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Journal of Virology, March 2000, p. 2930-2935, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The SU and TM Envelope Protein Subunits of Bovine
Leukemia Virus Are Linked by Disulfide Bonds, both in Cells and
in Virions
Elizabeth R.
Johnston
and
Kathryn
Radke*
Department of Animal Science and Graduate
Group in Biochemistry and Molecular Biology, University of
California, Davis, California 95616-8521
Received 30 September 1999/Accepted 8 December 1999
 |
ABSTRACT |
After the polyprotein precursor of retroviral envelope proteins is
proteolytically cleaved, the surface (SU) and transmembrane (TM)
subunits remain associated with each other by noncovalent interactions
or by disulfide bonds. Disulfide linkages confer a relatively stable
association between the SU and TM envelope protein subunits of Rous
sarcoma virus and murine leukemia virus. In contrast, the noncovalent
association between SU and TM of human immunodeficiency virus leads to
significant shedding of SU from the surface of infected cells. The SU
and TM proteins of bovine leukemia virus (BLV) initially were reported
to be disulfide linked but later were concluded not to be, since TM is
often lost during purification of SU protein. Here, we show that SU and
TM of BLV do, indeed, associate through disulfide bonds, whether the
envelope proteins are overexpressed in transfected cells, are produced
in virus-infected cells, or are present in newly produced virions.
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TEXT |
Retroviral envelope proteins confer
infectivity on the virus. These proteins are first synthesized as a
polyprotein precursor whose amino terminus is inserted through the
membrane of the endoplasmic reticulum. In the lumen of the endoplasmic
reticulum, the precursor protein becomes glycosylated. Protein folding
and disulfide bond formation are aided by protein disulfide isomerase
and other chaperone proteins (reviewed in references
8 and 10). Oligomers of precursor
proteins formed within this compartment are transported to the Golgi
apparatus, where carbohydrates are further processed. Cleavage of an
envelope precursor protein by a cellular dibasic endoprotease yields a
surface glycoprotein (SU) that is anchored to the lipid bilayer of
cellular membranes by covalent or noncovalent association with a
transmembrane protein (TM). Transport to the plasma membrane of
oligomers made up of cleaved envelope subunits places SU outside the
cell and makes the envelope protein complex available for incorporation
into the viral envelope during the budding of particles from the cell.
Infection of the host cell is initiated when SU mediates binding of
virions to cell surface receptors and TM induces fusion of viral and
cellular membranes.
To function properly, SU and TM envelope protein subunits must remain
associated with one another either through disulfide bonds linking two
cysteine residues or through noncovalent interactions. The gp85-SU and
gp37-TM envelope subunits of Rous sarcoma virus are covalently linked
by disulfide bonds (18). After purified viral particles are
lysed in sodium dodecyl sulfate (SDS), Rous sarcoma virus envelope
subunits migrate together as a large complex on nonreducing
SDS-polyacrylamide gels but migrate separately as discrete polypeptides
on reducing gels. In contrast, the gp120-SU and gp41-TM envelope
subunits of human immunodeficiency virus (HIV) associate noncovalently;
the two separate on sucrose density gradients whether or not reducing
agents have been used to break disulfide bonds (21). Lack of
covalent association with TM means that gp120-SU is easily shed into
culture medium after cleavage of the precursor protein and transport of
the envelope proteins to the cell surface (9, 17, 30, 34).
Substitution of amino acids other than cysteine within the N termini of
SU and TM can release even more gp120-SU (13, 17),
indicating that amino acids other than those directly forming disulfide
bonds affect the ability of HIV SU and TM to associate.
Whether the SU and TM proteins of bovine leukemia virus (BLV) are
disulfide bonded has been unclear. A 1978 review (2) stated
that the two envelope subunits are linked by disulfide bonds in
virions, but more recent reviews (3, 16) have said that they
are not. Dietzschold et al. (7) and Rohde et al. (31) showed in 1978 that glycosylated proteins of 60 and 32 kDa were disulfide bonded when BLV virions were disrupted either with
nonionic detergent or with SDS in the presence of the alkylating agent
iodoacetamide. However, the two proteins shared a number of tryptic
peptides (7), calling into question their identification as
distinct envelope subunits. Bex et al. (1) reported in 1979 that under nonreducing conditions, a 94-kDa complex of 60- and 30-kDa
glycoproteins was purified by gel filtration from virions solubilized
with nonionic detergent. Uckert et al. (39) later demonstrated by two-dimensional polyacrylamide gel electrophoresis that
glycoproteins of 60 and 30 kDa were linked if no reducing agent was
present during isolation of viral particles. However, using virion
lysates prepared in the absence of reducing agents, Schultz et al.
(35) purified 60- and 30-kDa proteins as separate entities
and showed that their respective amino-terminal sequences were distinct
and identical to those predicted for SU and TM by the nucleotide
sequences of a BLV provirus (29). Gatot et al. (12) recently stated that TM is lost during purification of SU, citing results of earlier experiments (27) in which
virions were lysed with 1% Triton X-100 and the viral proteins were
purified by ion-exchange chromatography. Under those conditions, SU was immunoprecipitated from the eluate without coprecipitation of TM.
Disulfide-linked SU and TM subunits were shown some years ago to be
present in both murine leukemia virus (MuLV)-infected cells and
virions, although only a small fraction of the mature envelope proteins
was involved (19, 22, 25, 33, 40). That fraction could be
increased upon addition of the thiol-reactive alkylating agent
N-ethylmaleimide (NEM). The disulfide linkage between
subunits has recently been shown to involve virtually all
cell-associated SU and TM (20). Two cysteine motifs, CXXC in
SU and CX6CC in TM, are highly conserved in avian and
murine C- and D-type retroviruses, as well as in BLV and its relative human T-cell leukemia virus (24; R. Patarca and
W. A. Haseltine, Letter, Nature 312:496, 1984). The
cysteine motif CWLC in gp70-SU of MuLV is involved in the linkage to
p15E-TM (24). Based on the disulfide bonding pattern of the
bacterially produced extracellular domain of p15E-TM (11),
the third cysteine of the conserved CX6CC motif of MuLV TM
has been proposed to be involved in the intersubunit disulfide bond
(24). Since BLV envelope proteins contain these cysteine
motifs, we asked whether SU and TM of BLV are similarly linked by
disulfide bonds and if so, whether their association is stabilized by
blocking free sulfhydryl groups during preparation of lysates.
Linkage of SU and TM in transfected cells overexpressing BLV
envelope proteins.
We initially investigated the nature of SU and
TM association using protein expressed from a cloned BLV env
gene. The construct pcDNA3-ENV was derived from env mRNA
present in cultured peripheral blood mononuclear cells obtained from a
BLV-infected sheep, and its sequence was shown to be wild type
(14). An expression plasmid (pcDNA3-CAT) encoding
chloramphenicol acetyltransferase (CAT) served as a negative control.
Antibodies recognizing epitopes within the BLV Env precursor protein,
mature SU, and mature TM were identified by their recognition on
immunoblots of glycoproteins expressed in COS-1 cells transfected with
Env or CAT expression vectors. Forty-eight hours after transfection, cells were disrupted in lysis buffer (25 mM Tris-HCl [pH 8.0], 150 mM
NaCl, 1% [wt/vol] Nonidet P-40, 1% [wt/vol] sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mg of ovalbumin per
ml, 0.5 µg of leupeptin per ml, 2 µg of aprotinin per ml, 10 µg
of pepstatin A per ml). Lysates were incubated on ice for 20 min, and
then insoluble material was removed by centrifugation at
16,000 × g for 30 min at 4°C. To enrich glycosylated
proteins, cleared lysates (1 ml) were gently rocked for 4 h at
4°C with 60 µl of 50% [wt/vol] Sepharose 4B conjugated to lentil
lectin (Sigma). The beads were washed once with lysis buffer, the
glycoproteins were eluted with an equal volume of double-strength gel
sample buffer (125 mM Tris-HCl [pH 6.8], 4% [wt/vol] SDS, 20%
[vol/vol] glycerol, 0.01% [wt/vol] bromophenol blue, 100 mM
dithiothreitol [DTT]), and the eluates were boiled for 4 min.
Following electrophoresis on an SDS-12% polyacrylamide gel,
glycoproteins were transferred to polyvinylidene difluoride membranes
by electrophoresis for 16 h at 15 V. Sections of the blot were
probed with serum from BLV-infected sheep 410 (28), with
hyperimmune rabbit serum raised against the 16 C-terminal amino acids
of TM (6), or with monoclonal antibody specific for the
linear D epitope (4) of SU (BLV2; Veterinary Medical
Research and Development, Inc., Pullman, Wash.). Washed strips were
incubated with biotinylated, species-specific anti-immunoglobulins and
then washed again before being incubated with biotinylated alkaline
phosphatase linked to avidin. To reveal sites of antibody binding,
strips were incubated with Lumi-Phos Plus substrate (GIBCO-Bethesda
Research Laboratories) for 1 min and then realigned and exposed to
X-ray film.
Serum from the BLV-infected sheep reacted weakly with gp51-SU (Fig.
1); the D-specific monoclonal anti-SU
antibody reacted
strongly with gp51-SU, as well as with the
glycosylated, 72-kDa
envelope precursor protein (gPr72-Env) that
contains SU determinants.
Anti-TM serum strongly recognized
gp30-TM and gPr72-Env, both
of which contain the C-terminal amino
acids of TM. In addition,
the anti-TM serum recognized a faint band
slightly larger than
gp51-SU, which could represent a dimer of TM.
Others have demonstrated
that multimers of HIV TM can persist in
SDS-polyacrylamide gels
(
23).
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FIG. 1.
Antigenic specificity of antibodies (Ab) recognizing BLV
envelope proteins. Strips of a membrane containing glycoproteins
enriched from lysates of COS-1 cells transfected with a BLV Env
expression vector (+) or a CAT expression vector ( ) were probed with
serum from a BLV-infected sheep (I) diluted 1:250, with rabbit anti-TM
antibody (T) diluted 1:4,000, or with monoclonal antibody specific for
the D epitope of SU (S) diluted 1:2,000. Binding sites were revealed
with luminescence created by reaction of alkaline phosphatase with
substrate. An X-ray film exposed for 4 h was developed and scanned
at 300 pixels/in., and the image was printed using Adobe Photoshop. The
molecular masses of marker proteins are indicated to the left, and BLV
proteins are identified on the right. The line between gPr72 and
gp51-SU marks the mobility of a band that may represent a dimer of TM,
as indicated in the text. Note that when the membrane was cut into
sections, the segment to be probed with anti-TM extended partway into
the Env-containing lane that was probed with anti-SU. Careful
examination of the blot shows that the intermediate band recognized by
anti-TM migrated more slowly than the large, heavy band representing
gp51-SU.
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To determine whether SU and TM could be retrieved together by
antibodies specific for one subunit, we immunoprecipitated radiolabeled
Env proteins from lysates of transfected cells. Cells that had
been
transfected with plasmid 24 h beforehand were washed and
incubated
for 16 h at 37°C in cysteine-free medium containing
[
35S]cysteine (80 µCi/ml; >1,000 Ci/mmol; Amersham).
Washed cells
were disrupted with cold lysis buffer. Glycoproteins were
enriched
by adsorption to lentil lectin and eluted by two 4-h
incubations
with 400 µl of 0.2 M
methyl-
-
D-mannopyranoside (Sigma) (
26).
Eluates were precleared with normal sheep serum and protein G-Sepharose
or normal rabbit serum and protein A-Sepharose.
Immunoprecipitates formed using immune sheep serum contained the two
BLV Env proteins having SU determinants, gp51-SU and
gPr72, but also
contained a protein with the mobility expected
for gp30-TM (Fig.
2A, lane 3). Antibodies present in the
immune
sheep serum were unlikely, at the concentration used, to have
cleared all envelope glycoproteins from the eluate. The remaining
supernatant was therefore exposed to rabbit anti-TM serum to learn
which proteins would be retrieved. The resulting immunoprecipitates
included the 30-kDa TM glycoprotein and gPr72, both of which contain
the C-terminal TM peptide (Fig.
2A, lane 4). Significantly,
gp51-SU
was also present, indicating that SU could be retrieved
in association
with TM. Thus, antibodies recognizing determinants in
either SU
or TM appeared to coprecipitate the other subunit from
lysates
of cells overexpressing Env protein, suggesting that a fairly
stable association exists between the two subunits. However, such
an
association could be created by disulfide bond formation in
lysates.
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FIG. 2.
Disulfide linkage of SU and TM proteins in transfected
cells overexpressing BLV envelope protein. COS-1 cells transfected with
a BLV Env expression vector (+) or a CAT expression vector () were
radiolabeled with [35S]cysteine, and glycoproteins
concentrated from cell lysates were subjected to immunoprecipitation.
(A) Cells were lysed in the absence of NEM. Glycoproteins recovered
from 2 × 105 transfected cells were precipitated with
serum (diluted 1:266) from a BLV-infected sheep (I), and the
supernatants were then precipitated with rabbit anti-TM (T) antibody
(Ab) diluted 1:1,000. Bound proteins were eluted from protein G- or
A-Sepharose with double-strength SDS gel sample buffer containing 100 mM DTT. The eluates were boiled for 4 min, and proteins were separated
by electrophoresis on SDS-12% polyacrylamide gels. The gel was fixed
with 30% methanol-10% acetic acid, impregnated with
En3Hance (DuPont), dried, and then exposed to X-ray film
for 14 days. The developed film was scanned at 300 pixels/in., and the
image was printed using Adobe Photoshop. (B) Before being lysed in
buffer containing 10 mM NEM, cells were incubated in cold
phosphate-buffered saline containing 10 mM NEM. Each lane represents
glycoproteins from 105 transfected cells that were present
in immunoprecipitates obtained using infected sheep serum (I) diluted
1:133, monoclonal antibody specific for the D epitope of SU (S) diluted
1:200, or anti-TM antibody (T) diluted 1:1,000. Prior to
electrophoresis, precipitated proteins were heated with or without DTT,
as indicated. A PhosphorImager screen was exposed to the
fluor-impregnated gel for 4 days before being scanned on a Storm 860 (Molecular Dynamics). Images digitized using Image-Quant software were
printed using Adobe Photoshop. The values to the left are molecular
sizes in kilodaltons.
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To learn whether SU and TM are disulfide linked in cells, formation of
disulfide bonds has to be prevented during cell lysis,
glycoprotein
enrichment, and immunoprecipitation. Prior to lysis,
cells were treated
for 15 min with cold phosphate-buffered saline
containing 10 mM NEM,
which is membrane permeant. Any cysteines
that are part of a disulfide
linkage will not react with NEM,
and once all free cysteines have
reacted with NEM, they are not
available to form disulfide bonds. NEM
was also included at 10
mM in the lysis buffer. Again, immune sheep
serum precipitated
the 72-kDa glycosylated envelope precursor protein,
as well as
the SU and TM proteins (Fig.
2B, lane 2). Rabbit anti-TM
serum
immunoprecipitated all three proteins as well (lane 6), again
indicating that SU was retrieved by its association with TM or
gPr72.
To determine whether the converse was true, the monoclonal
antibody
specific for the D epitope of SU was used to precipitate
envelope
proteins. It, too, retrieved TM protein in addition to
the two proteins
containing SU antigenic determinants (Fig.
2B,
lane 4). The heavy chain
of the monoclonal antibody did not compress
SU (lane 4) as did the
abundant heavy chain in sheep serum (lane
2). Proteins precipitated by
both the immune sheep serum and the
SU-specific monoclonal antibody
from control lysates of CAT-expressing
cells included a cellular
protein (lanes 1 and 3) that migrated
in the same area as the uncleaved
envelope precursor; this background
protein augmented the apparent
abundance of gPr72 in the adjoining
positive samples (lanes 2 and 4).
However, this cellular background
protein was not present in
precipitates of the peptide-specific
TM antibody (lane 5). In summary,
antibodies specific for determinants
found in either SU or TM could
retrieve the other envelope subunit
under conditions preventing new
disulfide bond formation during
manipulation of cellular lysates,
indicating that these proteins
are disulfide bonded in Env-expressing
cells.
If SU and TM are disulfide linked but the bonds are not reduced prior
to electrophoresis, the subunits should migrate on gels
as a complex
with greater mass. Without DTT in the gel sample
buffer, a
high-molecular-weight complex of approximately 94 kDa
was present when
either anti-TM antibody (lane 7) or anti-SU monoclonal
antibody (lane
8) was used. The gPr72 precursor band migrated
just ahead of the 94-kDa
complex; bands representing gp51-SU and
gp30-TM were no longer present
in the precipitates (lanes 7 and
8). Hence, all gp51-SU and gp30-TM
molecules retrieved in the
absence of a reducing agent were engaged in
high-molecular-weight
complexes, reinforcing the conclusion that SU and
TM of BLV are
linked by disulfide bonds when overexpressed in
cells.
Linkage of SU and TM in BLV-producing cells and in virions.
Since these experiments were done with envelope protein that was
overexpressed in the absence of other viral proteins, we asked whether
SU and TM would similarly exhibit disulfide bonding in BLV-infected
cells and in viral particles. BLV-negative B cells of the BL3 tumor
cell line (37) and BLV-infected B cells of the BL3* line
(32) were radiolabeled with [35S]cysteine for
7 h. NEM (10 mM) was then added to half of the cultures, and all
were incubated at room temperature for 15 min. After cells were
harvested by centrifugation at 110 × g for 4 min, the
radioactive medium was saved for isolation of virions. Cell pellets
were washed twice with cold phosphate-buffered saline and disrupted in
2 ml of cold lysis buffer, and the lysates were cleared. To remove
residual cells, the radioactive medium was centrifuged at
3,000 × g for 5 min and then to pellet virus, the resulting supernatant was centrifuged at 47,000 × g
for 20 min in the cold. Viral pellets were resuspended in 1 ml of lysis
buffer (with or without NEM) and cleared.
Anti-TM antibody immunoprecipitated envelope precursor protein and TM
from lysates of NEM-treated BL3* cells; SU was also
present (Fig.
3, lane 6). Only SU and TM were retrieved
from lysates
of viral pellets (lane 8). Without reduction of disulfide
bonds
by DTT (lanes 11 and 12), SU and TM migrated together as a
high-molecular-weight
94-kDa complex and no free SU or TM was detected.
To determine
whether initial treatment with NEM was necessary to
preserve disulfide
bonds, lysates were prepared in its absence. Even
without NEM,
both SU and TM were present in precipitates prepared with
the
anti-TM antibody (lanes 5 and 7). Interestingly, the amounts of
TM
and SU present in precipitates of cells and viral pellets were
less
than those present when cells had first been treated with
NEM (lanes 6 and 8), suggesting that stabilization of disulfide
bonds enhances the
binding of this antibody to TM. Callebaut et
al. (
5) have
similarly reported that binding of antipeptide
antibody to BLV SU is
enhanced when free sulfhydryl groups are
alkylated by treatment with
iodoacetamide.
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FIG. 3.
Recovery of SU in immunoprecipitates of TM from lysates
of BLV-infected cells and viral pellets. Radiolabeled BL3 and BL3*
cells (C) and virions (V) were incubated with or without 10 mM NEM for
15 min at room temperature before being lysed with or without NEM.
Glycoproteins recovered from lysates of 5 × 107 cells
or from virions produced by that number of cells were precipitated with
anti-TM serum, and then the immune complexes were bound to protein
A-Sepharose beads and eluted with double-strength sample buffer with or
without DTT. The image is of a 4-day exposure of a dried,
fluor-impregnated gel to a PhosphorImager screen. The values on the
left are molecular sizes in kilodaltons.
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|
These results indicate that gp51-SU and gp30-TM of BLV are linked
covalently through bonds that are reduced by treatment with
DTT.
Furthermore, all detectable BLV envelope protein subunits
that are
retrieved by binding to lentil lectin appear to be disulfide
bonded
since no free SU or TM was detected under nonreducing conditions.
Recently obtained evidence indicates that when MuLV Env is expressed
in
the absence of other viral proteins, all proteolytically processed
SU
and TM is disulfide bonded (
20). When proteins are
solubilized
in nonionic detergent, the linkage is disrupted in a time-
and
temperature-dependent fashion unless disulfide exchange reactions
are blocked by adding NEM or acidifying the solution. The envelope
subunits of BLV are likely to dissociate under similar circumstances,
which may explain why some previously obtained results indicated
that
these proteins are not linked by disulfide bonds. Under the
conditions
of the experiments reported here, treatment with NEM
was not required
to preserve disulfide bonds. However, in our
own previous work, we did
not retrieve TM when SU was immunoprecipitated
(
15); one
difference is that those immunoprecipitations were
done from cell
extracts rather than from enriched glycoproteins.
In the absence of
NEM, some isomerization of the cysteine residues
around the disulfide
bond may occur. Treatment with NEM or iodoacetamide
may stabilize
envelope protein conformation and allow better recognition
by
peptide-specific antibodies, as shown here and by others for
BLV
envelope protein (
5) and for HIV envelope protein
(
17).
Attachment of the maleimide group of NEM through a thioether linkage to
free cysteine residues prevents them from participating
in a thiol
exchange reaction with cysteines in existing disulfide
bonds. Pinter et
al. (
24) have proposed that such a reaction
can occur during
solubilization of MuLV envelope protein, affecting
the two cysteine
motifs proposed to be involved in the disulfide
bond between SU and TM.
They point out that the CXXC motif within
SU is similar to the
active-site sequence of thiol exchange enzymes
and have proposed that
an isomerization catalyzed by CXXC is necessary
for the conformational
changes required for viral entry into cells
(
24). All five
of the cysteine residues within the two motifs
are important for
correct folding of MuLV envelope protein; mutating
any one diminishes
proteolytic processing of envelope precursor
(
38). HIV
envelope protein has two conserved cysteine residues
in the external
portion of TM in a position analogous to that
of the CX
6CC
motif of oncogenic retroviruses. Changing either
of these cysteines in
HIV TM also impairs proteolytic processing
(
36). We have
found that the third cysteine of the CX
6CC motif
in BLV TM
is essential for proteolytic processing, as substitution
of arginine at
this position results in almost completely unprocessed
protein that
does not support syncytium formation (
14). Knowing
that SU
and TM are linked by a disulfide bond is important for
understanding
how these proteins interact in vivo. This linkage
may play a key role
in conformation changes during receptor binding
and the initiation of
fusion.
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ACKNOWLEDGMENTS |
We thank Glen Cantor for the kind gift of antipeptide antibodies
specific for BLV TM and David Sanders for discussions about intermolecular disulfide bonds in retroviruses. Debbie Grossman provided expert technical assistance.
This work was supported by Public Health Service grant CA-46374 from
the National Cancer Institute and by the UC Davis Cancer Center. E.R.J.
was a predoctoral trainee supported by Public Health Service grant
GM-07377 from the National Institute of Medicine. Instrumentation at
the NSF-funded Plant Genetics Facility, UC Davis, was used to acquire images.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Animal Science, University of California, One Shields Ave., Davis, CA 95616-8521. Phone: (530) 752-9025. Fax: (530) 752-0175. E-mail: KLRadke{at}ucdavis.edu.
Present address: Division of Infectious Diseases, School of
Medicine, Stanford University, Stanford, CA 94305-5107.
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Journal of Virology, March 2000, p. 2930-2935, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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