©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Photolabeling Identifies a Putative Fusion Domain in the Envelope Glycoprotein of Rabies and Vesicular Stomatitis Viruses (*)

(Received for publication, April 17, 1995; and in revised form, May 12, 1995)

Peter Durrer , Yves Gaudin (1), Rob W. H. Ruigrok (2), Roland Graf , Josef Brunner (§)

From the  (1)Laboratorium für Biochemie II, Eidgenössische Technische Hochschule Zürich, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich, Switzerland, the Laboratoire de Génétique des Virus, CNRS, F-91198 Gif-sur-Yvette, Cedex, France, and the (2)EMBL Grenoble Outstation, c/o Institut Laue-Langevin, B. P. 156, F-38042 Grenoble, Cedex 9, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Vesicular stomatitis and rabies viruses enter cells through receptor-mediated endocytosis, followed by fusion of the viral with the endosomal membrane. The latter step is catalyzed by the viral envelope glycoprotein, which, in the low pH environment of the endosome, undergoes a conformational transition to a fusion-competent state. To investigate whether fusion competence involves the low pH exposure of a hydrophobic fusion region(s), we have applied hydrophobic photolabeling using the recently developed phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phosphocholine ([I]TID-PC/16) (Weber, T., and Brunner, J.(1995) J. Am. Chem. Soc. 117, 3084-3095). Rosettes of rabies virus glycoprotein, whole rabies virus, or vesicular stomatitis virus were incubated with large unilamellar vesicles containing [I]TID-PC/16. Following reagent activation, the labeled glycoprotein was isolated and analyzed. In all cases, labeling of the glycoprotein strongly increased as the pH was lowered from 7.0 to 6.0, suggesting the exposure at acidic pH of a domain capable of interacting with membranes. To identify the labeled region(s), CNBr fragments were generated and analyzed by SDS-polyacrylamide followed by autoradiography. In rabies glycoprotein, the labeled segment was found to be contained within fragment RCr5 (residues 103-179). Glycoprotein from vesicular stomatitis virus was labeled within fragment VCr1 (residues 59-221). These results demonstrate that rhabdovirus glycoprotein contains a domain that at low pH is capable of interacting with a target membrane in a hydrophobic manner. This domain may play a role similar to that of the fusion peptide found in many other viral fusion proteins.


INTRODUCTION

Membrane fusion, a key step in the entry of enveloped animal viruses into their host cells, is catalyzed by virus envelope glycoproteins (for reviews, see (1, 2, 3, 4) ). The fusion protein best characterized to date is influenza virus hemagglutinin (HA)()(5) . It forms a homotrimer, each subunit of which is composed of two polypeptide chains, HA1 and HA2, linked together through a single disulfide bond(6) . HA has a dual function. Initially, it mediates viral attachment to sialic acid-containing glycolipids or proteins on the surface of the host cell, whereupon the bound virus is internalized. In the mildly acidic environment of the endosome, HA undergoes a major structural transition to a fusion-competent state, which then promotes the fusion of the viral membrane with the membrane of the endosome. Although the mechanism of the fusion reaction is far from understood, it is clear that a conserved hydrophobic sequence of 23 amino acids at the N terminus of the HA2 subunit, called the fusion peptide, plays an essential role(7) . Site-specific mutations within this conserved segment slow down or abolish fusion. In the native fusion-inactive form of HA, this segment is largely buried within the hydrophobic interface of the trimer(6) . At the pH of fusion, the fusion peptide becomes exposed and, prior to the actual fusion step, inserts into the target membrane(8, 9, 10, 11) . Fusion peptides structurally related to that of HA are found in many other viral fusion proteins, including the F protein of Sendai virus (a parainfluenza virus), the E-protein complex of Semliki Forest virus (a togavirus), and gp41 of human immunodeficiency virus (a retrovirus). A putative fusion peptide is also present in PH-30, a protein involved in sperm-egg fusion(12) .

VSV and rabies virus are members of the rhabdovirus family, which also enter cells through the endocytic pathway(13, 14) . Their fusion factor, the major envelope glycoprotein, contains neither a typical fusion peptide nor any other obvious hydrophobic sequence other than the (cleaved) signal sequence and the carboxyl-terminal membrane-anchoring domain. This raises the question of whether a hydrophobic tertiary structure element, a fusion ``patch,'' provides the equivalent function of the fusion peptide. Alternatively, one could also imagine a different mechanism of fusion. Various regions within the glycoprotein have been suggested to be essential for the fusogenic activity. Thus, peptides of 26 amino acid residues corresponding to the amino-terminal segment of VSV glycoprotein can cause hemolysis at low pH, with a pH dependence similar to that of the whole virus(15, 16) . Rose and co-workers (17) reported the loss of fusion activity for a mutant glycoprotein with an additional N-linked oligosaccharide at amino acid 117.()That this residue may be close to or within a fusogenic domain is also consistent with more recent work showing that mutations within a segment of uncharged amino acids, comprising residues 117-136, resulted in a significant decrease or loss of fusion activity(18, 19) . Although this segment is highly conserved within glycoproteins of several strains of vesiculoviruses, the degree of homology is low when compared with glycoproteins from various strains of rabies virus (see ``Discussion''). For this latter virus, a segment (FNGIILG) comprising residues 360-366 shows strong homology to the fusion peptide of paramyxoviruses and has also been proposed to play a role in the fusion process(20) .

There is evidence that mildly acid conditions induce a conformational transition in VSV and rabies virus glycoproteins to an activated state that is more hydrophobic than the native state(21, 22, 23) . Here, using the newly developed photoactivable lipid [I]TID-PC/16 ((24) ; for a review of the labeling technique, see (25) ), we show that during this transition, a segment within the glycoprotein ectodomain acquires the ability to interact with membranes. Cyanogen bromide fragmentation of the labeled protein has been applied to identify this membrane-active segment.


MATERIALS AND METHODS

Chemicals

[I]TID-PC/16 (>2000 Ci/mmol) was synthesized according to the procedures described previously(24) . In a similar way, [I]TID-BE was prepared by radioiododestannylation of 4`-(3-trifluoromethyl-3H-diazirin-3-yl)-2`-tributylstannylbenzyl benzoate.()The radioiodinated compounds were stored as solutions in ethanol/toluene (1:1, v/v). Egg lecithin (grade I) was from Lipid Products (South Nutfield, United Kingdom); phosphatidylethanolamine from Avanti Polar Lipids, Inc. (Birmingham, AL).

Viruses

The Indiana laboratory strain (Orsay) of VSV and the PV strain of rabies virus were grown in BSR cells (a clone of BHK-21) and purified as described(26, 27) . The virions were resuspended in TD buffer (137 mM NaCl, 5 mM KCl, 0.7 mM NaHPO, 25 mM Tris-HCl (pH 7.5)) and stored at -80 °C.

Glycoprotein Rosettes

Glycoprotein from rabies virus was solubilized and purified as described previously(27) . The protein was extensively dialyzed against 150 mM NaCl, 20 mM Tris-HCl (pH 7.5) containing Bio-Beads SM-2 (Bio-Rad) to eliminate the detergent. To the protein micelles (1-1.5 mg of protein/ml) were added NaN (final concentration of 0.02%) and a mixture of protease inhibitors (leupeptin (2 µg/ml), antipain (2 µg/ml), pepstatin (2 µg/ml), chymostatin (2 µg/ml), and aprotinin (16 µg/ml)). The glycoprotein rosettes were stored at 4 °C and used within 1 week. As judged by electron microscopy, the morphology of the spikes was the same as on intact virions; at pH 6.4 and 0 °C, rosettes aggregated without changes in morphology. At pH 6.0 and 37 °C, changes occurred as in intact virus. In all, glycoprotein rosettes behaved as viral glycoprotein.

Phospholipid Exchange Protein

This protein was isolated following the procedure of Kamp and Wirtz(28) . The last step in the purification was omitted. Prior to use, the protein (stored in 50% glycerol at -20 °C) was dialyzed against the appropriate buffer containing 10 mM -mercaptoethanol and then concentrated using a Centricon microconcentrator (Amicon, Inc.).

Preparation of LUVs

LUVs were prepared from phosphatidylcholine/phosphatidylethanolamine (4:1 molar ratio) containing [I]TID-PC/16 (up to 100 µCi/µmol of total lipid) according to the protocol of Weber et al.(29) . Either citrate/phosphate buffer (prepared by mixing 0.1 M citric acid and 0.2 M NaHPO to a final pH of 7.0) or 137 mM NaCl, 25 mM Tris (pH 7.5) was used.

Photolabeling Experiments

For labeling of rabies glycoprotein rosettes, 1 volume each of LUVs (in citrate/phosphate buffer) and glycoprotein (in TD buffer) were mixed. After 1 min, 0.1 M citric acid was added for adjustment to the desired pH. For labeling of viruses, LUVs and viruses, both in TD buffer (pH 7.4), were mixed. The pH was then adjusted by the addition of 2 volumes of 0.1 M citrate, 0.2 M phosphate buffer of the desired pH. For labeling of glycoprotein rosettes and VSV, incubation mixtures (in Eppendorf tubes) were irradiated for 30 s in a Pyrex vessel mounted 10 cm from a SUSS LH 1000 lamphouse equipped with a 350-watt high pressure mercury lamp. For photolabeling of intact rabies virus, a 100-watt high pressure mercury lamp was used; irradiation times were 2 min.

Isolation of Photolabeled Glycoprotein

To the photolabeled glycoprotein rosettes or viruses were added 3 volumes of chloroform/methanol (1:2, v/v). After 1 h at room temperature, the precipitated protein was sedimented in an Eppendorf centrifuge (10 min, 14,000 rpm). Following removal of the supernatant, the protein pellet was dried under reduced pressure, dissolved in sample buffer (3 min in boiling water), and subjected to 12% SDS-PAGE(30) . After brief staining with Coomassie Brilliant Blue R-250, the glycoprotein-containing band was excised, and the protein was electroeluted using a homemade apparatus. The protein (typically in 1-2 ml of 180 mM glycine, 25 mM Tris, 0.1% SDS) was concentrated in a Centricon 30 microconcentrator (Amicon, Inc.) to a final volume of 30-40 µl. To remove (most of) the salt, the sample was diluted with 2 ml of water and concentrated again.

Preparation and Separation of CNBr Fragments

SDS was first removed by ion-pair extraction(31) . To 100 µg of glycoprotein were added 0.8 mg of CNBr dissolved in 70% formic acid. The solution was flushed with nitrogen and kept in the dark for 20 h. Excess CNBr and formic acid were removed by evaporation on a Speed-Vac. The residue was dissolved in 20 µl of formic acid. Water (0.2 ml) was added, and the sample were subjected to lyophilization. The CNBr peptides were then dissolved in sample buffer and separated by SDS-PAGE using a 14.5% polyacrylamide gel containing 6 M urea(32) . The gel was stained with Coomassie Brilliant Blue R-250 and subjected to autoradiography.

Sequence Analyses

CNBr peptides separated by SDS-PAGE were transferred electrophoretically onto a PVDF membrane using the semidry blot technique (Sartoblot II-S apparatus) and the buffer system of Laurière(33) . After transfer, the PVDF membrane was rinsed with water, and the peptides were visualized by brief staining (5 min) with Coomassie Brilliant Blue R-250. Following destaining, the membrane was washed with water, and the peptide-containing bands were excised and subjected to Edman sequence analyses. The latter analyses were performed in the Eidgenössische Technische Hochschule protein chemical laboratory (directed by Dr. Peter James).


RESULTS

Labeling of Rabies Glycoprotein Rosettes

Since the glycoprotein cannot easily be obtained in a water-soluble form, lacking the hydrophobic membrane anchor, initial experiments were performed with intact detergent-solubilized protein. In the absence of detergent, the protein forms rosette-like aggregates, which, upon acidification, undergo a structural rearrangement similar to that seen with virus-bound glycoprotein. When glycoprotein rosettes and LUVs doped with [I]TID-PC/16 were incubated at different pH values and then exposed to UV light, the protein became labeled in a pH-dependent manner (Table 1). Labeling consistently increased as the pH of the incubation mixture was lowered from 7.0 to 6.4 and 6.0. Not surprisingly, some labeling was also seen at pH 7.0, presumably due to exchange of glycoprotein between rosettes and liposomes. In any case, at pH 7.0, labeling could be expected to be restricted to the C-terminal membrane anchor. In contrast, labeling at low pH may partly be due to conformation-specific interactions of the glycoprotein ectodomain with the membrane.



Generation, Separation, and Identification of CNBr Fragments from Rabies Virus Glycoprotein

To identify the region(s) in the glycoprotein labeled at low pH, it was first necessary to establish procedures for the generation and separation of suitable fragments. On the basis of the amino acid sequence of the glycoprotein of the PV strain and previous CNBr fragmentation studies with the structurally related ERA strain(34) , we decided to examine CNBr fragmentation.

PV strain glycoprotein contains 14 methionine and 15 cysteine residues. Of the latter, 14 are located within the ectodomain portion and form disulfide bridges, two of which are thought to connect CNBr fragments (Fig. 1A and 2A). Most of the fragments, accounting for >95% of the protein mass, are of reasonable size for separation by SDS-PAGE.


Figure 1: Fragmentation of rabies virus (PV strain) glycoprotein by CNBr (reducing conditions). A, linear arrangement of the polypeptide chain. The numbers refer to the positions of the 14 methionine residues in the mature protein. The putative positions of N-glycosylation are indicated (CHO). The transmembrane hydrophobic anchor is shown (hatchedbox). The model and nomenclature are based on previously published sequence data (46) and CNBr fragmentation studies with ERA strain glycoprotein(34) . B, SDS-polyacrylamide gel of CNBr fragments electrophoresed under reducing conditions (stained with Coomassie Brilliant Blue R-250). Also shown are the positions of molecular mass standards. For identification of the CNBr fragments, they were transferred onto PVDF membranes and subjected to N-terminal sequence analyses. The results of these analyses along with the assignments made are also depicted. In cases where the C-terminal cleavage site is uncertain, the corresponding residue position is in parentheses.



Fig. 1B shows the pattern of CNBr fragments separated on an SDS-polyacrylamide gel under reducing conditions together with the results from N-terminal sequence analyses. The pattern of bands is very similar to that reported previously(34) , except that RCr5 (corresponding to Cr4 in (33) ) appears here as a doublet of bands (RCr5 and RCr5). The sequencing results not only confirmed the assignments made on the basis of electrophoretic mobilities, but also provided an explanation for the apparent splitting of RCr5 into two bands: while the upper band (RCr5) shows the expected sequence AGDPRY, RCr5 lacks the first three amino acid residues and therefore is likely to be derived from RCr5 by acid cleavage of the susceptible Asp-3-Pro-4 bond. Sequence analyses of the diffuse band corresponding to 7-8 kDa suggest the presence of at least four different peptides (RCr1b, RCr6, RCr7, and RCr8), possibly contaminated by RCr3c. In view of the results presented below, it is important to note that both RCr5 and RCr5 gave clean sequencing results and therefore represent unique peptides.

Separation of the CNBr fragments under nonreducing conditions and Edman sequencing of the individual peptides gave the results summarized in Fig. 2B, which are also in agreement with the data reported by Dietzschold et al.(34) . RCa1a and RCa1b showed the same N-terminal sequences, a result pointing to the presence of an uncleaved methionine in one of the peptides contained in RCa1a. Re-electrophoresis of RCa1b under reducing conditions yielded two dominant bands with electrophoretic mobilities corresponding to RCr2 and RCr7 and a weakly stained band at the position of RCr1a (data not shown).


Figure 2: Fragmentation of rabies virus (PV strain) glycoprotein by CNBr (nonreducing conditions). A, linear arrangement of the polypeptide chain as described for Fig. 1A. Also shown are the two disulfide bridges assumed to cross-link CNBr fragments. The model and nomenclature are based on previously published sequence data (46) and CNBr fragmentation studies with ERA strain glycoprotein(34) . B, SDS-polyacrylamide gel of CNBr fragments electrophoresed under nonreducing conditions (stained with Coomassie Brilliant Blue R-250). For identification of the CNBr fragments, the nonstained peptides were transferred onto PVDF membranes and subjected to N-terminal sequence analyses. The results of these analyses along with the assignments made are also depicted. In cases where the C-terminal cleavage site is uncertain, the corresponding residue position is in parentheses.



Identification of Photolabeled Segments in Rabies Virus Glycoprotein

In addition to the fact that not all CNBr fragments could be cleanly separated, further complications were expected to arise from the likely labeling of multiple segments comprising both the membrane anchor and fusion domain. To cope with this problem, we first determined the radioactivity pattern resulting from fragmentation of glycoprotein labeled within the C-terminal anchor segment. This material was prepared by UV irradiation of a mixed micellar solution containing glycoprotein, [I]TID-PC/16, and the nonionic detergent CE in pH 7.5 buffer. This radioactivity pattern was then compared with those obtained upon fragmentation of glycoprotein labeled in the presence of LUVs at either pH 7.0 or 6.0 (Fig. 3, A and B). CNBr fragments derived from anchor-labeled glycoprotein gave rise to two rather diffuse radioactive bands, which show the same mobilities on the reducing and nonreducing gels (lanes1 and 4). While the lower band presumably corresponds to labeled RCr3c (this fragment is also generated under nonreducing conditions, but could not be identified by sequence analysis), the upper band is likely to be due to labeled RCr3a/RCr3b (lane1) and RCa3 (lane4).()As expected, the same pattern was also obtained upon labeling of glycoprotein rosettes in the presence of LUVs at pH 7.0 (lanes2 and 5). When, however, labeling was carried out at pH 6.0, a clearly different radioactivity distribution pattern was obtained. Under both reducing (lane3) and nonreducing (lane6) conditions, a major proportion of the radioactivity now appeared at 11 kDa, corresponding to RCr5/RCr5 (lane3) and RCa4 (lane6), two identical peptides. A minor amount of radioactivity also appeared to be associated with the membrane anchor.


Figure 3: Distribution of radioactivity among CNBr fragments derived from PV strain rabies virus glycoprotein following labeling of rosettes under different conditions. CNBr fragments were separated on 14.5% SDS-polyacrylamide gels containing 6 M urea under reducing (lanes 1-3) and nonreducing (lanes 4-6) conditions. Each lane corresponds to 40 µg of protein. The slab gels were stained with Coomassie Brilliant Blue R-250 (A). The radioactivity patterns were then determined using the phosphoimaging technique (B). Analyzed were CNBr fragments derived from glycoprotein (rosettes) labeled with [I]TID-PC/16 in the presence of detergent CE (1.7 mM) at pH 7.5 (lanes1 and 4) or labeled following incubation with [I]TID-PC/16-containing LUVs (lanes2, 3, 5, and 6). The incubation conditions (time, temperature, and pH) prior to reagent activation are specified in A.



Labeling of the Glycoprotein in Intact Rabies Virus

Next, we investigated the interaction of rabies virus with LUVs at different pH values (7.0, 6.4, and 6.0) and temperatures (0 and 23 °C) and after different periods of incubation (10 s, 1 min, and 10 min). Following photolabeling, the virus proteins were separated by SDS-PAGE, glycoprotein-containing bands were excised, and the protein was electroeluted. As shown in Table 2, the extent of labeling of the glycoprotein was strongly dependent upon the incubation conditions. Only very faint labeling was found after incubation at pH 7.0, and by far the strongest labeling was seen following fusion (pH 6.0 and 23 °C). Interestingly, at pH 6.4, labeling at 0 °C was four to six times stronger than at 23 °C.



To identify the regions in the glycoprotein labeled under the various conditions, CNBr fragments were generated and analyzed by SDS-PAGE and autoradiography (Fig. 4, A and B). Only very faint and diffuse radioactivity can be seen in samples corresponding to incubations at 23 °C and pH 7.0 (lane1) or pH 6.4 (lane2). Samples from incubations at 0 °C and pH 6.4 show two radioactive bands, the more prominent of which correlates with RCr5 and RCr5 and the other with a peptide in the region of RCr2/RCr3a/RCr3b (lanes4 and 5). To facilitate interpretation of these data, we also analyzed a sample of glycoprotein labeled in intact virus (pH 7.3) with [I]TID-BE (an improved version of [I]TID(35, 36) ; for chemical formula, see Fig. 5), a reagent expected to label the C-terminal anchoring domain (peptides RCr3a, RCr3b, and RCr3c). As shown in lane6, the resulting pattern is different from those in lanes4 and 5 and closely resembles that in lane1 of Fig. 3, also reflecting membrane anchor labeling. When labeling was performed following fusion of viruses with LUVs (lane3), radioactivity was found in fragments representing the membrane anchor (same as in lane6) as well as in RCr5/RCr5 (as in lanes4 and 5). The weakly labeled upper band in the RCr2/RCr3a region of lanes4 and 5 (and lane3, but not lane6) has not been characterized further. It may be due to an incompletely cleaved fragment containing the RCr5 region or labeled RCr2. In summary, whereas under prefusion conditions (0 °C and pH 6.4), the main sites of labeling are within RCr5, postfusion labeling results in incorporation of radioactivity into both RCr5 and peptides containing the membrane anchor.


Figure 4: Radioactivity distribution among CNBr fragments of glycoprotein labeled in intact rabies virus. After photolabeling of viruses in the presence of LUVs, the glycoprotein was isolated and subjected to CNBr cleavage. The CNBr fragments were separated by reducing SDS-PAGE, and the slab gel was stained with Coomassie Brilliant Blue R-250 (A), dried, and subjected to autoradiography (B). The incubation conditions prior to reagent activation for each of the six samples analyzed are specified in A.




Figure 5: Chemical structure of [I]TID-BE.



Labeling of the Glycoprotein of VSV

This study was extended to include also experiments with VSV. The design of the experiments was similar to that described above for rabies virus. The glycoproteins of both viruses share only limited sequence homology (37) as apparent also from the rather different distribution of methionine residues (Fig. 6A).


Figure 6: Fragmentation of VSV glycoprotein by CNBr under reducing conditions. A, positions of the methionine residues within the glycoprotein polypeptide chain. The sequence data were taken from (47) . CHO refers to N-glycosylation sites; the hatchedbox represents the hydrophobic transmembrane segment anchoring the glycoprotein in the viral membrane. B, SDS-polyacrylamide gel of CNBr fragments electrophoresed under reducing conditions (stained with Coomassie Brilliant Blue R-250). Also shown are the positions of molecular mass standards. For identification, the CNBr fragments (nonstained peptides) were transferred onto PVDF membranes and subjected to N-terminal sequence analyses. The results of these analyses along with the assignments made are also depicted. In cases where the C-terminal cleavage site is uncertain, the corresponding residue position is in parentheses.



When incubation mixtures of VSV and LUVs containing [I]TID-PC/16 were subjected to photolysis, the extent of labeling of the glycoprotein was also found to depend on the incubation conditions (Table 3). The pattern is similar to that obtained with rabies glycoprotein: no or negligible labeling was seen at pH 7.0, and comparably strong labeling occurred upon fusion of the virus with the label-containing LUVs (23 °C and pH 5.8). Likewise, labeling upon a short incubation at pH 6.4 and 0 °C was substantially stronger than that measured at pH 6.4 and 23 °C.



Generation, Separation, and Identification of CNBr Fragments from VSV Glycoprotein

VSV glycoprotein isolated from SDS-polyacrylamide gel was subjected to CNBr cleavage. The resulting peptides were separated on a reducing SDS-polyacrylamide gel (Fig. 6B), and the most prominent bands were subjected to N-terminal amino acid sequence analysis. The results of these analyses together with the assignments made are also summarized in Fig. 6B. With the exception of five small predicted fragments (residues 46-58, 222-239, 344-346, 347-386, and 387-391), all major peptides, accounting for 85% of the protein mass, could be identified.

Identification of Photolabeled Segments in VSV Glycoprotein

The above CNBr fragmentation scheme was used to determine the region(s) within the glycoprotein labeled in VSV in the presence of [I]TID-PC/16-containing LUVs (Fig. 7, A and B). Although the separation of the peptides was not completely satisfactory, it is again clear that upon incubation under mildly acidic conditions (pH 6.4 and 0 °C), the segment first labeled (lane7) does not correspond to the membrane anchor (VCr2), but rather to VCr1. Selective labeling of the membrane anchor (VCr2) was accomplished following phospholipid exchange protein-catalyzed transfer of [I]TID-PC/16 into the viral membrane (lane11) and resulted in several radioactive bands, most likely reflecting the tendency of VCr2 to aggregate (particularly apparent from the radioactivity that did not enter the gel). Although VCr2 has been identified within a region corresponding to 20 kDa, this is likely to correspond to a dimer (or trimer). VCr2 monomers, as predicted, may correspond to the band at 11 kDa seen only on the autoradiograph (lanes5, 6, and 11). Again, we noted that under conditions expected to result in increased fusion, there is also an increase in labeling of VCr2 (compare lanes5 and 6 and lanes 7-9). It is difficult to judge whether the extent of labeling of VCr1 increases during fusion or remains at a level comparable with that in lane7.


Figure 7: Radioactivity distribution among CNBr fragments of glycoprotein derived from labeled VSV. Incubation mixtures containing virions and LUVs were subjected to photolysis. Photolabeled glycoprotein was isolated and subjected to CNBr cleavage. The resulting fragments were separated by SDS-PAGE (reducing conditions), and the slab gel was stained with Coomassie Brilliant Blue R-250 (A) and, after drying, subjected to autoradiography (B). The incubation conditions prior to reagent activation for each of the 12 samples analyzed are specified in A. PLEP, phospholipid exchange protein.




DISCUSSION

The structural transitions of rabies virus glycoprotein have been described previously(23) .()Immediately after acidification below pH 6.7, the virions appear to be more hydrophobic and are able to interact with the target membrane in a manner different from that at neutral pH. It has been proposed that this hydrophobic interaction with the target membrane, mediated by glycoproteins in an activated state, may represent a first step in the fusion process. However, further protonation at a pH below 6.3 is necessary for fusion. In the absence of a target membrane, the activation state induces the formation of viral aggregates that are stabilized at low pH and low temperature(23) . Prolonged incubation at low pH leads to a subsequent conformational change of the glycoprotein to its inactive state, which is highly sensitive to proteases(38) , antigenically distinct from and of longer shape than the native structure. In the case of VSV, the same three states have been postulated from kinetic studies(39) . For this virus, the trimeric structure of the protein is stabilized in the inactive state(40) , which is associated with fusion inactivation (39) .()

In this report, we show that immediately after acidification, segments within the ectodomain of the glycoprotein are capable of interacting with model target membranes. Since labeling with the carbene-generating [I]TID-PC/16 can be assumed to be confined to the apolar phase of the membrane, the interaction of the glycoprotein with the membrane involves hydrophobic effects. It is tempting therefore to speculate that the corresponding region plays a functional role similar to that of the fusion peptide of other viral fusion proteins.

All the results obtained from photolabeling are consistent with previous results of fusion assays monitoring lipid mixing(23) . In particular, both techniques showed the same pH dependence for fusion. Analyses of the labeled glycoprotein revealed that upon fusion conditions (pH 6 for rabies virus; pH 5.8 for VSV), the main sites of labeling are contained within the transmembrane region and within a distinct segment of the ectodomain identified as RCr5 (residues 103-179) and VCr1 (residues 59-221) for rabies virus and VSV, respectively. Prefusion conditions (pH 6.4 and 0 °C) led to preferential or exclusive labeling of RCr5 and VCr1. Therefore, as for influenza virus(10, 11) , a domain of rhabdovirus glycoprotein, prior to fusion, interacts with the target membrane. Thus, our results confirm results of previous studies (23) suggesting that it is the activated state that is able to interact with the target membrane. Consistent with this view is the finding that incubation at pH 6.4 and 0 °C of the virus with the probe-containing target membrane results in a markedly stronger labeling of the glycoprotein than incubation at pH 6.4 and 23 °C, where inactivation occurs faster.

The approach taken here provides qualitatively different information than site-directed mutagenesis(17, 18, 19) . While our approach is designed to identify segments interacting directly with membranes, the role of the functional critical region (VSV glycoprotein residues 102-120 (118-136) identified by mutagenesis has not been defined further. Rather than representing a fusogenic region per se, it could be critical for the low pH exposure of a nearby element with fusogenic properties. In this context, it is important to note that mutations in other regions also affected the fusion properties of VSV (41) .

Although the segment identified by Zhang and Ghosh (18) is highly conserved in vesiculoviruses, there is less homology to the corresponding region in rabies glycoprotein (Fig. 8) (37) . Sequence comparison between RCr5 and the homologous region of glycoproteins of other rhabdoviruses reveals that the highest degree of homology is found within a region from Val-144 to Trp-197 (PV numbering). In the putative fusion domain of VSV (residues 102-120 (118-136)), only Gly-102 (118), Pro-110 (126), and Val-119 (135) appear to be conserved in both vesiculo- and lyssaviruses (Fig. 8). Finally, inspection of the RCr5 sequence does not reveal any extended hydrophobic segment. This could suggest that the membrane-interacting region adopts either the conformation of a sided -helix, as proposed originally for the HA fusion peptide(42) , or that of a -structure, as suggested for the putative fusion peptide of PH-30(43) .


Figure 8: Sequence alignment of the RCr5 fragment of rabies virus (PV strain) and the homologous region of glycoproteins of other rhabdoviruses. The amino acid numbering corresponds to the proteins before signal sequence cleavage. The shadedregion indicates the putative fusogenic domain of VSV(18) . Boxes indicate conserved residues. Conservative changes are indicated in boldfacetype. MOK, Mokola virus; IN, Indiana VSV; NJ, New Jersey VSV; CHA, Chandipura virus; PIR, Piry virus. The sequences have been published: PV strain rabies virus(46) , Mokola virus(48) , Indiana VSV(49) , New Jersey VSV(50) , and Chandipura virus(51) . The sequence of Piry virus glycoprotein can be found in GenBank (accession code PVPPMG). PV and Mokola virus are lyssaviruses; the others are vesiculoviruses.



Although there are obvious parallels in the behavior of rhabdovirus glycoprotein and influenza virus HA, it is also important to refer to striking differences that may be relevant in defining viral fusion mechanisms. First, in the case of rhabdoviruses, the conformational changes from the native toward the active and inactive conformations at low pH are completely reversible after readjusting the pH to above 7.0 (23, 38, 39, 40) . Moreover, fusion and fusion inactivation are kinetically distinct processes. In the case of influenza virus, the conformational transition leading to the exposure of the fusion peptide is irreversible, and fusion and fusion inactivation are kinetically related processes(44) .

Second, despite the fact that the basic designs of this study and previous experiments with influenza virus were similar, there are major differences between the quantitative aspects of labeling influenza virus HA and rabies virus glycoprotein. It appears that labeling of the glycoprotein at pH 6.4 and 0 °C is 1-2 orders of magnitude more efficient than labeling of HA under prefusion conditions. Although different intrinsic reactivities of the glycoprotein and HA fusion domains could, in principle, account for such differences in labeling, they could also reflect some more fundamental differences in behavior. This is especially apparent when labeling in the pre- and postfusion states of the two viruses is compared. In the case of rabies virus, the extent of labeling of the glycoprotein increases by a factor of 5 (see Table 2) when going from the activated prefusion state (pH 6.4 and 0 °C) to postfusion conditions (pH 6.0, 10 min). Analysis of the label distribution patterns (Fig. 4) indicates that this increase resulted mainly, or exclusively, from incorporation of label into the membrane-anchoring domain (a consequence of membrane fusion). In contrast, for influenza virus (strain PR8/34), the transition from the pre- to the postfusion state is accompanied by an increase in labeling of HA by at least a factor of 200(11) . Moreover, after fusion, the HA2 N-terminal fusion peptide is labeled at least 50 times more strongly than in the prefusion state, a result that can be ascribed to the ability of the HA fusion peptide to penetrate the viral/fused membrane, independent of whether the fusion peptide initially interacted with the target membrane. The absence of any significant increase in labeling of the glycoprotein ectodomain during fusion of rabies virus could imply that membrane insertion of the fusogenic domain of the glycoprotein occurs only during prefusion complex formation and that, unlike influenza virus HA, no fusion-independent pathway for membrane insertion exists. This issue is of considerable interest since fusion of influenza virus has been suggested to be linked mechanistically with the ability of the HA fusion peptide to penetrate the viral/fused membrane(29) .


FOOTNOTES

*
This work was supported by a grant (to J. B.) from the Swiss National Science Foundation (Bern, Switzerland) and by CNRS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-1-632-3003; Fax: 41-1-632-1269; jbrunner{at}bc.biol.ethz.ch

The abbreviations used are: HA, hemagglutinin; VSV, vesicular stomatitis virus; [I]TID-PC/16, 1-O-hexadecanoyl-2-O-[9-[[[2-[I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]sn-glycero-3-phosphocholine; [I]TID-BE, [I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl benzoate; PV, Pasteur Vaccin; ERA, Evelyn Rokitnicki Abelseth; LUVs, large unilamellar vesicles; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; CE, octaethylene glycol monododecyl ether.

Amino acid numbering corresponds to the protein before signal sequence cleavage. Unless stated otherwise, plain numbers (without Footnote 2) correspond to amino acid residues of the mature protein (after signal sequence cleavage).

The synthesis and characterization of this labeling reagent will be described elsewhere.

As indicated in Figs. 1 and 2, RCr3a/RCr3b and RCa3 may represent peptides of incomplete cleavage with identical N termini. Alternatively, they could represent products of dimerization of RCr3c.

Y. Gaudin, C. Tuffereau, P. Durrer, A. Flamand, and R. W. H. Ruigrok, submitted for publication.

Y. Gaudin and R. W. H. Ruigrok, unpublished results.


ACKNOWLEDGEMENTS

We thank Anne Flamand for constant support, Christine Tuffereau for helpful discussions, and Xuan Nguyen for excellent technical assistance. We also acknowledge the support by Professor G. Semenza.


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