§
§
* Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; Department of
Biochemistry and Biophysics, § Howard Hughes Medical Institute, University of California, San Francisco, California 94143; and
Department of Microbiology and Molecular Genetics, Harvard Medical School, Cambridge, Massachusetts 02138
It is not known how membrane fusion proteins that function at neutral pH, for example the human immunodeficiency virus envelope (Env) glycoprotein and intracellular fusion machines, are activated for target bilayer binding. We have addressed this question using a soluble oligomeric form of an avian retroviral Env glycoprotein (API) and soluble forms of its receptor. Binding of soluble receptor to API induces API to bind to liposomes composed of phosphatidylcholine and cholesterol at neutral pH. Liposome binding only occurs at fusion permissive temperatures (T > 20°C), is complete between 2 to 5 min at 37°C, and is stable to high salt, carbonate, and urea. Liposome binding is mediated by the ectodomain of the transmembrane subunit of API, and a mutant with a Val to Glu substitution in the Env fusion peptide (located in the ectodomain of the transmembrane subunit) shows significantly reduced liposome binding. Moreover, under conditions of equivalent binding to API, a mutant receptor that does not support infection (Zingler, K., and J.A.T. Young. 1996. J. Virol. 70:7510-7516) does not induce significant liposome binding. Our results indicate that a highly specific interaction between an avian retroviral Env and its receptor activates the retroviral glycoprotein for target bilayer binding at neutral pH in much the same way as low pH activates the influenza hemagglutinin. Our findings are discussed in terms of the mechanisms of viral and cellular fusion proteins that function at neutral pH.
PROTEIN-MEDIATED membrane fusion is a step in many
important biological processes (Rothman and Warren, 1994 Conversion to a hydrophobic form has been demonstrated for the ectodomains of several low pH-dependent
viral fusion proteins including the hemagglutinin (HA)1 of
influenza virus and the E1 protein of Semliki Forest virus (SFV; Harter et al., 1989 Many viruses do not require low pH in order to fuse with
host cells, and appear to be able to fuse directly at the
plasma membrane. This includes members of approximately half of the known families of enveloped viruses
including serious pathogens such as the human immunodeficiency virus (HIV) and respiratory syncytial virus (Hernandez et al., 1996 A number of receptors for enveloped viruses that fuse at
neutral pH have now been identified (Weiss, 1992 Like most retroviruses, the avian leukosis and sarcoma
virus (ALSV) fuses at neutral pH (Gilbert et al., 1990 Differing host range, receptor binding, and interference
patterns have divided ALSV into five major subgroups
designated A-E. The host cell receptor for ALSV-A is a
small transmembrane protein termed Tva (Bates et al.,
1993 Construction of API-Glu2
The KpnI-AflII fragment from pCB6-Env-API (GPI-linked Env A; Gilbert et al., 1993 Tissue Culture
The pCB6-Glu2-PI plasmid was transfected into NIH 3T3 cells by the calcium phosphate precipitation method (Wigler et al., 1977 Antibodies and Reagents
The anti-Tva antiserum used was that described previously (Bates et al.,
1993 Biotinylation and Coimmunoprecipitation
Binding Assay
After induction with sodium butyrate (5 mM for API and 10 mM for
Glu2-PI) for 16-18 h, cells expressing GPI-anchored proteins were labeled
with the membrane-impermeant reagent NHS-LC-biotin (Pierce) and
treated with PI-PLC as described previously (Gilbert et al., 1993 For measuring sTva dissociation from API, unlabeled PI-PLC-released
API was immunoprecipitated with anti-DAF/protein A-agarose as described above. Samples were washed four times in calcium- and magnesium-free PBS (PBS Liposome Binding Assay
Liposomes were freshly prepared before each experiment as described
previously (White and Helenius, 1980 Dissociation of the SU and TM Subunits
Biotinylated PI-PLC-released API was incubated with or without s47 on
ice as described above. Liposomes were added and the mixture was incubated for 5 min at 37°C. 75 µl of 8 M urea and 17.5 µl of 1 M DL-dithiothreitol (DTT) were then added and the solution was incubated at RT for
an additional 20 min before being placed in the bottom of a sucrose step
gradient and processed as described above. Fractions were immunoprecipitated with either anti-DAF or a mixture of both the anti-DAF and
anti-hr2 antibodies.
sTva Induces API Liposome Binding
Exposure of influenza HA or SFV E1 to acidic conditions
triggers the conversion of each ectodomain to a hydrophobic form capable of binding to liposomes (Skehel et al.,
1982 For the first liposome binding assays, biotinylated API
was incubated with sTva on ice to allow formation of the
sTva-API complex (Gilbert et al., 1994
Because Tva is related to the LDL receptor (Bates et al.,
1993
sTva contains the entire 83-amino acid extracellular domain of Tva. Within this domain the 40-amino acid LDL
receptor repeat binding motif suffices to mediate virus entry (Rong and Bates, 1995 Stability of Liposome Binding
We examined the stability of the receptor-induced API-
liposome interaction by treating API-liposome complexes
with 6 M urea, high salt (1 M sodium chloride) or carbonate, pH 11.0 ,before centrifugation. As seen in Fig. 3, none
of these treatments affected the attachment of API to liposomes. The interaction of API with liposomes is therefore
nonionic, hydrophobic, and stable.
Time and Temperature Dependence of
Liposome Binding
We next examined the time course of liposome binding by
varying the length of the 37°C incubation with liposomes
from 1 to 30 min. Samples were subjected to sucrose flotation gradients and analyzed as described above. API binding to liposomes occurs rapidly and is complete between 2 to 5 min at 37°C (Fig. 4, A and B). This is similar to BHA
which binds to liposomes within 1 to 2 min after exposure to
low pH (Doms et al., 1985
ALSV binds to chicken embryo fibroblasts at 4°C but
only fuses with them at temperatures >22°C (Gilbert et al.,
1990
TM Subunit Binds Liposomes
We next asked which subunit of API (SU or TM) mediates
liposome binding. SU and TM are associated through a
disulfide bond(s). We found that they could be dissociated
but only by treatment with 100 mM DTT and 6 M urea.
Under these conditions the anti-DAF antibody efficiently
immunoprecipitates TM and an antibody against the hr2
region of Env efficiently immunoprecipitates SU (Fig. 6
A). It is interesting that the anti-hr2 antibody only recognized SU after treatment with urea/DTT, suggesting that
the hr2 epitope is obstructed or in a different conformation in native API. Similar results were obtained with full
length Env A (Delos, S., and J. White, unpublished results).
Biotinylated API bound to receptor (s47) was incubated
with liposomes for 5 min at 37°C. The sample was then
treated with urea and DTT for 20 min at room temperature, and analyzed by a flotation gradient. Gradient fractions were immunoprecipitated with either anti-DAF (Fig.
6 B, top) or a combination of anti-DAF and anti-hr2 antibodies (Fig. 6 B, bottom). Immunoprecipitation with
anti-DAF (Fig. 6 B, top) showed that TM and SU are efficiently dissociated from each other by the urea/DTT treatment; only TM was detected in the immunoprecipitates.
Despite this, with s47-pretreated API the majority of TM
remained bound to the liposomes (migrated to the top of
the gradient). The TM subunit is more spread out in the
gradient (Fig. 6 B) than in samples that had not been
treated with urea/DTT (e.g., Fig. 1). This is likely due to
the combined action of urea and DTT, since treatment
with urea alone did not cause TM to spread out on the gradient (Fig. 3, middle). When samples of the API-liposome
complexes were incubated with urea/DTT, SU stayed at
the bottom of the gradient (Fig. 6 B, bottom). Conversely TM from the same sample migrated to the top of the gradient (Fig. 6 B, bottom). These results show that TM, and
not SU, mediates API binding to liposomes.
Liposome Binding Properties of a Fusion
Peptide Mutant
Studies with the influenza HA have demonstrated that liposome binding (at low pH) is mediated by the fusion peptide (Harter et al., 1989
Mutant Tva Does Not Induce Significant
Liposome Binding
Residues 46-48 in the Tva ectodomain are crucial for virus
entry (Zingler et al., 1995 s47WA, a soluble form of the W48A Tva mutant analogous to s47, was expressed in E. coli, purified, and refolded
(Peters, R., and D. Agard, unpublished results). By our
coimmunoprecipitation assay, s47WA appeared to bind to
API approximately 100-fold less well than wild-type (wt)
s47 (Fig. 8 A). This apparent 100-fold reduction in binding
differs from the sevenfold reduction reported by Zingler
and Young (1996)
In this study we present the first evidence that receptor
binding to a neutral pH fusion protein transforms its
ectodomain into a hydrophobic entity capable of binding
to target membranes. Moreover, we show that soluble
forms of the receptor are fully competent to elicit this response. This is the first demonstration of its kind for any
fusion protein that functions at neutral pH. After interaction with soluble receptor, the ALSV Env A ectodomain binds to liposomes composed solely of phosphatidylcholine and cholesterol. Binding is stable and nonionic in nature, temperature dependent (T > 20°C), and it is complete between 2 to 5 min at 37°C. Somewhat surprisingly,
the Env A-receptor complex appears to dissociate after
Env A binds to liposomes. Liposome binding is mediated
by the TM subunit of Env A and, at least in part, by the fusion peptide; a mutant in the fusion peptide region is significantly impaired in its ability to bind to liposomes. Furthermore, a mutant receptor that is competent to bind Env
A but is unable to support virus infection (Zingler and
Young, 1996 HA, the most extensively characterized viral fusion protein, is a homotrimer composed of two disulfide bonded subunits: HA1, which confers receptor binding activity, and
HA2 which contains the fusion peptide. When exposed to
low pH, the previously buried fusion peptide of HA2 is revealed and repositioned to interact with membranes, and
the HA1 globular head domains separate from each other
and from HA2 (White and Wilson, 1987 As for HA, a drastic change in the hydrophobicity of the
ALSV-A ectodomain occurs in response to a trigger. In
the case of HA the trigger is low pH; in the case of Env A
the trigger is a temperature-dependent sequence-specific
interaction with the viral receptor, Tva. Because Tva is sufficient to induce a change in the hydrophobicity of Env A,
it appears highly likely that Tva is the only host factor required for fusion. This differs from HIV, which needs at least
two receptors, CD4 and a chemokine receptor, to mediate
virus entry (reviewed in Bates, 1996 Previous studies suggested that Trp-48 of Tva may be an
important determinant for triggering postbinding events
necessary for fusion (Zingler et al., 1995 In response to its trigger, low pH, the fusion peptide of
HA is exposed and the ectodomain becomes hydrophobic.
Here we have clearly shown that in response to its trigger In the case of HA, exposure to low pH also causes the
globular head domains (HA1) to separate from each other,
and this dissociation is necessary for fusion (Godley et al.,
1992 Recently, CAR1 has been identified as the receptor for
members of two cytopathic subgroups of ALSV, ALSV-B
and ALSV-D (Brojatsch et al., 1996 While relatively little is known about viral fusion proteins
that function at neutral pH, even less is known about endogenous proteins that mediate cell-cell fusion and intracellular fusion events, all of which are thought to occur at
neutral pH. Several candidate cell-cell fusion proteins
have been identified, including fertilin (Blobel et al., 1992; Hernandez et al., 1996
). Due to its relative simplicity, virus-cell fusion is an attractive system with
which to study the molecular basis of membrane fusion.
Fusion between an enveloped virus and a target cell is mediated by viral surface glycoproteins. For many viruses, fusion takes place in the endosomal compartment where the
mildly acidic pH triggers structural rearrangements that
convert the fusion protein to a fusogenic form. The most
significant consequence of the low pH-induced conformational change is exposure of the fusion peptide and concomitant conversion of the previously hydrophilic ectodomain of the fusion protein to a hydrophobic form capable of binding membranes.
; Bron et al., 1993
; Klimjack et al., 1994
; White, 1995
). A water-soluble ectodomain of the influenza HA (BHA) can be prepared by treating influenza
virus particles with bromelain. BHA aggregates if exposed
to low pH in aqueous solution (Skehel et al., 1982
). If,
however, target membranes are present during the low pH
treatment, BHA associates with membranes (Skehel et
al., 1982
; Doms et al., 1985
). Photolabeling experiments have shown that this interaction is mediated by the fusion
peptide (Harter et al., 1989
). In addition, a mutant HA
with a Gly to Glu substitution at position 1 of the fusion
peptide, which displays no fusion activity, shows reduced
(~50%) liposome binding (Gething et al., 1986
). The E1
ectodomain of SFV also binds to target membranes when
exposed to low pH (Klimjack et al., 1994
), and mutations in the SFV fusion peptide that impair fusion also affect liposome binding (Kielian et al., 1996
).
). In contrast to what is known about the
mechanisms of viral fusion proteins that function at low
pH, little is known about the mechanisms of viral fusion
proteins that function at neutral pH. Unlike viruses that
fuse at low pH, those that fuse at neutral pH appear to require host cell receptors or additional factors (Weiss, 1992
).
Because of this we and others have proposed that interaction of the neutral pH viral fusion protein with its host cell
receptor(s) triggers conformational changes in the viral fusion protein that activate it for fusion (White, 1990
; Weiss,
1992
). By analogy to the low pH-induced activation of the
influenza HA and the SFV E1, the transition to a fusogenic state would include exposure of the previously buried fusion peptide whose interaction with the target membrane would initiate fusion. As far as we know, all cellular
fusion reactions such as trafficking of endocytic and exocytic vesicles, egg fertilization, and myotube formation
occur at neutral pH. Therefore cellular membrane fusion proteins/machines may use similar receptor-mediated strategies to initiate fusion.
; Brojatsch et al., 1996
; Deng et al., 1996
; Feng et al., 1996
).
Studies with the HIV envelope glycoprotein (Env) and its
primary receptor, CD4, have provided preliminary support for the basic theory of "receptor-induced" activation.
Upon binding to CD4, HIV Env undergoes a number of conformational changes, including exposure of the V3
loop as well as changes in sensitivity to proteolysis and antibody binding (Sattentau et al., 1993
; Matthews et al.,
1994
; Stamatatos and Cheng-Mayer, 1995
; Sullivan et al.,
1995
). In addition, structural data suggest that there may
be similarities between fusion-related forms of the influenza HA (Bullough et al., 1994
) and HIV Env (Chan et al.,
1997
; Weissenhorn et al., 1997
). Attainment of the former
structure does (White and Wilson, 1987
; Bullough et al., 1995) and attainment of the latter structure most likely involves large conformational changes. Despite these noted
changes, neither exposure of the fusion peptide nor
changes in hydrophobicity have yet been demonstrated for
HIV Env upon binding of either CD4 or a chemokine receptor, two host cell factors required for HIV entry.
). Its
Env is synthesized as an inactive precursor that is cleaved
intracellularly by a host protease into a surface (SU) and a
transmembrane (TM) subunit. The SU and TM subunits remain associated with each other through a disulfide
bond(s) and form a trimer of SU/TM heterodimers (Einfeld and Hunter, 1988
). The SU subunit has determinants
for receptor binding whereas the TM subunit anchors the
protein in the viral membrane and contains the fusion peptide. For most retroviruses the fusion peptide is located at
the NH2 terminus of the TM protein, but ALSV is unique
among known retroviruses in that its fusion peptide is internal to the NH2 terminus of the TM subunit (Hernandez, L.D., and J.M. White, manuscript submitted for publication). Structural models suggest a strong similarity between the ALSV TM and the Ebola virus glycoprotein implying that aspects of their fusion mechanisms may be
similar (Gallaher, 1996
).
; Young et al., 1993
); Tva binds specifically to the subgroup A envelope glycoprotein (Connolly et al., 1994
; Gilbert et al., 1994
). The extracellular domain of Tva has a
single copy of a cysteine-rich motif, seven of which are
present in the low density lipoprotein (LDL) receptor.
This 40-amino acid repeat motif is sufficient to mediate virus entry (Rong and Bates, 1995
). We have previously
shown that upon interaction with sTva, a soluble form of
the Tva ectodomain, Env A undergoes a temperature-
dependent conformational change in the SU subunit that
correlates with the activation of its fusion function (Gilbert et al., 1995
). Here we show that binding of Tva at fusion-permissive temperatures renders the Env A ectodomain hydrophobic as measured by a liposome binding
assay. Liposome binding is mediated by the TM subunit
and, at least in part, by the fusion peptide. Liposome binding
also appears to require a specific sequence in the receptor. This is the first example of receptor-induced conversion of
the ectodomain of a fusion protein that functions at neutral pH from a hydrophilic to a hydrophobic entity.
Materials and Methods
) was replaced with the corresponding KpnI-AflII fragment of pCB6-Glu2 yielding pCB6-Glu2-PI. The resulting Env gene has
the single amino acid substitution Val to Glu at residue 30 of the TM subunit (in the fusion peptide) as well as the signal for glycosylphosphatidylinositol (GPI) addition in place of the transmembrane domain and cytoplasmic tail.
). 24 h after
transfection, cells were placed in selective medium (DME, 10% supplemented calf serum; [Hyclone, Logan, UT], and 500 mg/Liter geneticin
GIBCO BRL, Gaithersburg, MD) for 14 d. Geneticin-resistant clones were
picked and screened for expression of envelope protein after treatment
for 16-18 h with 10 mM sodium butyrate (Sigma Chemical Co., St. Louis,
MO), and the highest expressing clones were amplified. The NIH 3T3 cell
line expressing Env-API was maintained in the above medium and has
been described previously (Gilbert et al., 1993
). Env-API cells were
treated for 16-18 h with 5 mM sodium butyrate to induce higher Env-API expression levels. The secreted form of Tva (sTva) was a gift of P. Bates
(University of Pennsylvania, Philadelphia, PA)
) and was a gift of P. Bates (University of PA, Philadelphia, PA). Polyclonal antisera were raised in rabbits against peptides corresponding to
part of the host range 2 (hr2) domain of the SU subunit of Env A (amino
acids 206-220), and the decay-accelerating factor glycosylphosphatidylinositol (DAF-GPI) signal peptide. These latter antibodies were affinity
purified by chromatography over their respective peptides coupled to SulfoLink resin (Pierce, Rockford, IL) according to the manufacturer's instructions. Phosphatidylinositol-specific phospholipase-C (PI-PLC) was
purified from an overexpressing bacterial strain (Koke et al., 1991
). A gift
of P. Bjorkman (California Institute of Technology, Pasadena, CA) and
O. Griffith Hayes (University of Oregon, Eugene, OR). Two additional
soluble forms of Tva, s47 and s47WA, were purified and refolded from
overexpressing bacterial strains.
). sTva,
s47, and s47WA proteins were biotinylated on ice for 45 min in PBS (pH
7.8) containing 0.5 mM MgCl2 and 1 mg/ml NHS-LC-biotin. The excess biotin was quenched by the addition of 50 mM glycine for 1-18 h at 4°C. For
coimmunoprecipitation of s47 and API, the indicated amount of biotinylated s47 or s47WA protein was incubated with unlabeled PI-PLC-
released API for 1 h on ice in a total volume of 200 µl. Anti-DAF antibody was added and incubated for 1 h at 4°C followed by another 1-h incubation after the addition of protein A-agarose (Boehringer Mannheim
Biochemicals, Indianapolis, IN). Samples were washed four times in lysis
buffer (Hepes-buffered saline with 1% Nonidet P-40), boiled, and reduced in Laemmli sample buffer, and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and probed with streptavidin coupled to
HRP (strep-HRP; Pierce) to detect biotinylated proteins. The HRP signal
was detected by enhanced chemiluminescence (ECL; Amersham Corp.,
Arlington Heights, IL).
). Biotinylated sTvA was added to the immunoprecipitates on ice for 20 min. Liposomes or PBS
was added and samples
were incubated for 5 min at 37°C. An additional 100 µl PBS
was added
and the samples were centrifuged at 14,000 rpm in a microcentrifuge for
10 s at 4°C. Supernatants were chloroform-methanol precipitated as described below. The immunecomplexes and the supernatant proteins were
analyzed by SDS-PAGE and blotted with strep-HRP.
) and contained phosphatidylcholine and cholesterol (Sigma Chemical Co.) in a 4:1 molar ratio. Biotinylated PI-PLC released GPI-linked envelope glycoproteins were incubated with soluble receptor (either sTva, s47, or s47WA) or diluted fetal calf serum on
ice for 15 min in a total volume of 60 µl. PI-PLC released material from
~2 × 106 cells was used for each sample. 40 µl of liposomes were then added and the samples were incubated at the indicated temperature for
the indicated amount of time. 300 µl of 67% sucrose was added to bring
the final concentration of sucrose to 50%. This solution was overlaid with
300 µl of 25% sucrose and 200 µl of 10% sucrose in a clear polycarbonate
ultracentrifuge tube (Beckman Instruments Inc., Fullerton, CA). After
centrifugation at 200,000 g for 3 h at 4°C, 9 × 100 µl fractions were collected from the top of the gradient, brought up to a final volume of 400 µl
with lysis buffer, immunoprecipitated with the anti-DAF antibody, separated by SDS-12.5% PAGE and processed for the detection of biotinylated proteins as described above. For quantitation of liposome binding, blots were probed with 125I-labeled streptavidin, dried, and exposed to a
phosphorimaging screen. Exposed phosphorimaging screens were scanned
into a PhosphorImager workstation using the ImageQuant program (Molecular Dynamics, Inc., Sunnyvale, CA). Protein band intensities were determined by summing the pixels in a constant volume rectangle. The top three fractions are defined as liposome bound. For 6 M urea, 1 M NaCl,
and 15 mM carbonate (pH 11.0) treatments, liposomes with bound API
were incubated with the appropriate solution for 10 min at room temperature
(RT) before the addition of the sucrose solution. For the detection of sTva,
fractions were chloroform-methanol precipitated: 10 µl of salmon sperm
DNA (1 mg/ml) was added to each 100 µl fraction. 550 µl methanol, 150 µl
chloroform, and 550 µl H2O were added. Samples were vortexed and centrifuged at 14,000 rpm in a microcentrifuge for 3-5 min at RT. The top layer was aspirated and 550 µl of methanol was added to the bottom layer. Samples were again vortexed and centrifuged at 14,000 rpm in a microcentrifuge. Pellets were resuspended in Laemmli sample buffer, boiled, and then
processed by SDS-PAGE and Western blotting with the anti-Tva antibody.
Results
; Doms et al., 1985
; Gething et al., 1986
; Harter et al.,
1989
; Klimjack et al., 1994
). We performed a liposome binding assay using the water-soluble Env A ectodomain, API,
to test whether binding of sTva to Env A causes an analogous change in the hydrophobicity of the Env A ectodomain. API is a GPI anchored form of Env A that contains the signal for GPI addition from DAF in place of the Env
A transmembrane and cytoplasmic tail domains. It is expressed, oligomerized, and transported to the cell surface
in a manner similar to full-length Env A. It can be removed
from the cell surface with PI-PLC yielding a water-soluble
oligomer (Gilbert et al., 1993
). The soluble oligomer binds
specifically to the subgroup A receptor (Gilbert et al., 1994
),
and undergoes a temperature-dependent conformational
change in the SU subunit upon this interaction in a manner
similar to full-length Env A (Gilbert, 1995
; Hernandez,
L.D., unpublished results). In this paper, the term API refers
to the PI-PLC-released soluble oligomeric form of API.
). After 15 min, liposomes composed of phosphatidylcholine and cholesterol were added and the samples were incubated at 37°C
for 30 min, returned to 4°C, placed in the bottom of a centrifuge tube, overlaid with a sucrose step gradient, and
subjected to ultracentrifugation. The gradient was fractionated and the position of API was determined by immunoprecipitation with an anti-DAF antibody that recognizes the first nine residues at the COOH terminus of the
engineered TM subunit. In the absence of sTva, API did
not bind liposomes and remained at the bottom of the gradient (Fig. 1, top). In the presence of sTva, however, API
was found at the top of the gradient indicating it had associated with the liposomes (Fig. 1, bottom).
Fig. 1.
Receptor-induced liposome binding. Biotinylated PI-PLC-released API was incubated with or without sTva for 15 min
at 4°C incubated with liposomes at 37°C for 30 min and then analyzed on flotation gradients as described in Materials and Methods. Gradients were fractionated, and samples were immunoprecipitated with anti-DAF antibody, boiled and reduced, separated
by SDS-12.5% PAGE, transferred to nitrocellulose, probed with
streptavidin-HRP, and visualized by ECL to identify API.
[View Larger Version of this Image (56K GIF file)]
), it was possible that sTva itself has an affinity for liposomes. We therefore asked which component of the
sTva-API complex is responsible for liposome binding. sTva
alone or the sTva-API complex was incubated with liposomes at 37°C for 30 min and analyzed for liposome binding by a sucrose flotation gradient as described above.
Fractions were chloroform-methanol precipitated and Western blotted with anti-Tva serum. sTva that had not been
incubated with API remained at the bottom of the gradient showing that sTva itself does not have strong affinity
for liposomes (Fig. 2 A, top). sTva that had been prebound
to API and incubated with liposomes also remained at the
bottom of the gradient (Fig. 2 A, bottom) indicating that it
had dissociated from API, which floats to the top of the
gradient (Fig. 1). We used a more sensitive method for detecting receptor by using biotinylated sTva in the liposome binding assay and determining where it ran in the gradient.
Again sTva was found only at the bottom of the gradient
(data not shown). This was somewhat unexpected since
sTva binds to Env A with a Kd of 1.5 nM (Zingler and
Young, 1996
; Rong et al., 1997
) and remains bound to Env
A through sucrose density centrifugation and coimmunoprecipitation in detergent-containing solutions (Gilbert et
al., 1994
). As shown in Fig. 2 B by a coimmunoprecipitation assay, a significant fraction of sTva dissociates from
API in the presence, but not in the absence, of liposomes.
Fig. 2.
sTva dissociates from API. (A) sTva or sTva-API was
incubated with liposomes and subjected to centrifugation as in
Fig. 1. Gradient fractions were chloroform-methanol precipitated, separated by SDS-12.5% PAGE, transferred to nitrocellulose, and Western blotted with anti-Tva serum. (B) Unlabeled
API was immunoprecipitated with the anti-DAF antibody and
protein A agarose. Biotinylated sTva was added to the immunoprecipitates on ice for 20 min. Liposomes or PBS were added and
samples were incubated for 5 min at 37°C. Beads were centrifuged and the supernatants collected and chloroform-methanol precipitated. Beads and supernatant proteins were reduced,
boiled, separated by SDS-12.5% PAGE, transferred to nitrocellulose, and blotted with streptavidin-HRP.
[View Larger Versions of these Images (43 + 39K GIF file)]
). We therefore generated in Escherichia coli and refolded a 47-amino acid fragment of
Tva that contains the 40-residue LDL receptor repeat motif (Peters, R., and D. Agard, unpublished results). s47
binds to Env A and induces the same structural changes in Env A that sTva does (Hernandez, L., and J.M. White, unpublished results). Since we found s47 to be a more stable
reagent than sTva, we used s47 as the "receptor" for all
subsequent experiments.
Fig. 3.
Stability of the API-liposome interaction. API-liposome complexes were incubated with either 1 M NaCl, 6 M urea,
or 15 mM carbonate, pH 11.0, for 10 min at RT. Samples were
processed as described in the legend to Fig. 1.
[View Larger Version of this Image (39K GIF file)]
; Gething et al., 1986
).
Fig. 4.
Time course of liposome
binding. The API-s47 complex was
incubated with liposomes at 37°C
for varying amounts of time and analyzed on sucrose flotation gradients. (A) Sucrose gradient fractions
were immunoprecipitated and processed as in Fig. 1. (B) Sucrose gradient fractions were immunoprecipitated and processed as in Fig. 1
with the exception that the blots
were probed with 125I-labeled
streptavidin and subjected to quantitation by phosphorimager analysis
as described in Materials and Methods. (% Bound) The percent API in
the top three fractions.
[View Larger Versions of these Images (58 + 12K GIF file)]
). We therefore proposed that activation of the ALSV
fusion protein must be temperature-dependent. Our previous studies on Tva-Env interactions are consistent with
this hypothesis; sTva causes a change in the sensitivity of
the SU subunit of Env A to thermolysin at 37°C but not at
4°C (Gilbert et al., 1995
). We therefore asked if receptor-induced liposome binding is temperature-dependent. To
do this API-s47 complexes were incubated with liposomes
for 5 min at 4°, 10°, 22°, or 37°C, returned to 4°C, and analyzed for liposome binding (Fig. 5). Little liposome binding was detected at 4° or 10°C. Liposome binding then increased as the temperature was further increased with a
high degree of binding (72%) occurring at 37°C. These results show that liposome binding is temperature-dependent similar to the sTva-induced conformational change in
SU and ALSV fusion with chicken embryo fibroblast cells.
Fig. 5.
Temperature dependence of liposome binding. The
API-s47 complex was incubated with liposomes for 5 min at the
indicated temperatures and analyzed on sucrose flotation gradients. Sucrose gradient fractions were immunoprecipitated, processed, and quantitated as described in the legend to Fig. 4 B.
[View Larger Version of this Image (10K GIF file)]
Fig. 6.
API binds liposomes through the TM subunit. (A) Biotinylated API was incubated with either PBS, 6 M urea, or 6 M urea/100 mM DTT for 20 min at RT in a total volume of 100 µl. Samples were brought up to 400 µl with PBS and immunoprecipitated with anti-DAF or anti-hr2 antibody and processed as described in the legend to Fig. 1. (B) The s47-induced API-liposome complex was treated with 6 M urea and 100 mM DTT before centrifugation to dissociate the TM and SU subunits. Each sucrose gradient fraction was immunoprecipitated with anti-DAF or a mixture of anti-DAF and anti-SU antibodies and processed as described in the legend to Fig. 1.
[View Larger Versions of these Images (64 + 64K GIF file)]
). We therefore hypothesized that
the ALSV fusion peptide mediates liposome binding. We
have recently shown that mutations in the candidate fusion peptide of ALSV Env A (residues 21-42 of TM) significantly impair the ability of Env A to mediate both virus-
cell and cell-cell fusion without affecting Env processing, folding, oligomerization, cell surface expression, receptor
binding, or ability to undergo a receptor-induced conformational change (Hernandez, L.D., and J.M. White, manuscript submitted for publication). We therefore constructed
a GPI-linked form of one of these mutants (Val30Glu)
and tested it in the liposome binding assay. The GPI form
of this mutant is referred to as Glu2-PI. Glu2-PI was impaired in receptor-induced liposome binding (Fig. 7 A,
middle) when compared to wt API (Fig. 7 A, bottom). The
Glu2-PI protein was spread out in the gradient suggesting
that its interaction with liposomes was relatively weak.
Quantitation of the data indicated that Glu2-PI binds to liposomes with ~50% the efficiency of wt API (Fig. 7 B).
This phenotype is similar to the behavior of the Gly1 to Glu HA fusion peptide mutant (Gething et al., 1986
). These results support our hypothesis that the fusion peptide mediates liposome binding (see Discussion).
Fig. 7.
Liposome binding capability of Glu2-PI. The position 2 glutamate mutant protein (Glu2-PI) was assayed for liposome
binding as in Fig. 4. Fractions were immunoprecipitated with
anti-DAF, processed by SDS-12.5% PAGE and transferred to nitrocellulose. Blots were probed with (A) streptavidin-HRP (visualized by enhanced chemiluminescence), or (B) 125I-labeled streptavidin (quantitated by phosphorimager analysis as described in
Materials and Methods). (% Bound) Percent API or Glu2-PI in
the top three fractions.
[View Larger Versions of these Images (53 + 13K GIF file)]
; Zingler and Young, 1996
). A
substitution that replaced the tryptophan at position 48 with alanine reduced infectivity titers approximately three
to four orders of magnitude, but only decreased cell surface Tva binding affinity for an ALSV-A SU-Ig fusion
protein approximately sevenfold (Zingler and Young, 1996
).
These findings suggested that the W48A mutant has a defect in a postbinding step(s) necessary for virus entry. We
therefore asked if W48A could induce liposome binding.
, and may be due to differences in the
systems used. Nonetheless equal binding to API could be
achieved using 100 times more s47WA protein than wt s47
(Fig. 8 A). Under conditions of comparable binding, the
s47WA receptor (100×) did not induce significant liposome binding when compared to (1×) wt s47 (Fig. 8 B).
With 100× mutant receptor the majority of TM stays at
the bottom of the gradient. If the amount of mutant receptor is further increased (250×) to binding levels that exceed the wt (Fig. 8 A), API now appears to spread on the
gradient. Nonetheless, even using 250× mutant protein,
API does not display a clear cut liposome binding phenotype as occurs with 1× of the wt receptor (Fig. 8 B).
Fig. 8.
Ability of a mutant receptor, s47WA, to (A) bind to
API and (B) induce liposome binding. (A) Indicated amounts of
biotinylated s47 or s47WA protein were incubated with equal
amounts of PI-PLC-released API protein in a total volume of
200 µl for 1 h on ice. Samples were immunoprecipitated with the
anti-DAF antibody, separated by SDS-15% PAGE and transferred to nitrocellulose. Blots were probed with streptavidin-HRP
and visualized by enhanced chemiluminescence. 1× = 0.36 µg
protein. (B) Biotinylated PI-PLC-released API was incubated
with 0.36 µg (1×) s47 or 3.6 µg (10×), 36 µg (100×), or 90 µg
(250×) s47WA protein at 4°C for 20 min. Liposomes were added
and samples were incubated at 37°C for 5 min before centrifugation. Fractions were analyzed as described in the legend to Fig. 1. Similar results were obtained with the s47WA mutant (100×)
when the 4°C incubation step was increased to 90 min.
[View Larger Version of this Image (44K GIF file)]
Discussion
) is compromised in its ability to induce liposome binding. We propose that this receptor-induced
change in hydrophobicity of a neutral pH fusion protein
ectodomain is analogous to the low pH-induced activation of fusion proteins such as the influenza HA (Doms et al.,
1985
; Gething et al., 1986
) and the SFV E1 protein (Klimjack et al., 1994
).
; Bullough et al., 1994
; Hernandez, 1996). Along with our previous findings,
the results presented here suggest that ALSV Env A may
undergo similar changes during fusion activation.
). Whereas CD4 binding has been shown to induce some structural changes in HIV Env, interaction of HIV Env with a chemokine receptor may be necessary to induce changes that expose the
HIV fusion peptide and transforms its ectodomain into a
hydrophobic form. Because soluble forms of Tva are able
to induce Env A binding to liposomes, it is clear that, at
least in the ALSV system, the proteinaceous fusion trigger
does not need to be embedded in the target membrane to
elicit the first steps of the fusion process. In this respect, it
will be interesting to see if soluble Tva can induce virus- liposome fusion.
; Zingler and Young,
1996
). Our results are consistent with this prediction; the
W48A Tva mutant, s47WA, is impaired in its ability to induce liposome binding even under conditions in which its
binding to the Env A ectodomain is comparable to or exceeds that of wt Tva. Our results therefore show that binding of Tva to Env is necessary, but not sufficient, to effect
a change in the hydrophobicity of the Env ectodomain; the
two proteins must interact in a highly specific and temperature-dependent manner in order to induce the structural
changes necessary for target bilayer binding. Trp-48 appears to be an important contact point in this interaction.
specific Tva binding
the Env A ectodomain becomes
hydrophobic. We propose that the increase in hydrophobicity is due at least in part to exposure of the fusion peptide. In previous work we have shown that an antibody
against the Env A fusion peptide reacts preferentially with
Env A following binding to Tva at T > 22°C (Gilbert et al.,
1995
). Here we show that a mutant in the Env A fusion
peptide (Val30 to Glu) that is abolished in fusion activity
(Hernandez, L.D., and J.M. White, manuscript submitted
for publication) is significantly compromised in its ability
to bind to liposomes: binding of the Glu-substituted mutant to liposomes was ~50% and appeared unstable compared to that of the wt Env ectodomain. We believe that
the phenotype of Glu-substituted mutant is caused by the
presence of a charged residue, glutamate, in the hydrophobic surface of the Env A fusion peptide. In this respect the
phenotype of the Glu-substituted Env is highly reminiscent of the first fusion peptide mutant: Gly1
Glu of the
HA fusion peptide (Gething et al., 1986
). This original HA
fusion peptide mutant binds to liposomes less well (~50%)
than wt HA (see Fig. 8 in Gething et al., 1986
). Future studies using techniques such as labeling with photoactivatable phospholipids (Harter et al., 1989
; Durrer et al.,
1995
) are necessary to test whether the hydrophobic interaction between the Env A ectodomain and target bilayers
is an exclusive property of the fusion peptide. Computer
modeling has predicted that the Ebola virus glycoprotein
is structurally very similar to the ALSV TM subunit (Gallaher, 1996
) with an analogous region postulated to act as
the fusion peptide. Our results suggest that the corresponding region in the Ebola glycoprotein may act in a
similar manner to attach the fusion protein to target membranes under fusion-inducing conditions.
; Kemble et al., 1992
). We have observed two structural changes in the receptor binding subunit (SU) of Env
in response to its fusion trigger: SU becomes sensitive to
thermolysin (Gilbert et al., 1995
) and it dissociates from
sTva in the presence of liposomes (Fig. 2). Analogous to HA, the structural changes in the Env SU subunit may reflect separation of the receptor binding domains of Env
and they may be necessary for fusion.
). CAR1 is a member
of the tumor necrosis factor receptor family and contains
two extracellular cysteine-rich domains and a death domain in its cytoplasmic tail that has been implicated in virus-induced cell death. CAR1 is not related to Tva or to any other known retroviral receptor. It will now be possible to determine if different ALSV subgroups, cytopathic
and noncytopathic, share a common mechanism for activation of their fusion proteins despite using highly unrelated
receptors. For example, it will be interesting to determine
if CAR1 binding to Env B or D induces analogous structural changes to those seen in Env A upon Tva binding, particularly conversion of the Env B or D ectodomain to a
hydrophobic entity.
)
and meltrin (Yagami-Hiromasa et al., 1995
), members of
the ADAM gene family (Wolfsberg and White, 1996
) involved in sperm-egg and myoblast fusion, respectively. N-ethylmaleimide-sensitive factor (NSF), soluble NSF-
attachment proteins, and soluble NSF-attachment proteins
receptors, are involved in intracellular fusion reactions such
as vesicular transport between Golgi compartments (for
reviews see Rothman and Warren, 1994
; Denesvre and
Malhotra, 1996
; Monck and Fernandez, 1996
). The large
number of proteins involved in cell-cell and intracellular fusion events suggests that these latter fusion events are
more complex than the apparent two component ALSV
system. Nonetheless, the overall general fusion mechanism may be similar: specific protein-protein interactions
cause conformational changes that expose a hydrophobic
region(s) in one or more of the members of the fusion protein complex. This conformational change would, in turn,
enable the fusion protein(s) to bind hydrophobically to
target membranes and initiate membrane merger (Hernandez et al., 1996
). In view of the findings presented here,
it is now reasonable to consider, for example in the case of
intracellular fusion events (Rothman and Warren, 1994
;
Denesvre and Malhotra, 1996
; Monck and Fernandez,
1996
), that a soluble protein may act as the fusion trigger.
Received for publication 11 August 1997 and in revised form 19 September 1997.
Work in Dr. White's laboratory was supported by a grant from the National Institutes of Health (A122470). L.D. Hernandez was supported in part by a predoctoral fellowship from the Howard Hughes Medical Institute. D.A. Agard is an Investigator of the Howard Hughes Medical Institute.We thank Paul Bates for baculovirus produced sTva and the anti-Tva antiserum; Pamela Bjorkman and O. Griffith Hayes for providing the PI-PLC overexpressing bacterial strain; and Joanna Gilbert for generating the anti-DAF antibody.
ALSV, avian leukosis and sarcoma virus; API, GPI-linked ALSV subgroup A Env glycoprotein; BHA, bromelain-released hemagglutinin; DAF, decay-accelerating factor; DTT, dithiothreitol; ECL, enhanced chemiluminescence; Env, envelope glycoprotein; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; HIV, human immunodeficiency virus; LDL, low density lipoprotein; NSF, N-ethylmaleimide-sensitive factor; PI-PLC, phosphatidylinositol-specific phospholipase-C; RT, room temperature; SFV, Semliki Forest virus; sTva, soluble ALSV subgroup A receptor; SU, surface; TM, transmembrane; Tva, ALSV subgroup A receptor; wt, wild-type.
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