(Received for publication, April 17, 1995; and in revised form, May 12, 1995)
From the
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-[
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)
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.
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
[
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.
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.
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
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
[
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.
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.
The structural transitions of rabies virus glycoprotein have
been described previously(23) .
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
[
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
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)
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.
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
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)(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) .
(
)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) .
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.
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
Na
HPO
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).
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.
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.
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 C
E
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.
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
C
E
(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.
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).
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.
(
)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) .
(
)
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.
) 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) .
), 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) .
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) .
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) .
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; C
E
,
octaethylene glycol monododecyl ether.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.