INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Penetration of enveloped viruses into host cells involves the fusion of viral and cellular membranes and the subsequent transfer of genetic material into the target cell (1-3). Some of the enveloped viruses, including single-stranded RNA viruses such as influenza viruses, and alphaviruses, are internalized into the target cell by receptor-mediated endocytosis at acidic pH (4). However, the viral envelopes of e.g. paramyxoviruses and retroviruses including human immunodeficiency virus, fuse directly with the cellular plasma membrane at neutral pH (2, 5).

Sendai virus, a member of the paramyxovirus family, contains two types of glycoproteins, hemagglutinin neuraminidase and fusion (F) glycoprotein (5, 6). Although the hemagglutinin neuraminidase glycoprotein is responsible for the attachment of virions to the receptor of the target cell (7, 8), it may also play some as yet undefined role in the fusion process (9). The fusion glycoprotein is believed to disrupt the target cell membrane and induce membrane fusion. The fusion protein monomer, synthesized as an inactive precursor (F0), is cleaved by a host proteolytic enzyme to form the biologically active protein consisting of two disulfide-linked subunits, F1 and F2 (7, 8, 10-12). This processing exposes a hydrophobic region at the N terminus of the F1 subunit, which is highly conserved among paramyxovirus F proteins. This domain, designated "fusion peptide", is considered to be directly involved in promoting fusion with the target membrane (12, 13). Evidence supporting this hypothesis includes: (1) site-directed mutagenesis in the fusion peptide regions of several enveloped viruses including influenza virus (1, 14), HIV¹ (15, 16), simian immunodeficiency virus (17), and SV5 (18), which severely affect the fusogenic activity of the corresponding viruses; and (2) studies with synthetic peptides that mimic the fusion peptide region of several viruses including influenza virus (19, 20), HIV (21-23), simian immunodeficiency virus (24), and Sendai virus (25). In addition, the photoactive lipid probe preferentially labeled the fusion peptide domain of influenza hemagglutinin (26). The role of the F2 subunit in the paramyxovirus-mediated membrane fusion process is not yet clearly understood. Reduction of disulfide bonds on the F1-F2 fusion protein results in loss of fusion activity, which may mean that F2 is required for the function of the fusion protein (27).

Although the role of the fusion peptide of the viral envelope glycoprotein has been substantiated, it is difficult to envision that the entire fusion process is determined solely through the small N-terminal region of the fusion protein. Indeed, recent reports point to the possible involvement of heptad repeats located adjacent to the fusion peptide region and the transmembrane anchor domain in the mediation of membrane fusion. Heptad repeats have been assumed to play a role in the assembly of the fusion protein of the influenza virus (28), HIV (29), and Sendai virus (30). Point mutations in the heptad repeats of the fusion proteins of the measles virus (31), the transmembrane gp41 of HIV (32, 33), and Newcastle disease virus (34) severely affect viral activity. Furthermore, synthetic peptides corresponding to heptad repeats of HIV (35-37), Sendai virus (30), and several paramyxoviruses (38) can inhibit viral-induced cell fusion by their corresponding viruses.

Hydropathy plot of Sendai virus fusion protein shows that in addition to the N-terminal fusion peptide and the transmembrane anchor domain, there are other significantly hydrophobic regions within the ectodomain of the fusion protein (39). This may indicate that additional regions may be involved with the membrane fusion process. To study this possibility, we selected SV-201 (amino acids 201-229), a domain with intermediate hydrophobicity, preceding the N-terminal heptad repeat and which potentially can form an amphipathic -helix structure. This region is not only extremely conserved in the Sendai virus family but also has appreciable amino acid homology with the corresponding regions of other paramyxoviruses (Fig. 1). We have synthesized SV-201 and two mutant peptides, Mu1SV-201 and Mu2SV-201. In Mu1SV-201, 2 conserved amino acids were substituted by 2 negatively charged amino acids to retain the peptide with a zero net charge, and in Mu2SV-201, 7 amino acids at the N terminus were randomized keeping the hydrophobicity of the peptide unaltered. We found that SV-201 but not the two mutants and the hydrophobic 33-amino acid fusion peptide of the Sendai virus was a potent inhibitor of virus-induced hemolysis. SV-201 inhibited lysis only when it was incubated with the virions prior to, but not after, their attachment to red blood cells. However, both the wild type and mutant peptides could not inhibit virus-mediated agglutination of human red blood cells. These results, together with data obtained using fluorescently labeled wild type and mutant peptides, are discussed in terms of the possible role of the SV-201 domain of the Sendai virus fusion protein in the assembly of the fusion protein during the initial step of the fusion process and in membrane apposition.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Butyloxycarbonyl-amino acid phenylacetamidomethyl resin and dimethylformamide (peptide synthesis grade) were purchased from Applied Biosystems (Foster City, CA), and butyloxycarbonyl-amino acids were obtained from Peninsula Laboratories (Belmont, CA). Other reagents for peptide synthesis included trifluoroacetic acid (Sigma), N,N-diisopropylethylamine (Aldrich, distilled over ninhydrin), dicyclohexylcarbodiimide (Fluka), and 1-hydroxybenzotriazole (Pierce). Egg phosphatidylcholine (PC) and phosphatidylserine (PS) from bovine spinal cord (sodium salt, grade I) were purchased from Lipid Products (South Nutfield, United Kingdom). NBD-F (4-fluoro-7-nitrobenz-2-oxa-1,3-diazole) was obtained from Molecular Probes (Eugene, OR). All other reagents were of analytical grade. Buffers were prepared in doubly glass-distilled water.

Peptide Synthesis, Fluorescent Labeling, and Purification-- The peptides were synthesized by the solid phase method on the corresponding resin (0.15 meq) as described previously (40, 41). Double coupling was carried out with freshly prepared hydroxybenzotriazole-active esters of butyloxycarbonyl-amino acids. Labeling of the N terminus of a peptide was achieved as reported previously (42). Briefly, 15 mg of a resin-bound peptide in its fully protected form was treated with trifluoroacetic acid (50% v/v in methylene chloride) to remove the butyloxycarbonyl-protecting group from the N-terminal amino groups of the linked peptides. The resin-bound peptides were then treated with either (i) tetramethylrhodamine succinimidyl ester (3-4 eq) in dry dimethylformamide containing 5% v/v diisopropylethylamine or (ii) NBD-fluoride (2-3 eq) in dry dimethylformamide, which led to the formation of resin-bound N¹-Rho or N¹-NBD peptides, respectively. After 48 h, the resins were washed thoroughly with N,N-dimethylformamide and then with methylene chloride. The peptides were then cleaved from the resins by hydrogen fluoride and finally precipitated with ether. All the peptides were purified using reverse phase-high performance liquid chromatography on a C₄ reversed phase Vydac column (300 Å pore size). The column was eluted in 40 min using a linear gradient of 25-80% acetonitrile in water (containing 0.05% trifluoroacetic acid (v/v)), at a flow rate of 0.6 ml/min. The purified peptides were shown to be homogeneous (~99%) by analytical high performance liquid chromatography. The peptides were subjected to amino acid analysis.

Preparation of Small Unilamellar Vesicles-- Small unilamellar vesicles were prepared by sonication of PC or PS/PC (1:1 w/w) as described previously (43). Briefly, dry lipids were dissolved in CHCl₃:MeOH (2:1 v/v). The solvents were evaporated under a stream of nitrogen, and the lipids were resuspended in PBS buffer (at a concentration of 7.2 mg/ml) by vortex mixing. The resulting lipid dispersions were sonicated (10-30 min) in a bath-type sonicator (G1125SP1 Sonicator, Laboratory Supplies Company Inc., NY) until the turbidity had cleared. The lipid concentrations of the resulting preparations were determined by phosphorus analysis (44). Vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan) by depositing a drop of vesicles on a carbon-coated grid and negatively staining with uranyl acetate. Examination of the grids demonstrated that the vesicles were unilamellar with an average diameter of 20-50 nm.

Virus and Erythrocytes-- Sendai virus (Z strain) was a generous gift of Prof. Michael Ovadia from Tel Aviv University, Israel. The virus was resuspended in buffer composed of 160 mM NaCl, 20 mM Tricine, pH 7.4, and stored at 70 °C. Human blood was obtained from a blood bank and used fresh. Prior to use, erythrocytes were washed twice with PBS, pH 7.3, and diluted to the desired concentration with the same buffer.

Sendai Virus-induced Hemolysis of Human RBCs and Its Inhibition by SV-201-- Virions, erythrocytes, and peptides were mixed in different orders of addition and in various amounts. Briefly, peptides were added at different concentrations to virions (35 hemagglutination units) and erythrocytes (2.2%) by three different methods: (i) Peptides were added to virions suspension followed by incubation (20 or 80 min, room temperature) to enable the binding of peptides to virions. Erythrocytes were then added and the suspension further incubated for 10 min. (ii) Virions were incubated with erythrocytes in an ice bath for 30 min followed by the addition of peptides, and further incubated for 20 min. (iii) Peptides were incubated with erythrocytes for 10 min at room temperature followed by the addition of virions, and further incubated for 20 min. At the end of each one of the three experiments, final incubation was always at 37 °C for 40 min, followed by centrifugation at 5700 × g for 8 min to remove intact cells. In all assays, duplicate samples were used, and two aliquots taken from the supernatant of each sample were placed in two wells of a 96-well plate. Each experiment was repeated 2-4 times. The amount of hemoglobin released was monitored by measuring the absorbance of the wells by using a plate reader at 540 nm.

Hemagglutinin Assay-- Hemagglutinating activity of Sendai virus in the presence and absence of peptides was determined using fresh human RBCs in a 96-well microtiter plate by standard methods (27, 45, 46). In a control experiment, 100 µl of 4% RBC were added to 100 µl of Sendai virus suspension. In another control experiment the same amount of RBCs was added to solutions of SV-201, Mu1SV-201, and Mu2SV-201 in PBS (concentrations ranging from 0 to 15 µM). In the actual experiment, Sendai virions were incubated with SV-201, Mu1SV-201, Mu2SV-201, and the fusion peptide (0-15 µM each) for 80 min to allow the binding of peptides to virions, followed by the addition of 4% RBCs (100 µl) to each suspension. One hour after the addition of RBCs, the degree of agglutination was observed as described by others (45, 46). The agglutinated RBCs settled in the bottom of the wells in aggregates to form a uniform carpet of cells, whereas unagglutinated RBCs formed a compact button of cells. All the experiments were done in duplicate using 2% RBCs and 35 hemagglutination units Sendai virions in a volume of 200 µl.

NBD and Rhodamine Fluorescence Measurements-- Fluorescence emission spectra of NBD- and Rho-labeled peptides either in PBS or in the presence of lipid vesicles were recorded at room temperature on a Perkin-Elmer LS-50B spectrofluorometer with the excitation monochromator set at 467 and 530 nm, respectively, with a 5-8-nm slit width. Measurements were performed in a 0.5-cm path length glass cuvette in a final reaction volume of 0.4 ml.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

We report the identification, synthesis, biological function, and possible site of action of a 29-residue peptide, designated SV-201, derived from a conserved region in the ectodomain of Sendai virus fusion protein (amino acids 201-229). Fig. 1 depicts the sequence, designation, and location of SV-201 in the F1 domain of the fusion protein. A possible structure of this sequence could be an amphipathic -helix, which could be involved in the assembly of proteins in solution as well as within membranes (47). A Shiffer and Edmundson wheel projection of the 18 most conserved amino acids of SV-201 reveals that the peptide has the potential to form an amphipathic -helical structure (Fig. 2, inner circle), in which most of the hydrophobic amino acids are segregated on one surface with the hydrophilic amino acids on the opposite surface. Indeed, CD studies showed that SV-201 has ~70% -helical structure in trifluoroethanol (48), a solvent which stabilizes the helical structure. Fig. 2 also shows the wheel structure of the three homologous regions of three other viruses. Interestingly, the most conserved regions face the hydrophobic surface. In addition, a net positive charge is conserved in the hydrophilic surface. The two mutant peptides, Mu1SV-201 and Mu2SV-201, served as controls (Fig. 1). In Mu1SV-201 a conserved glycine at the hydrophobic face (position 207) and a positively charged arginine in the hydrophilic face (position 205) were substituted with 2 negatively charged amino acids, rendering the peptide neutral. In Mu2SV-201, 7 amino acids at the N terminus were randomized, thus maintaining the same amino acid composition as in SV-201. The hydrophobic fusion peptide (25) served as a third control. CD experiments showed that Mu1SV-201 and Mu2SV-201 adopt 61 and 60% -helical structure, respectively, in trifluoroethanol indicating that the mutations did not alter significantly the secondary structure.² The fusion peptide has been shown previously to adopt a predominantly -helical structure (25). The peptides were then labeled with the fluorescent probes rhodamine and NBD to study their ability to assemble in solution and to bind phospholipid membranes.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A, sequence alignment of SV-201 (amino acids 201-229) of Sendai virus with homologous regions of Rinderpest, measles, and parainfluenza virus. Conserved residues are underlined. B, schematic representation of the F1 domain of the fusion protein of the Sendai virus; C, designations and sequences of the peptides used. Mutated amino acids are underlined.

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Shiffer and Edmundson wheel projections of 18 (amino acids 5-22) most conserved amino acids of SV-201 and the corresponding domains of Rinderpest, measles, and parainfluenza viruses (from inside to outside). Shaded areas represent hydrophobic amino acids.

SV-201 Is a Potent Inhibitor of Sendai Virus-mediated Hemolysis Only If Added Prior to the Attachment of the Virus to Erythrocytes-- The hemolytic activity of Sendai virus is associated with fusion of the virus with target RBCs (49). The assay was performed as follows. Virions were initially incubated with RBCs at room temperature to allow their attachment to the cells. Subsequent incubation at 37 °C resulted in RBCs lysis. The extent of lysis was measured by absorbance at 540 nm (characteristic of hemoglobin). Fig. 3 depicts the inhibitory effect of SV-201, Mu1SV-201, Mu2SV-201, and the fusion peptide on Sendai virus-mediated hemolysis. Column A shows the absorbance measured for a control experiment in the absence of a peptide. The hemolytic activity of the Sendai virus was drastically reduced when the same amount of virions was pretreated with SV-201 (4 µM) prior to the addition of RBCs (column B). The absorbance measured was only 23% of controls, indicating 77% inhibition of Sendai virus-induced hemolytic activity. In contrast, Mu1SV-201 (4 µM, column C) and the fusion peptide (4 µM, column D) did not exhibit any inhibition activity, and Mu2SV-201 (4 µM, column E) exhibited very little inhibition. Interestingly, by changing the order of addition of peptide and virions, SV-201 lost its inhibitory effect. If RBCs were first added to virions and incubated for 30 min to permit viral attachment to the RBCs, and then SV-201 was added, no inhibition was observed (column F). Thus, SV-201 could inhibit Sendai virus-induced hemolytic activity only if interacted first with virions. Column G shows that SV-201 (4 µM) also has no inhibitory effect when incubated with RBCs before virions were added. Since ~70% of the peptide remains free in the solution in the presence of RBCs (data not shown), the attachment of virions to RBCs is probably kinetically faster than binding of SV-201 to virions. It should be noted that the wild type, mutant, and fusion peptides had no hemolytic activity up to the maximum concentration tested (100 µM) (data not shown), thus eliminating the possibility that they contribute to the hemolytic activity during the fusion process. Overall, these results suggest that SV-201 inhibits Sendai virus-mediated hemolytic activity by interfering with some of the steps that take place during the attachment of virions to the target cells.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   The inhibition potential of wild type SV-201, the mutants Mu1SV-201 and Mu2SV-201, and the fusion peptide under various conditions as measured by absorbance at 540 nm. All experiments were done in duplicates in two to four separate experiments and the concentrations of peptides, Sendai virus, and RBCs were fixed at 4.0 µM, 35 hemagglutination units, and 2.2% (v/v), respectively. The final incubation was always at 37 °C for 40 min, followed by centrifugation at 5700 × g for 8 min. A, a control experiment. Virions were added to 100 µl of PBS followed by the addition of 125 µl of 4% RBC and incubated for 80 min at room temperature. SV-201 (B), Mu1SV-201 (C), fusion peptide (D), and Mu2SV-201 (E) were added to virions in 100 µl of PBS and incubated for 80 min at room temperature (similar results were obtained following incubation at 4 °C). 125 µl of RBC (4%) were then added and incubated for 10 min. Virions were added to 225 µl of 2.2% RBC at 4 °C and incubated on ice for 30 min (F). SV-201 was then added and incubation continued for 20 min at room temperature. Similar results were obtained at an incubation time of 80 min and therefore are not shown. Wild type SV-201 was added to 225 µl of 2.2% RBC and incubated for 10 min at room temperature (G). Virions were then added and incubated for an additional 20 min.

A Possible Mechanism of Inhibition-- The results of Fig. 3 suggest that SV-201 can interfere with a functional domain of the Sendai virus fusion protein only when it is allowed to interact with the virions before the addition of RBCs. To search for a possible mechanism for the inhibitory effect of SV-201, hemagglutinin activity of Sendai virions was examined in the presence of the peptides. Moreover, the wild type SV-201 and the two mutants were tested for their ability to self associate, to coassemble in solution, and to bind phospholipid membranes.

SV-201 Does Not Inhibit the Binding of Sendai Virions to Target Red Blood Cells-- Sendai virus-induced hemagglutinin assay was performed in the presence and absence of the peptides to examine whether they disturb the binding of virions to target RBCs. Binding of Sendai virions to human red blood cells causes their agglutination (27, 50, 51). In a control experiment agglutination of human red blood cells was readily observed upon their incubation with virions. In another control experiment the same amount of RBCs was added to solutions of SV-201, Mu1SV-201, Mu2SV-201, and the fusion peptide in PBS (in the concentration range used for the inhibition assay) to examine whether the peptides themselves agglutinate RBCs. No hemagglutination was observed, which ruled out hemagglutinin activity of the peptides (Table I). The peptides (0-15 µM, in duplicates) were then incubated with virions for 80 min to allow their binding to the virions, followed by the addition of RBCs and incubation for 1 h. We observed that Sendai virions could agglutinate the RBCs in the presence of SV-201, Mu1SV-201, Mu2Sv-201, and the fusion peptide to the same extent observed in the absence of the peptides (Table I). These results reveal that SV-201, Mu1SV-201, Mu2SV-201, and the fusion peptide do not disturb the binding of virions to target RBCs.

SV-201, but Not the Mutant Peptides, Self-Associates in Solution-- Viral fusion proteins are oligomeric in both native and fusogenic states (52). To examine the possible involvement of the 201-229 amino acid domain in the assembly of the fusion protein in the native state, the assembly of SV-201, Mu1SV-201, and Mu2SV-201 were studied in aqueous solution. For these measurements, the peptides were labeled at their N termini by either rhodamine or NBD. Identical results were obtained with both fluorescent probes, and therefore, only the data with the rhodamine-labeled peptides are presented. Since the fluorescence of rhodamine is quenched when several molecules are in close proximity, an increase in fluorescence is expected when an aggregated rhodamine-labeled peptide dissociates, a process that occurs when the peptide is cleaved by a proteolytic enzyme. When equal concentrations of Rho-SV-201, Rho-Mu1SV-201, and Mu2SV-201 (0.10 µM each) were dissolved in methanol, a solvent which does not promote peptides' aggregation, the three peptides exhibited the same fluorescence as expected (data not shown). However, in PBS the fluorescence intensity of Rho-SV-201 was much lower than that of Rho-Mu1SV-201 and Rho-Mu2SV-201 (Fig. 4, time point 2), suggesting that the former is in a higher oligomeric state than the other two peptides. Fig. 4 shows the time response profile of the changes of rhodamine fluorescence upon the addition of proteinase K. The data revealed that upon addition of proteinase K (time point 2), the fluorescence of Rho-SV-201 increased drastically (5 times), whereas that of Rho-Mu1SV-201 and Rho-Mu2SV-201 changed only slightly. It should be noticed that both Rho-labeled mutants dissociate when they are diluted in PBS, as evidenced from the spontaneous dequenching of rhodamine fluorescence prior to the addition of proteinase K. Eventually, the wild type and mutant Rho-labeled peptides (0.10 µM each) exhibited very similar fluorescence after degradation with proteinase K. This indicates that the lower fluorescence of Rho-SV-201 in PBS is due to its oligomerization in aqueous solution. The finding that Mu2SV-201 is not self-associated in solution although it has the same amino acid composition and similar structure as that of the wild type peptide indicates that the self-association of SV-201 is sequence specific and not due to its hydrophobic nature.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Determination of the aggregation state of the peptides by enzymatic digestion of rhodamine-labeled peptides. Rho-SV-201 (), Rho-Mu1SV-201 (- - - -), and Rho-Mu2SV-201 (... . .) were dissolved in PBS (0.1 µM final concentration) at time point 1, and proteinase K (0.5 mg/ml) was added at time point 2.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Detection of assembly of SV-201, Mu1SV-201, and Mu2SV-201 in aqueous solution from dequenching of fluorescence of Rho-labeled peptides following the addition of different unlabeled peptides. Curves, represented by a continuous line, a dashed line, a dashed dotted line, and a dotted line, indicate the addition of 0.4 µM unlabeled SV-201, Mu1SV-201, Mu2SV-201, and SV-473, respectively, to Rho-labeled SV-201 (0.035 µM).

Binding Site of SV-201 in the Sendai Virus Fusion Protein-- SV-201 cannot inhibit Sendai virus-mediated hemolysis after the attachment of virions to RBC. This suggests that the mechanism of the inhibitory effect of SV-201 involves its interaction with a functional domain in the Sendai virus fusion protein, which is not associated with the merging of the host and target cell membrane bilayers. Since SV-201 self-associates in aqueous solution, it is possible that SV-201 binds to its counterpart in the Sendai virus fusion protein. Fig. 5 demonstrates that the self-association of SV-201 in aqueous solution is a slow kinetic process, and that Mu2SV-201 interferes only slightly with the assembly process with fast kinetics. Therefore, if the interaction of SV-201 with its counterpart in the Sendai virus fusion protein is indeed required for its inhibitory effect, then the level of inhibition should be dependent on the incubation time of virions with SV-201 but not with Mu2SV-201 or Mu1SV-201. We therefore assayed the inhibitory effects of SV-201 and the control peptides after three incubation periods: (i) at almost zero incubation time, i.e. RBCs were added to virions immediately after the addition of SV-201 to the virions; (ii) after 20 min of incubation; and (iii) after 80 min of incubation. Fig. 6A demonstrates that the inhibitory effect of SV-201 increases with longer incubation time with virions, whereas the lower inhibitory effect of Mu2SV-201 is not increased with longer incubation time (Fig. 6C). Both Mu1SV-201 (Fig. 6B) and the fusion peptide (data not shown) were inactive. These data suggest that the binding site of SV-201 is probably located on its counterpart in the Sendai virus fusion protein. In other words, the synthetic peptide SV-201 most probably interacts with the functional domain consisting of amino acids 201-229 in the fusion protein to exhibit its inhibitory effect. However, we cannot rule out the possibility that SV-201 interacts with some other functional domains in the Sendai virus fusion protein in a kinetically slow process.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Percentage of hemolysis as a function of peptide concentration and time of incubation. A longer incubation period of SV-201 with virions decreases the degree of hemolysis, indicating increase in inhibition potential, whereas the inhibition potential of Mu1SV-201 and Mu2SV-201 remains the same at a lower or longer incubation time. Panel A, SV-201; panel B, Mu1SV-201; and panel C, Mu2SV-201. The histogram found with the fusion peptide was identical to that in panel B and therefore not shown. Various amounts of peptides were incubated with virions in 100 µl of PBS for variable times at room temperature, followed by the addition of 125 µl of 4% RBC for 10 min and finally for 40 min at 37 °C. RBCs were added to virions immediately after the addition of peptides (); RBCs were added to virions 20 min after the addition of peptides (); and RBCs were added to virions 80 min after the addition of peptides (). After the final incubation, solutions were centrifuged at 5700 × g for 8 min. Percentage of hemolysis was calculated from the absorbance of the supernatants at 540 nm.

Membrane Binding of NBD-labeled SV-201-- The sensitivity of the NBD moiety to the dielectric constant of its surroundings facilitates the determination of the environment of the NBD-labeled polypeptide in its membrane-bound state. NBD has been used previously in polarity and binding experiments (53-56). The fluorescence emission spectra of NBD-labeled SV-201, Mu1SV-201, and Mu2SV-201 were measured in aqueous solutions and in the presence of zwitterionic PC vesicles. All of the three NBD-labeled peptides exhibited fluorescence emission maxima around 540 nm in PBS (Fig. 7), pointing to the location of the NBD-labeled segments in hydrophilic environments (55-57). However, in the presence of PC vesicles in PBS, the fluorescence emission maxima of the three NBD-labeled peptides shifted to 531 ± 1 nm, concomitant with large increases in fluorescence intensities (Fig. 7), indicating their binding to membranes. The three NBD-labeled peptides exhibited similar blue shifts and fluorescence enhancement also in the presence of negatively charged PS/PC vesicles (data not shown). The NBD-labeled fusion peptide of Sendai virus has a similar magnitude of blue shift and fluorescence enhancement in the presence of vesicles (42). Similar magnitudes of blue shifts are observed when surface-active NBD-labeled peptides interact with lipid membranes (54-56) and are consistent with location of the NBD on the surface of the membrane (57). It might be speculated from the ability of SV-201 to bind lipid vesicles that the corresponding 201-229 amino acid domain of the Sendai virus fusion protein binds to the target cell membrane and assists in membrane apposition. NBD-labeled Mu1SV-201, Mu2-SV-201, as well as the fusion peptide exhibited the same emission maximum and almost identical fluorescence enhancement compared with wild type peptide in the presence of PC and PS/PC vesicles. Therefore, the inhibitory activity of SV-201 is most likely not the result of a nonspecific effect resulting from membrane modulation as has been seen with derivatives of hydrophobic peptides (58-60).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Fluorescence emission spectra of NBD-labeled peptides in PBS or in the presence of phospholipid vesicles. Fluorescence spectra of 0.113 µM NBD-SV-201 (- - -), 0.112 µM NBD-Mu1SV-201 (), and 0.110 µM NBD-Mu2SV-201 (-··-··-) in PBS before and after the addition of 288 µM SUV composed of PC; (-·-·-) wild type NBD-SV-201, (····) mutant NBD-Mu1SV-201 and (- - · - - ·) NBD-Mu2SV-201. The excitation wavelength was set at 467 nm.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

We have described here the biological function and a possible site of interaction of a synthetic segment SV-201, corresponding to amino acids 201-229 in the Sendai virus fusion protein adjacent to the heptad repeat SV-163, which is located near the fusion peptide (30). A summary of the results of the functional studies is shown in Table I. Recent studies have demonstrated the inhibitory effects of synthetic peptides modeled after heptad repeat regions in several viruses including HIV, Sendai, and other paramyxoviruses (30, 35, 36, 38, 61). However, the peptides induced similar inhibitory effects whether they were incubated with the virions before or after their attachment to the target cell. Unlike the reported heptad repeats, SV-201 specifically inhibited the hemolytic activity of Sendai virus if interaction with the virions took place prior to their attachment to RBCs (Fig. 3). When virions were added to a mixture of RBCs and SV-201, Sendai virus-induced hemolysis was not inhibited (Fig. 3). This may be due to two reasons: (i) a major portion of the peptide is bound to RBCs, leaving only a small amount of the peptide to interact with the virions; and (ii) viral attachment to RBCs is faster than SV-201 binding to virions. The first possibility was ruled out following our observation that more than 70% of SV-201 remains free in solution in the presence of RBCs (data not shown). Therefore, it is probable that the faster kinetics involved in attachment of virions to RBCs protects the virions from the interference of SV-201. The data presented here suggest a possible binding site and a stage of the fusion process that might be affected by SV-201.

A Possible Binding Site for SV-201-- Fluorescence dequenching experiments with rhodamine-labeled and unlabeled peptides demonstrated that SV-201 self-associates in aqueous solution, which is not the case for both of its mutants (Fig. 5). The fact that Mu2SV-201 cannot self-associate in aqueous solution despite having the same amino acid composition (same hydrophobicity) as the wild type SV-201, suggests that the oligomerization of SV-201 is a sequence-specific process and not due merely to nonspecific hydrophobic interactions. Moreover, Mu1SV-201 cannot coassemble with wild type SV-201, and Mu2SV-201 can only coassemble slightly at high concentrations, although Mu1SV-201 contains two negative charges that are located in a position which should favor attraction to the positive charges in SV-201, assuming parallel assembly. Fig. 5 also shows that SV-201 cannot associate with SV-473, a heptad repeat positioned near the transmembrane anchor domain that possesses potent inhibitory effects, both before and after virion attachment to RBCs (30). The self-association of SV-201 led us to speculate that its counterpart (amino acids 201-229) in the Sendai virus fusion protein might be the functional domain with which it interacts to exhibit its inhibition potential. This possibility was strengthened by experiments in which longer incubation time of SV-201 with virions yielded increased levels of inhibition (Fig. 6) due to the slow kinetics of monomer displacement in the aggregate (Fig. 5). In contrast, longer incubation time of Mu2SV-201 with virions did not enhance its inhibition potential supporting the non-time-dependent fluorescence dequenching experiment of Rho-SV-201 in the presence of unlabeled Mu2SV-201. Presumably, the Mu1SV-201 mutant peptide does not inhibit lysis because it cannot recognize the functional domain (probably amino acids 201-229) on the Sendai virus fusion protein as demonstrated by the inability of Mu1SV-201 to interfere with SV-201 assembly (Fig. 5).

A Step in the Fusion Process That Might Be Affected by SV-201-- Membrane fusion has been postulated to involve four steps (reviewed in Ref. 6): 1) adhesion of the membranes involved in fusion; 2) close approach of the lipid bilayers of the membranes; 3) destabilization of the bilayers at the point of fusion; and 4) the actual fusion event, i.e. mixing of the two bilayer membranes. SV-201 can inhibit the fusion process by interfering directly with one of these steps, or alternatively, the interaction of SV-201 with amino acids domain 201-229 (as suggested above) can disrupt the functional structure of the fusion protein, which in turn can affect one of the fusion steps.