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Journal of Virology, June 2005, p. 7319-7326, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7319-7326.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the ß3-Integrin Receptor

Richard S. Larson,* David C. Brown, Chunyan Ye, and Brian Hjelle

Department of Pathology, Infectious Diseases and Inflammation Program, UNM School of Medicine, Albuquerque, New Mexico

Received 4 October 2004/ Accepted 11 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the {alpha}vß3 integrin, and transfection of human ß3 integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-ß3 antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified {alpha}vß3, we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 µg/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use ß3 integrins and PHV uses a different receptor, ß1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via ß3 integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The term hantavirus encompasses any of the more than 20 distinct agents within the genus Hantavirus in the family Bunyaviridae. All of the pathogenic hantaviruses are carried by one or two species of wild rodents of the family Muridae in any of the subfamilies Murinae, Arvicolinae (voles), or Sigmodontinae. About half of the known members of the genus Hantavirus are pathogenic; the rest are known through their isolation from or genetic detection in wild rodent specimens. Hantaviruses cause two human diseases: hemorrhagic fever with renal syndrome in the Old World and hantavirus cardiopulmonary syndrome in the New World (34).

All members of the viral family Bunyaviridae consist of enveloped spherical particles with a helical nucleocapsid and use a genome consisting of three negative-stranded or ambisense RNAs. The three fully negative-sense RNA segments of the hantaviruses are designated L (large; about 6,500 nucleotides [nt]), M (middle; about 3,600 to 3,700 nt) and S (small; 1,700 to 2,100 nt). The proteins they encode are an RNA-dependent RNA polymerase (RdRp, from the L segment mRNA); a glycoprotein precursor, GPC, that is processed into G1 and G2 transmembrane glycoproteins from the M segment mRNA; and a nucleocapsid protein/RNA chaperone (N), expressed from the S segment mRNA.

In vivo and in vitro, hantaviruses show tropism for the vascular endothelium (2, 6, 12-14, 43, 44). Hantavirus entry into cells is dependent upon the cell-surface expression of integrins bearing the ß3 subunit (10, 11). Perhaps paradoxically for these rodent-borne viruses, human integrins such as {alpha}vß3 confer susceptibility to infection to CHO (Chinese hamster ovary) cells, whereas their murine (Mus musculus) counterparts do not. The pathogenic hantaviruses examined thus far, which include Hantaan virus (HTNV), Seoul virus (SEOV), Sin Nombre virus (SNV), New York virus (NYV), and Puumala virus (PUUV), are able to enter cells that have been transfected by human {alpha}vß3, whereas two hantaviruses that have not yet been linked conclusively to human disease, Tula virus and Prospect Hill virus (PHV), do not (21). The entry of SNV and other pathogenic viruses can be prevented by treatment with neutralizing antibodies directed either against the virus or against the integrin receptor; entry is also inhibited by vitronectin but not by fibronectin. That antibodies against the ß3 subunit are capable of preventing hantavirus entry is particularly interesting because one such humanized monoclonal antibody, ReoPro, is a Food and Drug Administration-approved antithrombotic that is already widely available to clinicians.

The viruses that cause hantavirus cardiopulmonary syndrome have an unusual potential applicability as bioweapons because of their high lethality, known aerosol route of transmission, and lack of specific therapies (25). This potential threat could create demand for compounds that can be administered either prophylactically or therapeutically to victims of a bioweapon attack. Thus, there has been increased interest in the development of specific antiviral inhibitors against threat agents. One approach that has proven effective in reducing the morbidity of viral infections has been preventing the virus from entering into the host cells through their cell-surface receptors, such as through prophylactic or therapeutic administration of specific antiviral immunoglobulins. We sought to develop strategies for inhibiting viral entry based upon relatively inexpensive technologies that we hoped could be generalized to other threat agents. One approach we investigated was the development of compounds that mimic the known ability of anti-ß3 antibodies to prevent the entry of hantaviruses into cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and monoclonal antibodies. A phage display library expressing cysteine-constrained nonapeptides was purchased from New England Biolabs (Cambridge, MA). The octyl-ß-D-glucopyranoside-solubilized formulation of human integrin {alpha}vß3 was purchased from Chemicon International (Temecula, CA). The purity of {alpha}vß3 is >95% as verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using silver stain, and the functional integrity of the {alpha}vß3 formulation was verified by assays for binding to biotinylated fibronectin. ReoPro, the Fab fragment of the chimeric human-murine monoclonal antibody c7E3, was purchased from Eli Lilly and Company (Indianapolis, IN). ReoPro was suspended in 0.01 M sodium phosphate, 0.15 M sodium chloride, and 0.001% polysorbate 80, pH 7.2. Cysteine-constrained peptides were synthesized by Biopeptide (San Diego, CA). Peptides were resuspended at a concentration of 10 mM in phosphate-buffered saline (PBS) containing 5% dimethyl sulfoxide, pH 7.4, and stored at –80°C until use. The purity of the peptides was >95% as verified by high-performance liquid chromatography. The peptides were isolated using high-performance liquid chromatography, and the correct mass was confirmed by mass spectrometry. Vero E6 cells (ATCC CRL 1586) were purchased from the American Type Culture Collection (Manassas, VA). Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG) was purchased from Boehringer Mannheim (Indianapolis, IN).

Isolation of {alpha}vß3 binding phage. Phage display was performed as previously described with minor modifications, using a cyclic nonapeptide phage library (7). For the first round of panning, 0.5 ml of {alpha}vß3 at a concentration of 100 µg/ml in 0.1 M NaHCO3 (pH 8.6) buffer was coated onto 60- by 15-mm petri dishes (Nalge Nunc International, Naperville, IL) overnight in a humidified chamber at 4°C. The following day, the plates were blocked for 1 h at room temperature with 5 mg/ml bovine serum albumin in 0.1 M NaHCO3 (pH 8.6) buffer. The plates were washed six times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% [vol/vol] Tween 20). The phage library was added to the plate at a phage concentration of 2 x 1011 in 1 ml TBST. The plate was gently rocked for 1 h at room temperature. Unbound phage was removed by washing 10 times with 1 ml of TBST. Bound phage was then eluted by adding 500 µl of ReoPro (42 µM) for 1 h with gentle rocking at room temperature. The eluate containing the bound phage was removed and saved. Phage was amplified in Escherichia coli ER2738 bacteria and partially purified by polyethylene glycol precipitation. The binding, elution, and amplification steps were repeated using 100 µg/ml of {alpha}vß3. In the third and fourth rounds of panning, 1 ml of {alpha}vß3 at a concentration of 50 µg/ml and then 2 ml of {alpha}vß3 at a concentration of 10 µg/ml were coated onto the petri dish. In the final four rounds of panning, 3 ml of {alpha}vß3 at a concentration of 5 µg/ml was coated onto the petri dish. As a positive control, the phage library was also panned on streptavidin-coated dishes, and a peptide sequence known to bind streptavidin was obtained after five rounds [C-G-X-(F/Y/W)-(S/N)-H-P-Q-C] (7).

Nucleotide sequencing and sequence analysis. After selection and amplification of the phage library on {alpha}vß3, automated nucleotide sequencing derived the peptide sequence on the surface of the phage (DNA Research Services, Department of Pathology, University of New Mexico, Albuquerque). Double-stranded DNA was prepared according to the manufacturer's specifications using the QIAGEN QIAprep spin miniprep kit (Valencia, CA). DNA was amplified according to the manufacturers' specifications using the ABI Prism BigDye Terminator 3.1 kit (Applied Biosystems, Foster City, CA) and the –96 gIII sequencing primer (New England Biolabs, Cambridge, MA). The reactions were purified using Centrisep spin columns (Princeton Separations, Adelphia, NJ). The samples were then dried, resuspended in formamide, and denatured. Sequencing was performed on a Hitachi 3100 gene analyzer (Applied Biosystems, Foster City, CA).

Sequence alignments of each peptide to SNV strain SN77734 glycoproteins (6), Hantaan glycoprotein (40), and Prospect Hill virus (24) were done in a pairwise fashion. Alignments were performed using the Gap program, which is based on the algorithm of Needleman and Wunsch (23). We employed a Blosum62 scoring matrix which has been previously shown to be effective at detecting protein sequence similarity based on evolutionary rates and analyzed amino acid pairs in blocks of aligned protein segments rather than individually as in earlier Dayhoff rule-based programs (17). The gap shift limit for SNV glycoproteins was set at 12 and that for the peptide was set at 10. A quality score with a standard deviation was generated. The quality score for the alignment to any point is equal to the sum of the scoring matrix values of the matches in that alignment, less the gap-creation penalty times the number of gaps in that alignment, less the gap-extension penalty times the total length of all gaps in that alignment. We generated P values by performing 100 randomized alignments while preserving the amino acid composition (22). The t test was used to generate a P value for each peptide alignment to SNV.

Focus reduction test. We plated duplicate wells of 105 Vero E6 cells/well in minimal essential medium (MEM) (GIBCO, Grand Island, NY) with 10% fetal bovine serum onto Lab-Tek 16-well chamber slides (Fisher Scientific, Pittsburgh, PA) 24 h before beginning the assay. After washing the cells once with PBS, we added 108 to 1012 (serially diluted) transducing units of phage or a ReoPro dilution series in a volume of 50 µl directly to the now-confluent cells for 1 h at 37°C in MEM with 2.5% fetal bovine serum and then added 50 µl of SNV strain SN77734 (6) (500 to 1,000 focus-forming units/ml) in MEM-2.5% fetal calf serum and allowed the virus to incubate for 1 h at 37°C at a biosafety level 3 facility, Centers for Disease Control registration number C20041018-0267. We then removed the virus by aspiration, and after two washes in PBS, we replaced the media with MEM supplemented with 2.5% fetal calf serum. After 24 to 36 h, we fixed the cells in 300-µl/well ice-cold methanol/acetone (50:50) and then maintained the slide at 4°C before staining. We washed the cells twice with PBS before staining and then overlaid the cells with 100 µl of a 1:10,000 dilution of polyclonal rabbit anti-SNV recombinant N antibody (4) in PBS in a humidified chamber for 1 h at 37°C. We then added 50 µl of 1:500 FITC-conjugated anti-rabbit IgG in PBS, performed three washes in PBS, counterstained with 200 µl of 1:105 Evans Blue (wt/vol) for 3 to 5 min, and followed this by rinsing in water. After mounting the slides, we counted the foci by fluorescent microscopic examination. We used control wells containing no inhibitor to retrospectively verify the number of SNV foci that had been added to each well.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of {alpha}vß3 binding peptides that block entry of SNV. Repeated selection of phage from the cyclic nonapeptide phage display library resulted in the enrichment of phage that bound {alpha}vß3. We employed the Fab fragment of antibody c7e3 (ReoPro) to elute phage from the plate-bound {alpha}vß3, since this mAb has previously been shown to bind {alpha}vß3 and prevent SNV infection, implying that SNV and ReoPro might bind similar locations on {alpha}vß3 (9). As a result, we postulated that some nonapeptide-bearing phage might competitively bind the SNV-binding domain of {alpha}vß3. In addition, we eluted with mAb for 90 min in order to elute peptides with higher affinity and slower off rates (i.e., peptides likely to be potent inhibitors).

After six rounds of panning and amplification, the peptide sequences of individual plaques were deduced by determining the nucleotide sequences of the portion of the phage genome that encoded them (Table 1). The predicted peptide sequences of 72 plaques were determined. We arbitrarily sorted the peptides into four groups on the basis of their abilities to inhibit SNV entry (Table 1). Two peptide sequences were identified twice as independent clones, and both were in group 3 (CPPGKSSMC and CFPTTSRGC). Each phage was tested for its ability to block SNV infection in a focus reduction test, and their activity was compared to control media and concentrations of ReoPro that have been shown to result in maximal inhibition (10, 11). Group 4 contained 11 different peptide-bearing phages that showed greater peak inhibitory activity than did 80 µg/ml ReoPro (80 to 91% inhibition for the peptides versus 77% inhibition for ReoPro). Peptides with less than 30% inhibition (group 1) were not investigated further.


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  		TABLE 1. Inhibition with peptide-bearing phage

 
The 12 most potent peptides were further examined for their abilities to inhibit SNV entry in a dose-dependent manner (Fig. 1 and Table 2). The concentration of each peptide-bearing phage that produces 50% of its maximum potential inhibitory effect (IC50) was determined. IC50s ranged from 8 x 107 to 9 x 108 particles/µl (Table 2). ReoPro showed a dose response over a range from 1 to 25 µg/ml, but its activity was asymptotic at 80 µg/ml; a control phage bearing the sequence CQSLPTHNC (from group 1; Table 1) did not inhibit viral entry.



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  		FIG. 1. Focus reduction assay for Sin Nombre virus entry of Vero E6 cells. Dose-response curves showing the activities of four representative peptide-bearing phages in comparison to those of ReoPro and a phage bearing the control peptide CQSLPTHNC are shown.

 

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  		TABLE 2. IC50s of peptide sequences

 
Specificity of peptides homologous with SNV glycoproteins for inhibition of ß3-integrin-dependent hantavirus infection. In order to determine whether the peptides that bind {alpha}vß3 might have sequence similarity to SNV glycoproteins, we compared all of the 70 peptide sequences with those of SNV G1 and G2 (Fig. 2). Eight sequences among groups 2, 3, and 4 that had significant similarity were found, with alignment to discontinuous regions of G1 and G2 (P < 0.0001; Table 1; Fig. 2).



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  		FIG. 2. Alignment of peptide sequences with glycoproteins (G1 and G2) of SNV. Only those peptides showing statistically significant similarity (P ≤ 0.0001) with SNV G1 or G2 are shown above the glycoprotein sequence. The peptide sequences shown in bold are those that have inhibitory activities greater than that of 80 µg/ml ReoPro (Table 1; group 4). The proteolytic cleavage recognition site, WAASA, between G1 and G2, is boxed. HTNV and PHV sequences are shown below the glycoprotein sequence in areas where peptides have significant homologies (P < 0.0001) with either HTNV or PHV glycoprotein sequences.

 
We next compared the abilities of the peptides to inhibit SNV, HTNV, or PHV infection in a focus reduction assay. We tested two sets of peptides: one set represented peptides that displayed sequence similarity to the glycoproteins of SNV, and the other represented the 11 most potent peptide inhibitors of SNV entry (group 4).

Blocking of SNV entry into Vero E6 cells with the set of eight SNV-related peptides showed results similar to those shown in Table 1, with inhibition increasing from group 2 to group 3 and from group 3 to group 4 (Fig. 3A). HTNV infection was also inhibited by all eight of the peptides that showed sequence relatedness to SNV (39% to 85%) (Fig. 3B). The group 2 peptides inhibited HTNV infectivity (63 to 82%) to a greater degree than they inhibited that of SNV (39 to 59%). Two of the group 3 peptides (CPPGKSSMC and CHNLKPPTC) did not inhibit HTNV virus infectivity to the same degree as they inhibited SNV infectivity (77% and 79% for SNV and 39% and 60% for HTNV). In contrast to the relatively similar inhibition results for HTNV and SNV, PHV infectivity was not inhibited significantly (>30%) with seven of the eight SNV-related peptides (Fig. 3C). Notably, the peptide from group 2 (CEWTESSMMC) that inhibited PHV does not display significant similarity to PHV glycoproteins (data not shown).



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  		FIG. 3. Focus reduction assay demonstrating specificities of inhibition of ß3-integrin-dependent hantaviruses with peptide-bearing phage. Peptide sequences that were found to be significantly similar (P < 0.0001) to SNV G1 and G2 were tested for their abilities to block SNV (A), HTNV (B), or PHV (C) infection. Each peptide and its group (Table 1) is indicated. Virus (2,000 PFU) and 1 x 109 phage were added to each well. The degrees of inhibition are indicated, and standard deviations are shown. Each condition was repeated in duplicate. A nonblocking peptide bearing phage from group 1 (CQSLPTHNC) was used as a nonblocking control. ReoPro and fibronectin were used as blocking controls at final concentrations of 80 µg/ml and 40 µg/ml, respectively. Two types of buffer controls are shown: media is phage buffer without phage, and ReoPro buffer is the ReoPro phosphate buffer without ReoPro. The compositions of these buffers are indicated in Materials and Methods.

 
The 11 peptides from group 4 were also examined for their abilities to block SNV, HTNV, and PHV entry (Fig. 4). All 11 peptides inhibited SNV and HTNV to high levels (>67%) (Fig. 4A and B). In contrast, a generally much lower level of inhibition was seen with PHV (6%-71%), although high concentrations of 4 of the 11 peptides inhibited >50% of PHV infection (Fig. 4C). In both sets of experiments shown in Fig. 3 and 4, SNV and HTNV infections were inhibited by ReoPro, while PHV infection was inhibited by fibronectin, but not ReoPro, in agreement with observations made by others (10).



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  		FIG. 4. Focus reduction assay demonstrating inhibitory activities of the peptides with the greatest activities against SNV. The 11 peptides (group 4) that were most potent for the inhibition of SNV (A) in a focus reduction assay were tested in parallel for their abilities to inhibit infection by HTNV (B) and PHV (C). Virus (2,000 PFU) and 1 x 109 phage were added to each well. The degrees of inhibition are indicated, and standard deviations are shown. Each condition was repeated in duplicate. ReoPro and fibronectin were used as blocking controls at final concentrations of 80 µg/ml and 40 µg/ml, respectively. Two types of buffer controls are shown: media is phage buffer without phage, and ReoPro buffer is the ReoPro phosphate buffer without ReoPro. The compositions of these buffers are indicated in Materials and Methods.

 
In order to determine whether any of the eight SNV-related peptides had significant sequence similarity to HTNV or PHV as well, we compared the sequences of these eight peptides with HTNV and PHV. Only two of the eight peptides had significant sequence identity (P < 0.0001) with either HTNV or PHV at the site analogous to that in SNV (Fig. 2). No peptide with significant sequence similarity to HTNV and SNV but not PHV was identified.

Potency of synthetic peptides alone and in combination. We next chemically synthesized four cyclic peptides from group 4: the three that showed the most inhibition (CPFVKTQLC, CLHKPWSRC, and CRSLTDNQC) and the most potent that had significant sequence similarity to SNV surface glycoproteins (CPGHIHRTC) (Table 1). These four peptides were screened for their abilities to inhibit SNV entry in vitro (Fig. 5). At concentrations of 2 mM, the peptides were able to inhibit infectivity by 51%, 15%, 26%, and 14%, respectively. This degree of inhibition was more than those of control phage (CQSLPTHNC) or peptide (CLLRMRSIC). Nonetheless, three of the four peptides displayed quite modest levels of inhibition (<30% with CLHKPWSRC, CRSLTDNWC, and CPGGHIHRTC). We anticipated that peptides may have a shorter half-life in the presence of serum due to the proteases in the serum. For this reason, we performed the assay both in the presence and in the absence of serum and observed no difference in their abilities to inhibit SNV infection (data not shown).



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  		FIG. 5. Focus reduction assay demonstrating the inhibitory activities of peptide-bearing phage and selected synthetic peptides. Phage, peptides, and ReoPro were adsorbed onto confluent monolayers of Vero E6 cells (ATCC CRL 1586) on microscope slides for 1 h before the addition of 50 to 100 immunofluorescence assay foci of SNV strain SN77734; after 24 to 36 h, we stained the monolayers with polyclonal rabbit anti-SNV N antibody (1:1,000) followed by FITC-conjugated anti-rabbit IgG and counted foci. The percentages of inhibition using final concentrations of 1 x 109 phage/µl or 2 mM peptide are shown. Peptides were cyclized by the reduction of flanking cysteines at residues 1 and 9 before addition. Maximal peptide concentrations were chosen based upon their maximal solubilities. ReoPro was used at a final concentration of 80 µg/ml. Three types of buffer controls are shown: media is phage buffer without phage, PBS is phosphate-buffered saline without peptide, and buffer is the ReoPro phosphate buffer without ReoPro. The compositions of these buffers are indicated in Materials and Methods.

 
We next examined whether using peptides in combination could have additive effects (Fig. 5). The relatively high potency of CPFVKTQLC was not increased when combined with any of the other three peptides. However, combinations of peptides CLHKPWSRC and CRSLTDNWC (50%) or CRSLTDNWC and CPGHIHRTC (40%) showed enhanced inhibition in comparison to each of the three peptides alone (P < 0.05). The combination of all four in comparison to CPFVKTWLC alone did not result in a statistically significant increase in inhibition (P > 0.05). These findings indicate that among synthesized peptides, CPFVKTQLC is the most potent, but combinations of CLHKPWSRC and CRSLTDNWC or of CRSLTDNWC and CPGHIHRTC may approximate the same level of inhibition as that of CPFVKTWLC alone.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of combinatorial peptide libraries displayed on the surfaces of filamentous bacteriophage offers an efficient means of obtaining a diverse set of peptides that bind a target protein. In this study, we identified peptides that bind {alpha}vß3 and that inhibit SNV entry. A combinatorial library generated 70 different peptide sequences that bind {alpha}vß3 and were eluted by the Fab fragment of c7E3 mAb (ReoPro). Further testing showed that 11 of the 70 peptide sequences had inhibitory abilities that exceeded that of ReoPro (even when used at 80 µg/ml) when expressed on the phage surface. In addition, even though no virus was used to identify the peptides, 8 of the 70 peptides proved to have significant sequence relatedness to SNV glycoproteins. We compared all 19 peptide sequences (11 with maximum inhibitory ability and 8 homologous peptides) for their abilities to inhibit SNV, HTNV, and PHV infections. There was a distinct difference in the abilities of these peptides to inhibit SNV and HTNV in comparison to PHV, which supports the contention that SNV and HTNV use ß3 integrins and PHV uses a different receptor, ß1 integrin, to enter cells (10). We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV and showed that they could, singly and in combination, inhibit SNV infection when added as free peptides outside of the context of a phage. These peptides inhibited viral entry in vitro, albeit to a lesser degree than did phage expressing the same peptide. Thus, we have identified novel peptides that primarily inhibit infection by pathogenic hantaviruses (SNV and HTNV) and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

All of the evidence that hantaviruses such as SNV or HTNV require ß3 integrin to enter cells is based upon focus assays that measure downstream evidence that entry has occurred (8, 9, 31). Gavrilovskaya et al. (8, 9) first demonstrated that pathogenic hantaviruses appear to require expression of the human ß3 integrin in order to efficiently enter cells. Until the present work, this discovery had not been confirmed in other laboratories. Although it is possible to attribute the requirement for ß3-integrin expression to a direct binding interaction between SNV or HTNV and ß3 integrin, it had also been possible that ß3-integrin expression provided an unknown signal to cells that preconditions them in other ways for hantavirus entry via another surface receptor. Since the inhibitory peptides described here were identified by competition between a monoclonal antibody and {alpha}vß3, our findings support the concept that SNV is interacting directly with ß3 integrin.

There were clear differences in the levels of peptide inhibition among SNV, HTNV, and PHV, consistent with the concept that HTNV and SNV are using ß3 integrin for cell entry, while PHV uses ß1 integrin. In this context, it is interesting that some of the peptides block all three viruses. ß1 and ß3 integrin are highly homologous proteins (44% identity) (1) that have similar tertiary structures (41, 42). Furthermore, many members of the ß1- and ß3-integrin families bind identical ligands, although with different affinities (5, 15, 16, 42). Finally, peptide sequences containing RGD sequences have been described that block native ligand binding to both ß1 and ß3 integrins (18, 26-30, 32, 38). Thus, there is strong evidence that the ß1 and ß3 receptors are structurally related, can bind similar ligands, and can be inhibited by similar peptide antagonists. It is perhaps not surprising that viruses from the same group would bind to related integrin receptors and that binding to different integrins may be inhibited in some cases by peptides similar to those seen in our study. Nonetheless, understanding these fine structural differences will likely be an important contribution to further understanding the pathophysiologic differences among hantaviruses.

In this study, we identified peptides that block virus entry by competing phage with a mAb and the virus receptor. We chose this approach since we had previously used it successfully to identify inhibitory peptides of other integrin-ligand pairs (35-37). It is unclear what epitope on ß3 integrin is recognized by ReoPro, and it is likely that antibodies are large enough to prevent interactions with regions well beyond the antibody's own epitope or the virus binding site on ß3. As a result, when competing with a mAb, it is likely that peptides are identified that compete with the mAb but not the virus, as seen in our study. In fact, 10 of 11 peptides (group 4) with the greatest inhibition of SNV infectivity did not have significant sequence relatedness to SNV glycoproteins. Identification of inhibitory peptides that do not have significant sequence relatedness to native ligands has been described in other systems (35-37), and a large amount of work has indicated that nonhomologous amino acids may mimic the binding activities of each other (reviewed in reference 35). In addition, a comparison of the peptide sequences homologous with SNV to HTNV and PHV glycoproteins did not clearly identify possible contact points of virus glycoproteins with their receptors. The involvement of contact points between SNV glycoproteins and ß3 would be suggested if the peptides were found to bear amino acid sequence relationships to both SNV and HTNV glycoproteins but not to PHV glycoproteins. However, only one of the eight peptides that were homologous with SNV glycoproteins showed significant similarity with HTNV, although all eight blocked HTNV infection. Two of the eight peptides had sequence similarity to PHV, yet neither of these blocked PHV infection. While this analysis does not demonstrate that any of our eight peptides with sequences similar to those of SNV glycoproteins represent contact points between SNV and {alpha}vß3, the large number of peptides with statistically significant homology raises the possibility that at least a subset of sequences may represent such contact points. Further studies will be needed to determine whether the peptides we have identified can exhibit additive inhibitory effects on viral entry.

The factors that could contribute towards the inhibitor potential of a peptide expressed on phage are cyclicity, multivalency, steric hindrance due to size, and the possible conformation-stabilizing and -forming effects of the phage environment. All the peptides that we tested in this study were disulfide restrained in a cyclic form and were expressed on the pIII protein of the phage, indicating that there may be up to five copies of peptide per phage (8). As a result, the multivalency of the peptide on the phage may contribute to the significant improvement in the maximal inhibitory ability of peptide expressed on phage in comparison to that of synthesized peptides. Others have observed this phenomenon and have alternatively proposed that the phage environment provides conformation or structural stability to the peptides (19, 20, 33). Finally, the large size of the phage could contribute additional steric hindrance and cause accentuated inhibition of SNV binding with the cell surface receptor on that basis.

Although we identified peptides that in the context of presentation on the surface of phage were substantially more effective at blocking hantavirus entry than was an antibody to {alpha}vß3, our findings agree with those of others that inhibitors that bind {alpha}vß3 do not completely block hantavirus entry. In addition, even a combination of four inhibitory peptides showed only a 61% inhibition (Fig. 5). These findings are in agreement with those of other investigators, who have shown that even combinations of anti-{alpha} and anti-ß subunit antibodies cannot further improve the inhibition of SNV entry (10). Although some residual entry of viruses is nonsaturable and likely to be nonspecific, our study as well as observations made by others suggests that the glycoproteins of hantaviruses are highly folded and may have multiple discontinuous epitopes (39).

Hantavirus interactions with {alpha}vß3 integrins provide a potentially contributory mechanism for the observed alterations in vascular permeability during infection and also provide potential targets for therapeutic intervention. ReoPro is a humanized mouse-derived Fab fragment from the original monoclonal antibody c7E3, which recognizes both {alpha}iibß3 and {alpha}vß3 integrins and is used therapeutically to inhibit platelet aggregation. However, mAb therapy using even humanized antibodies may have significant side effects when compared to the side effects seen in patients after peptide administration (3, 9). This observation motivated our efforts to identify small peptides that may inhibit SNV infection. The peptides that we identified in this study may serve as lead compounds for further optimization. The optimization of peptide antagonist activity through alanine and homologous amino acid substitution has resulted in 2- to 3-log improvements in inhibitory activity in other systems and may be justified in this system as well (35, 37). Finally, structural information about peptides may be used as a guide to create orally available nonpeptide organics.


   ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grants R21 AI53334, R21 AI053400, and UO1 AI56618.


   FOOTNOTES
 
* Corresponding author. Mailing address: UNM School of Medicine, 2325 Camino de Salud, CRF 223, Albuquerque, NM 87131. Phone: (505) 272-9762. Fax: (505) 272-5186. E-mail: rlarson{at}salud.unm.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, June 2005, p. 7319-7326, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7319-7326.2005
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