocation of the IFNGR-1 subunit from within the cell.
Using the IFN- mimetic in in vitro binding assays and a standard in vitro nuclear transport assay system, we presented data to suggest that the IFN- NLS can play multiple roles in IFN- mimetic signaling. These include binding to the intracellular cytoplasmic domain of IFNGR-1 and simultaneous interaction with the nuclear import machinery to mediate the nuclear transport of IFNGR-1. Further, phosphorylated STAT1, which binds to phosphorylated IFNGR-1, is shown to be transported to the nucleus in an in vitro system by IFN-(95-133) and a phosphorylated IFNGR-1 subunit. Finally, the N-terminal -helix of the IFN- mimetic also plays an important role in IFNGR-1 binding and subsequent events. The mimetic data support the conclusion that the versatility of the C terminus of IFN-, involving both its -helix and its NLS, is important for IFN- mimetic-mediated STAT1 nuclear translocation and associated IFN--like biological activities. Details of the discussion on the mechanism of intracellular trafficking of IFN- and the IFN- mimetics are presented elsewhere (14, 21-23).
The presence of the N terminus receptor binding region in IFN- is key to its interaction with the receptor extracellular domain and the subsequent internalization and interaction with the cytoplasmic domain of the receptor subunit, IFNGR-1, via the IFN- C terminus domain (13). The failure to bind to the IFN- receptor extracellular domain prevents internalization and IFN- interaction with the IFNGR-1 cytoplasmic domain. The cytoplasmic interaction of IFN- via its C terminus is key to enhanced binding of JAK2 to IFNGR-1, its subsequent autophosphorylation, phosphorylation of IFNGR-1, and ultimately, phosphorylation of STAT1 for nuclear import and gene activation (13). The interaction of IFN- with B8R blocks the extracellular interaction and therefore subsequent events that result in induction of antiviral activity.
We have shown here that, in contrast to IFN-, the IFN- mimetics do not bind to the B8R protein, that the mimetics induce antiviral activity against EMC virus and VSV in the presence of B8R, and, finally, that mimetics inhibit vaccinia virus replication in the presence of endogenous B8R protein. An important conclusion is that the IFN- C terminus mimetics provide a proof-of-concept demonstration of antipoxvirus IFN- drugs that bypass the B8R virulence factor. The findings caution against focusing solely on the IFN- receptor extracellular domain in the development of IFN- antiviral drugs.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grant RO1AI056152 to H.M.J.
We gratefully acknowledge the Flow Cytometry Core Facility, University of Florida, for assistance with flow cytometry.
The paper is Florida Experimental Station Journal Series no. R-10766.
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ABSTRACT
We have developed peptide mimetics of gamma interferon (IFN-) that play a direct role in the activation and nuclear translocation of STAT1 transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN- 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- and prevents its interaction with the receptor. Human and murine IFN- mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN- 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-, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN- 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- was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN- mimetics was not affected by B8R protein, while IFN--induced upregulation was blocked. Finally, the mimetics, but not IFN-, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN- can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.
INTRODUCTION
There are several studies using mostly nucleoside analogs to test their efficacy against acute exposure to smallpox and other poxviruses (5, 6, 17). The most promising of these analogs appears to be cidofovir (5). However, undesirable toxic effects, particularly those involving kidneys, have been observed in animal studies (17). Perhaps this is because these drugs do not appear to be virus specific. Thus, there is a timely need for other approaches to the development of poxvirus therapeutic agents. Vaccinia virus, which is used worldwide to vaccinate against smallpox infections, is a prototype of the poxvirus family. These viruses are particularly effective in neutralizing host innate antiviral defense mechanisms, such as the interferon (IFN) system. A major reason for this is the production by these viruses of soluble secreted proteins that bind to and prevent alpha/beta IFN (IFN-/) and IFN- from binding to their respective receptors on the cell membrane (16). An important virulence factor of poxviruses is the B8R protein, which is a homolog of the extracellular domain of the IFN- receptor and can therefore bind to intact IFN- and prevent its interaction with the receptor (24).
We have discovered, characterized, and synthesized small peptide agonists/mimetics of IFN-. These peptides, consisting of IFN- C terminus and C terminus analogs, HuIFN-(95-134) (from amino acid 95 to 134) for humans and MuIFN-(95-133) (from amino acid 95 to 133) for mice, possess potent antiviral activity against vesicular stomatitis virus (VSV) (25-28). VSV is commonly used in the detection and quantitation of IFN activity. The structural motif required for the recognition of the extracellular domain of the IFN- receptor chain IFNGR-1 and that involved in intracellular signaling are contained in two separate domains on the N terminus and C terminus, respectively, of the IFN- molecule (28, 31). The peptide mimetics act intracellularly to activate the Jak/STAT signaling apparatus and, thus, do not recognize the IFN- receptor extracellular domain (28). Therefore, smallpox and vaccinia virus virulence factor B8R should not interact with the IFN- mimetics and, thus, should not block mimetic antiviral activity. The mimetics were delivered into the cell via penetration by attachment of a lipophilic residue to chemically synthesized peptides, as described earlier for the delivery of IFN- peptide mimetics (20, 29, 30). Intracellular expression of mimetic genes was carried out by introducing the coding sequence for the murine (amino acids 95 through 133) or human (amino acids 95 through 134) IFN- sequence driven by the human cytomegalovirus (CMV) promoter in an adenovirus vector that we have previously used for intracellular expression of both IFN-/ and IFN- (1, 2).
We demonstrate in this report that IFN- mimetic peptides possess antiviral activity against vaccinia virus, encephalomyocarditis virus (EMCV), as well as VSV, in both the absence and the presence of coexpressed B8R protein. By contrast, IFN- failed to show antiviral activity against vaccinia virus, because of its endogenous B8R protein. We also show that expression of B8R protein in cells inhibited the antiviral activity of intact IFN- against EMC virus and VSV. The results of this study provide proof of concept of the hypothesis that these IFN- mimetics can exert antiviral activity against viruses, including poxviruses, such as vaccinia, under conditions that inhibit the antiviral activity of intact IFN-.
MATERIALS AND METHODS
Cell lines and recombinant adenoviruses. Human embryonic kidney cell line 293 (American Type Culture Collection [ATCC], Manassas, VA), used for the propagation of recombinant adenoviruses and BSC-40 cells for the propagation of vaccinia virus, was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Human WISH and mouse L929 cells (ATCC) were grown in Eagle's minimal essential medium (EMEM) containing 10% fetal bovine serum. Vaccinia virus Western Reserve strain was a gift from Richard Condit (University of Florida). Vaccinia virus was grown and titrated on BSC-40 cells.
The AdEasy adenoviral vector system from Stratagene (La Jolla, CA) was used. The construction and propagation of adenoviral vectors were carried out according to the manufacturer's protocol. Plasmids containing human or murine IFN- (ATCC) were used to carry out PCR. To obtain the IFN- mimetic peptide sequence, forward primers containing an appropriate restriction site, an initiating methionine, and a coding sequence starting from amino acid 95 through 100 for human or murine IFN- were used. The reverse primer contained an appropriate restriction site, a termination codon, and the coding sequence from amino acid 134 to 128 for human IFN- and from amino acid 133 to 127 for murine IFN-. A plasmid carrying the complete coding sequence for the B8R gene of the vaccinia virus Western Reserve strain was provided by T. Yilma (University of California, Davis). This plasmid was used to obtain a PCR product that contained the entire coding sequence for the B8R protein and the desired restriction sites at their termini. PCR products were digested with appropriate enzymes and cloned in the multiple-cloning site in the plasmid, pShuttleCMV. For the control plasmid, pShuttle MCS, which does not have a transgene, was used. Linearized plasmids, as described above, were cotransformed with pAdeasy plasmid in BJ5183 cells to obtain the recombinant adenovirus sequence. Recombinant plasmids were used to infect human embryonic kidney 293 cells to obtain viruses. The purification of viruses was carried out by using two CsCl gradients. These viruses were characterized by restriction enzyme digestion and DNA sequencing across the coding sequence. Cells that were about 50% confluent were infected with different recombinant adenoviruses at the indicated particles per ml for 1 h, followed by growth in Dulbecco's modified Eagle's medium for the periods indicated.
Peptide synthesis. Peptides corresponding to residues 95 through 133 and 95 through 125 of murine IFN- were synthesized on an Applied Biosystems 9050 automated peptide synthesizer (Foster City, CA) using conventional fluorenylmethyloxycarbonyl chemistry as described previously (26). The addition of a lipophilic group (palmitoyl-lysine) to the N terminus of the synthetic peptide was performed as the last step by using semiautomated protocol. Peptides were characterized by mass spectrometry and purified by high-performance liquid chromatography. Peptides were dissolved in either deionized water or dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO).
Expression of MHC class I. Human WISH cells were transfected with different recombinant adenoviruses for 1 h, followed by growth in EMEM for 48 h. Cells were then washed and incubated with a monoclonal antibody to human major histocompatibility complex (MHC) class I molecules conjugated with R-phycoerythrin (R-PE). Mouse immunoglobulin G2a conjugated with R-PE was used as a control. Both of these R-PE-conjugated antibodies were from Ancell (Bayport, MN). Cells were analyzed for immunofluorescence (FL-2) in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Data were collected in list-mode format and analyzed using CellQuest software (Becton Dickinson Immunocytometry Systems).
Radioligand binding assay for 125I-IFN- and B8R protein. The solid-phase radioimmunoassay for the binding of 125I-IFN- and B8R protein was carried out as described earlier (23). Recombinant human IFN- was purchased from Endogen (Rockford, IL) and was labeled with 125I to a specific activity of approximately 40 μCi/μg. Supernatants from cells transduced with an empty-vector control or a B8R expression vector were harvested, passed through a Centricon-50 column (Millipore, Bedford, MA), and lyophilized for concentration. About 5 μg of the proteins from the supernatant was diluted in 0.1 M carbonate buffer, pH 9.6, and adsorbed to the wells in a microtiter plate overnight at 4°C. The plates were washed once with phosphate-buffered saline (PBS). Blocking buffer (1% bovine serum albumin in PBS) was added to the wells and incubated at room temperature for 1 h. The following competitors diluted in blocking buffer were added: cold IFN- at 10 U/ml, MuIFN-(95-133) at 10 μM, and 5 μg each of proteins from supernatants of B8R expression vector- or control vector-transduced cells. In one of the wells, the plate was coated with 5 μg of protein from supernatant of control vector-transduced cells and no competitor was added. After 30 min of incubation with the competitor, 125I-IFN- was added and incubated for 1 h. After four washings, the counts in each well were counted. All bindings were done in triplicate, and the results represent the means plus or minus standard deviations.
Antiviral assay. Antiviral assays were performed by using a cytopathic-effect reduction assay using VSV and EMCV or by plaque formation for vaccinia virus. For VSV and EMCV, WISH cells (4 x 103) were plated in a microtiter dish and allowed to grow overnight. These cells were then infected with different recombinant adenoviruses for 1 h at the concentration indicated, followed by growth in EMEM medium for 24 h. VSV or EMCV at multiplicity of infection of 0.1 was then added to these cells for 1 h, followed by growth in EMEM medium 24 h. Cells were then stained with crystal violet. The dye retained was extracted in methyl Cellosolve, and absorption at 550 nM was measured (8).
Western blot analysis. WISH cells transduced for 48 h with B8R-expressing vector or empty-vector control were washed with PBS and harvested in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 50 mM NaF, 5 mM EDTA, and protease inhibitor cocktail [Roche Biochemicals, Indianapolis, IN]). Protein concentrations were measured using a bicinchoninic acid kit from Pierce (Rockford, IL). Protein (10 μg or dilutions thereof) was electrophoresed on 12% Tris-glycine gel, transferred to a nylon filter, and incubated with human IFN-, followed by washings to remove unbound IFN-. The filter was next probed with an antibody to human IFN- (PBL Biomedicals, Piscataway, NJ). After washings, a horseradish peroxidase-conjugated secondary antibody was used. Detection was carried out by chemiluminescence.
RESULTS
The adenovirus vector used in these studies, pAdEasy, is a derivative of ad5, which is deleted in early regions I and III. A cassette of CMV promoter-driven transgene replaces early region I. These vectors are replication deficient, owing to the deletion of early region I. The coding sequence of the IFN- mimetic peptide or B8R protein was inserted in the multiple cloning site of pAd vector, driven by the CMV promoter. These vectors were first characterized by restriction enzyme digestion, followed by DNA sequencing across the transgene. In addition to intracellular expression of mimetics, peptide MuIFN-(95-133) was also chemically synthesized with a lipophilic residue, palmitic acid, for penetration across the plasma membrane (20, 29, 30). A peptide encompassing residues 95 through 125 of murine IFN-, [MuIFN-(95-125)], to which the lipophilic moiety was similarly added, was used as a negative control. We have previously shown that MuIFN-(95-125) lacked a required nuclear localization sequence for mimetic activity (26). Data presented below demonstrate that mimetics, whether expressed intracellularly or via delivery of chemically synthesized lipopeptides, possessed similar qualitative IFN- mimetic activity.
To follow the expression of IFN- mimetics, WISH cells were transduced with control or IFN- mimetic expression vectors. Proteins from cell lysate, obtained 2 days after transduction, were quantitated by an enzyme-linked immunosorbent assay using a polyclonal antibody raised in rabbits against the MuIFN-(95-133) peptide (Fig. 1). Vectors expressing nonsecreted mimetics showed the presence of MuIFN-(95-133) and HuIFN-(95-134) at 1.2 and 1.1 μg per 106 cells, respectively, in cell extracts, while the supernatants did not show any detectable IFN--related peptides. Human IFN-(95-134) peptide is recognized by the antibody raised against the murine IFN-(95-133), since the two peptides share sequence homology. No IFN--related peptide was detected in the cells transduced with an empty-vector control, indicating that transduction with recombinant adenovirus in itself does not induce endogenous IFN- expression. Thus, consistent with previously described studies on the mechanism of action of IFN- mimetics, the expressed mimetics reside in the cell (2).
Prior to the determination of IFN- mimetic antiviral activity in the presence and absence of the poxvirus B8R IFN--binding protein, we first established the ability of mimetics to upregulate MHC class I molecules. Accordingly, both MuIFN-(95-133) and HuIFN-(95-134) were expressed in WISH cells in the absence or presence of B8R protein, and MHC class I upregulation was determined. Human IFN- activity was similarly tested, since its biological activity is inhibited by B8R protein (24). As shown in Fig. 2, cells expressing MuIFN-(95-133) or HuIFN-(95-134) and cells treated with human IFN- all upregulated MHC class I molecules about twofold compared with the baseline expression seen for untreated and control-vector-transduced cells. Similarly, chemically synthesized lipo-MuIFN-(95-133) also upregulated MHC class I molecules, while a control peptide, lipo-MuIFN-(95-125), showed only the basal level of MHC class I expression. The expression of B8R protein in mimetic-transfected cells and lipomimetic-treated cells did not inhibit any of the mimetics in the upregulation of MHC class I. IFN--induced upregulation, by contrast, was inhibited by B8R protein. Expression of B8R protein itself did not have any effect on the baseline expression of MHC class I molecules. The data are consistent with mimetic interaction with the cytoplasmic domain of IFN- receptor IFNGR-1 subunit (25). The B8R IFN--binding protein is known to bind to the intact IFN- N terminus and prevent its interaction with the IFNGR-1 extracellular domain (24). Thus, IFN- activity was inhibited by B8R, while IFNGR-1 cytoplasmic-binding mimetic biological activity was not, since B8R should not recognize the mimetic.
To verify that the IFN- mimetics did not bind to the B8R IFN- receptor-binding protein, competition-binding experiments were carried out with 125I-labeled human IFN- and B8R protein in a solid-phase radioligand binding assay. Since B8R is a secreted protein, supernatants from WISH cells transduced with B8R expression vector were concentrated by lyophilization and used for the coating of microtiter plates. Supernatants from control-vector-treated WISH cells were processed similarly. 125I-IFN- was allowed to bind to B8R protein or protein from vector control-treated cells on microtiter plates, and competitions were performed with unlabeled IFN-, IFN- mimetic, and supernatants from B8R expression vector or empty-vector-transduced cells. As shown in Fig. 3, unlabeled intact IFN- reduced 125I-IFN- binding to B8R, while MuIFN-(95-133) at functional concentrations, as per Fig. 2 (10 μM), had no effect on 125I-IFN- binding. Supernatant from B8R-expressing cells inhibited 125I-IFN- binding, while the supernatant from control-vector-treated cells did not significantly affect solid-phase competition for 125I-IFN-. 125I-IFN- did not bind to supernatant from empty-control-vector-treated cells. The competition data are consistent with the MHC class I upregulation data of Fig. 2, where B8R protein competed with the extracellular domain of IFNGR-1 for binding to IFN- at its N terminus but failed to recognize the IFN- mimetics, which specifically bind to the cytoplasmic domain of IFNGR-1 (25). The binding data are also consistent with the secretion of B8R protein with the expression system used and the B8R protein recognition of intact IFN- but not IFN- mimetics.
EMC virus replication is known to be inhibited by IFN-. We therefore determined whether IFN- mimetics could inhibit EMC virus replication in WISH cells as well as the effect of B8R IFN--binding protein on such replication inhibition. Cells were either transfected with mimetic genes as described above for intracellular expression or treated with lipophilic mimetic for penetration of plasma membrane. Human IFN- was also added separately. Mimetic-transfected cells were also simultaneously transfected with the B8R expression vector. In addition, cells were treated with increasing concentrations of human IFN-, lipo-MuIFN-(95-133), or lipo-MuIFN- (95-125) 8 h after transduction with B8R-expressing vector. Cells were allowed to grow for 24 h and then challenged with a 0.1 multiplicity of infection of EMC virus, and the cytopathic effect was quantitated 24 h later by crystal violet staining and absorption measurement, as described previously (8). As can be seen in Fig. 4, cells transfected with a vector expressing MuIFN-(95-133) and HuIFN-(95-134) as well as those treated with lipo-MuIFN-(95-133) and intact IFN- protected WISH cells against EMC virus infection in a dose-dependent manner, while the empty-vector control, the peptide control, or B8R by itself did not have any protective effective against viral infection. Mimetic protection against EMC virus, either via transfection or via lipomimetic addition, was not affected by B8R transfection of the cells. IFN- protection, however, was inhibited by B8R, which is consistent with the anti-IFN- properties of the B8R IFN--binding protein. Thus, the IFN- mimetics possess antiviral activity against EMC virus that was not affected by the poxvirus virulence property of B8R protein.
We next determined the ability of the IFN- mimetics to inhibit replication of the rhabdovirus VSV and the effect of B8R protein on mimetic inhibition. EMC virus is a picornavirus, so the VSV experiment tests the generality of the antiviral properties of the mimetic as well as the effect of B8R protein on the antiviral activity. As can be seen in Fig. 5, WISH cells transduced with the MuIFN-(95-133) and HuIFN-(95-134) genes and those treated with lipo-IFN-(95-133) or intact human IFN- showed antiviral activity in a dose-dependent manner. Further, the mimetic antiviral activity was not blocked by B8R protein, while that of IFN- was blocked. We conclude that mimetics possess antiviral activity against VSV similar to that against EMC virus and that such activity was not affected by the poxvirus virulence factor B8R IFN--binding protein.
To test for the effect of the protein secreted by cells transduced with B8R-expressing construct on IFN- activity, supernatants from B8R expressing or control-vector-treated WISH cells were harvested and, in a second step, added to WISH cells in the presence of IFN- and then challenged with VSV. As shown in Fig. 6, IFN- activity was suppressed in the presence of supernatant from B8R-transduced cells, while supernatant from control-vector-transduced cells did not have any effect on IFN- antiviral activity. This demonstrated that the B8R gene expression resulted in secreted B8R protein that bound to and neutralized added IFN-.
The B8R protein produced in WISH cells was further characterized for its ability to bind to intact IFN- (Fig. 7). Cells were transduced with a B8R-expressing vector or an empty-vector control for 8 h. Cell extracts were electrophoresed on an acrylamide gel. Protein was transferred to a nylon filter and incubated with intact IFN-. Unbound IFN- was removed by washing, and the filter was probed with an antibody to IFN-. A band with an approximate molecular mass of 43 kDa, which corresponds to a dimer of B8R protein, was observed in B8R-expressing cells; its intensity decreased upon dilution. No such band was seen in the extracts of cells transduced with an empty-vector control. Thus, the cells transduced with the B8R expression vector secrete a protein with a molecular mass similar to the size of the B8R dimer (3).
Vaccinia virus is the prototype of the poxvirus family, and it has been used extensively in the study of the pathogenesis of this family of viruses (16). The IFN- mimetics were therefore examined for their antiviral effects against the Western Reserve strain of vaccinia virus, which expresses the intact B8R protein. WISH cells were first transduced with IFN- mimetic expression vectors or treated with lipo-MuIFN-(95-133), lipo-MuIFN-(95-125), or intact human IFN- followed by challenge with vaccinia virus. MuIFN-(95-133) and HuIFN-(95-134) mimetic peptides expressed intracellularly or by the addition of lipo-MuIFN-(95-133) conferred resistance to vaccinia virus infection, while intact human IFN- at 10 U/ml failed to protect WISH cells from vaccinia virus (Fig. 8). Empty vector control or the lipo-MuIFN-(95-125) did not exhibit any protection against vaccinia virus. These observations, combined with the B8R protein studies described above, indicate that human and murine IFN- mimetic peptides can circumvent the B8R IFN--binding protein virulence factor activity, providing proof of concept that IFN- mimetics are a potential approach to development of smallpox antivirals. Thus, the IFN- mimetics regulate virus replication differently from intact IFN-, suggesting unique antiviral properties of these mimetics.
DISCUSSION
Although smallpox was declared eradicated worldwide by the World Health Organization almost 20 years ago, recent events have generated renewed interest in developing new therapeutics for this class of virus. Smallpox or related viruses may be used in biological warfare, primarily through aerosolization (4). Since general vaccinations have been terminated for several decades, there is currently very low immunity in the general population, which leaves many vulnerable to infection. The likelihood of the emergence of new strains through natural recombination within the pox family of viruses, such as that seen in the recent outbreak of monkeypox, does exist (7). That the virulence of this family of viruses can be enhanced further was indicated by the recent demonstration that the introduction of interleukin 4 in the mousepox genome resulted in a virus that was 100% lethal, even in genetically resistant and previously immunized animals (12). Use of the existing vaccine, which is the same as that used 2 centuries ago, carries the risk of side effects, including encephalitis, myocarditis, and myopericarditis (11). Therefore, alternative antiviral treatments are being actively investigated.
Variola is the causative agent of smallpox. Vaccinia virus is a prototype of poxviruses and has been used as a vaccine to eradicate smallpox. It has been studied extensively for its pathogenesis and for its potential use as a gene delivery vehicle (16, 18). DNA sequencing has revealed that the genes common to vaccinia and variola viruses show greater than 90% similarity (10). Therefore, vaccinia virus represents the best system to study the effects of smallpox family of viruses. The genes responsible for viral replication are contained in the central region of the genome, while a large number of immune evasion genes are flanked on either side of this central region.
One of the immune evasion genes of vaccinia virus is the B8R gene, which codes for a soluble protein that resembles the extracellular surface of the IFN- receptor and can thereby neutralize the activity of IFN- from several species, in particular, human IFN- (24). The B8R protein exists as a homodimer naturally, and this apparently provides an advantage to the virus in the efficient binding and inhibition of IFN- in solution (3). It has been shown with rodent models that B8R is a virulence factor for vaccinia virus infection (24). Vaccinia virus vectors with an inactivated IFN- receptor homolog B8R gene are attenuated in vivo (32). An IFN mimetic that bypasses the IFN- receptor extracellular domain should also functionally attenuate the B8R gene. Further, the functional neutralization of B8R and the inhibition of the growth of vaccinia virus (or other poxviruses, such as smallpox) should help reduce the production of other poxvirus proteins that have an inhibitory effect on host defense. The other proteins encoded by vaccinia virus that interact with the IFN system (reviewed in reference 15) are as follows. E3L protein can bind to double-stranded RNA. K3L has structural similarity to the N terminus of eIF-2. VH1 can dephosphorylate STAT1. The B18R gene codes for a soluble IFN-/ receptor. We reason that if virus replication is inhibited by IFN- agonists/mimetics, then the vaccinia virus induction of other virulence factors will be inhibited. A similar situation exists with hepatitis virus C, which produces multiple proteins that can inactivate the IFN system (15), and yet IFN is the only therapy available to date to treat this disease.
We have previously shown that a polycationic sequence in the C terminus of human and murine IFN- is a nuclear localization sequence (NLS) and is responsible for the appearance of IFN- in the nucleus of treated cells (19, 20). Site-directed mutagenesis (2), or chemical synthesis of peptides (23), replacing positively charged amino acids with alanines resulted in loss of biological activity. While the N terminus of IFN- has been shown to bind to the extracellular receptor domains by peptide competition and crystallographic structure determination, no extracellular receptor binding site for the NLS-containing C terminus of IFN- has been demonstrated (reviewed in reference 13).
We have identified a high-affinity, species-nonspecific binding site (Kd, 10–8 M) for the IFN- C terminus on the cytoplasmic domain of both human and murine IFNGR-1 (9). This site, also demonstrable in whole cells, was identified as a membrane proximal region of the cytoplasmic domain within amino acid residues 253 through 287 (13). Consistent with this, we were able to demonstrate that peptides from the C-terminal NLS-containing domains of the human IFN-, HuIFN-(95-134), and the murine IFN-, MuIFN-(95-133), were sufficient to induce IFN--associated activities when internalized by murine macrophages (27). The intracellular peptide mimetics, which contain the NLS of IFN, were found to inhibit virus replication by 106- to 109-fold and upregulated MHC class II expression up to 10-fold in a fashion that depended strictly on the presence of the NLS in these peptide sequences (27). The peptide mimetics interacted exclusively with the cytoplasmic domain of IFNGR-1 within residues 253 through 287 and not with the extracellular region of IFNGR-1. An N-terminal -helix in these peptides within residues 108 through 121 was also found to be important for function by virtue of its requirement in the mimetics for binding to IFNGR-1 (amino acids 253 through 287) (26). Other studies showed that the peptides behaved as IFN- mimetics only when delivered intracellularly in cells and that their mimetic properties required the expression of IFNGR-1 in such cells showing the requirement of the cytoplasmic domain of IFNGR-1 (29, 30). HuIFN-(95-134) and MuIFN-(95-132) share approximately 80% homology and interact with the membrane-proximal IFNGR-1 cytoplasmic region (amino acids 253 through 287) in a non-species-specific manner (27). Therefore, the murine and human peptides were equally potent. The IFN- peptide mimetics, like intracellular IFN-, also were able to induce the activation and nuclear translocation of STAT1, as well as being able to induce the endocytosis and nuclear translocation of the IFNGR-1 subunit from within the cell.
Using the IFN- mimetic in in vitro binding assays and a standard in vitro nuclear transport assay system, we presented data to suggest that the IFN- NLS can play multiple roles in IFN- mimetic signaling. These include binding to the intracellular cytoplasmic domain of IFNGR-1 and simultaneous interaction with the nuclear import machinery to mediate the nuclear transport of IFNGR-1. Further, phosphorylated STAT1, which binds to phosphorylated IFNGR-1, is shown to be transported to the nucleus in an in vitro system by IFN-(95-133) and a phosphorylated IFNGR-1 subunit. Finally, the N-terminal -helix of the IFN- mimetic also plays an important role in IFNGR-1 binding and subsequent events. The mimetic data support the conclusion that the versatility of the C terminus of IFN-, involving both its -helix and its NLS, is important for IFN- mimetic-mediated STAT1 nuclear translocation and associated IFN--like biological activities. Details of the discussion on the mechanism of intracellular trafficking of IFN- and the IFN- mimetics are presented elsewhere (14, 21-23).
The presence of the N terminus receptor binding region in IFN- is key to its interaction with the receptor extracellular domain and the subsequent internalization and interaction with the cytoplasmic domain of the receptor subunit, IFNGR-1, via the IFN- C terminus domain (13). The failure to bind to the IFN- receptor extracellular domain prevents internalization and IFN- interaction with the IFNGR-1 cytoplasmic domain. The cytoplasmic interaction of IFN- via its C terminus is key to enhanced binding of JAK2 to IFNGR-1, its subsequent autophosphorylation, phosphorylation of IFNGR-1, and ultimately, phosphorylation of STAT1 for nuclear import and gene activation (13). The interaction of IFN- with B8R blocks the extracellular interaction and therefore subsequent events that result in induction of antiviral activity.
We have shown here that, in contrast to IFN-, the IFN- mimetics do not bind to the B8R protein, that the mimetics induce antiviral activity against EMC virus and VSV in the presence of B8R, and, finally, that mimetics inhibit vaccinia virus replication in the presence of endogenous B8R protein. An important conclusion is that the IFN- C terminus mimetics provide a proof-of-concept demonstration of antipoxvirus IFN- drugs that bypass the B8R virulence factor. The findings caution against focusing solely on the IFN- receptor extracellular domain in the development of IFN- antiviral drugs.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grant RO1AI056152 to H.M.J.
We gratefully acknowledge the Flow Cytometry Core Facility, University of Florida, for assistance with flow cytometry.
The paper is Florida Experimental Station Journal Series no. R-10766.
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