Characterization of the Biosynthesis of Human Immunodeficiency
Virus Type 1 Env from Infected T-cells and the Effects of Glucose
Trimming of Env on Virion Infectivity*
Markus
Dettenhofer and
Xiao-Fang
Yu
From the Department of Molecular Microbiology and Immunology, Johns
Hopkins University School of Hygiene and Public Health,
Baltimore, Maryland 21205
Received for publication, September 29, 2000, and in revised form, October 27, 2000
 |
ABSTRACT |
HIV (human immunodeficiency virus)-1 Env is
displayed on the surface of infected cells and subsequently
incorporated into virions, which is necessary for the initiation of a
viral infection by recognition of the CD4 and the chemokine receptors
(such as CCR5 or CXCR4) on the surface of new target cells. As a type 1 integral membrane glycoprotein, Env is cotranslationally translocated into the endoplasmic reticulum. In this report, we characterized the
synthesis of Env, which did not occur at a constant rate but by
translational/translocational pausing that has not previously been
shown with a viral encoded glycoprotein. Overall translation was not
impeded by the presence of the reducing agent dithiothreitol in
vivo, although this did influence the cleavage of the precursor gp160 into its mature form, gp120. Env interacts transiently with resident components of the endoplasmic reticulum such as calnexin, which had maximal association at a 10-min post-translation. Addition of
the glucosidase inhibitor, castanospermine, failed to significantly influence the association of Env with calnexin, consistent with the notion that calnexin recognizes components other than
-terminal glucose. Moreover, castanospermine treatment failed to affect the
infectivity of virions. Taken together, this report demonstrates the
existence of translational/translocational pausing for a viral glycoprotein and suggests that trimming of glucose from HIV-1 Env is
not essential for the initiation of virus infection.
 |
INTRODUCTION |
HIV-1,1 as with all
retroviruses, encodes gag, pol, and
env genes. Protein expression of Gag and Pol is directed
from full-length viral genomic RNA, which is also packaged as a dimer
into virions. Gag protein expression is sufficient to drive the budding
and assembly of virions from the plasma membrane (1), while the Pol
proteins act as the major enzymatic components of the retrovirus life
cycle. Env (gp160) traffics through the secretory pathway as a type I
integral membrane protein and is cleaved into its gp120 and gp41 forms
by a cellular protease that resides in the medial to trans Golgi
network (2-5). During the budding process, virions acquire a portion
of the host cell membrane containing the Env molecule, which is
composed of two subunits, the surface domain or gp120 and the
transmembrane protein or gp41. After gp120 successfully binds CD4 and
its respective coreceptor, it is thought that a conformational switch
occurs to expose the fusion peptide of gp41. This then leads to the
fusion of the viral membrane and the new target cell to initiate a new
round of infection.
The translation of Env (gp160) begins as a coupled event with ER
translocation. The N terminus of Env encodes a signal sequence, which
as it emerges from a translating ribosome is recognized by the signal
recognition particle complex. The binding of the signal recognition
particle to the leader sequence recruits the emerging nascent
polypeptide to the Sec61p/translocon of the ER (6). The ribosome then
forms a tight seal over the cytosolic side of the ER membrane at the
translocon pore as the protein is translated. Translocation then
proceeds with the proposed assistance of ER lumenal components such as
BiP (7-10). It has been demonstrated that certain proteins do not
undergo a translationally coupled translocation at a steady rate, but
rather that amino acid coding regions of certain proteins dictate
translocational pausing (11). These pause-transfer sequences mediate
the stopping and restarting of translocation during which the
protein-conducting channel becomes partially opened, exposing the
nascent chain polypeptide to the cytosol (12). These studies have
raised the notion that in addition to the ribosome and the lumenal
components of the ER, the nascent chain may regulate translocation.
During protein translocation into the ER, the signal peptide is cleaved
by the signal peptidase, and the growing nascent polypeptide chain is
modified by the addition of N-linked oligosaccharides within
the lumen of the ER. Alteration in the glycosylation of Env has been
shown to impact the folding of Env (13-15). In addition to the role of
the ER lumenal chaperones in translocation of nascent polypeptides,
BiP, protein-disulfide isomerase, and 94-kDa glucose-regulated protein
act to prevent the misfolding of these proteins (16, 17). The
lectin-like molecular chaperones, calnexin and calreticulin, have been
shown to promote the folding and assembly of glycoproteins within the
ER (18-20). Data have emerged suggesting that the association of the
oxidoreductase, ERp57, in a complex with either calnexin or
calreticulin regulates oxidation and isomerization of newly synthesized
glycoproteins (21, 22). It has been proposed that calnexin acts on
emerging glycoproteins through its association with their sugar
residues (23); however, this has been disputed in a study in which
calnexin was shown to prevent the aggregation of nonglycosylated
proteins in vitro (24). Several reports have shown that the
treatment of cells with drugs that inhibit
-glucosidase, such as
castanospermine (CST) and deoxynojirimycin (DNJ), interfere with the
ability of calnexin to associate with glycoproteins in the ER (25).
This results in the inability of these molecules to fold properly
leading to their retention within the ER (26). Following the misfolding
of proteins within the ER, they undergo a degradative process, probably
mediated by the cytosolic 26 S proteasome (27). The treatment of
HIV-1-infected cells with
-glucosidase inhibitors was shown to
interfere with syncytia formation (28, 29). However, gp120, expressed
in the presence of DNJ (13), and NB-DNJ demonstrated no difference in
their abilities to bind to CD4 or to bind to T-cells (30). These
studies brought us to re-examine the effects that
-glucosidase
inhibitors have on HIV-1 infection.
In this report, we examined the biosynthesis of gp160 Env as it is
translated and translocated into the ER. It was revealed that Env
undergoes several translational/translocational pauses, which are
ATP-dependent but not dependent on disulfide bond
formation. Additionally, Env associates with calnexin in HIV-1-infected
T-cells independent of the glucose trimming pathway. Moreover, the
treatment of HIV-1-infected T-cells with CST fails to interfere with
its secretory pathway trafficking or infectivity on susceptible target cells.
 |
EXPERIMENTAL PROCEDURES |
Cells, DNA Transfection, and Infection--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum and antibiotics and passaged upon confluence. H9 cell
lines were grown in RPMI 1640 with 10% fetal bovine serum and
antibiotics and maintained at a density of <1 × 106
cells/ml.
COS-7 cells were transfected by the DEAE-dextran method. Briefly, COS-7
cells were trypsinized and seeded at 50% confluence 24 h prior to
transfection. Cells (5 × 106) were then trypsinized,
pelleted, and resuspended in 1 ml of TD buffer (25 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, and 0.7 mM K2HPO4) containing 500 µg
of DEAE-dextran and 5 µg of HXB2 or HXB2NEO (31). Transfection was
carried out at 37 °C for 30 min, and the cells were then washed in 5 ml of complete medium and reseeded in T-75 flasks. CST was added
directly to the tissue culture medium at the time of transfection.
Cell culture supernatants containing viral particles were harvested 3 days after transfection, precleared by centrifugation in a Sorvall RT
6000B centrifuge at 3,000 × g for 30 min, filtered through a 0.2-µm pore size membrane, and used for infectivity analysis. Virions were pelleted through a 30% sucrose cushion at
100,000 × g for 90 min.
Pulse-Chase Labeling and Immunoprecipitation--
H9 cells
chronically infected with HXB2NEO were washed twice with PBS and
incubated for 30 min in cysteine-free RPMI 1640 supplemented with 2%
fetal bovine serum. When performing rapid pulse-chase, pulse labeling
was performed for 2 min in cysteine-free RPMI 1640 supplemented with 1 mCi/ml [35S]cysteine (PerkinElmer Life Sciences),
otherwise longer pulse labelings were performed in the presence of 200 µCi/ml [35S]cysteine. The pulse was ended by adding
prewarmed RPMI 1640 supplemented with 10% fetal bovine serum with 5 mM cysteine. After various chase time periods, the cells
were transferred to ice and washed twice in ice-cold PBS (32).
Alternatively, following the 2-min pulse labeling either
cycloheximide (CHX, 0.5 mM) or dithiothreitol (DTT,
5 mM) was added to the chase media, or ATP was depleted
from cells by the addition of prewarmed glucose-free media with 20 mM 2-deoxy-D-glucose and 10 mM
sodium azide (33). Additionally, H9 cells were incubated with 200 µM CST (Sigma) for 45 min prior to pulse labeling, and
the drug remained present throughout the pulse-chase period. For
immunoprecipitation of Env, cells were lysed in a buffer containing 1%
Triton, 10 mM Tris (pH 7.6), 300 mM NaCl, and
protease inhibitors. For coimmunoprecipitation of Env and calnexin,
cells were lysed in a buffer containing 2% CHAPS, 50 mM
Hepes (pH 7.6), 200 mM NaCl, and protease inhibitors. Nuclei were pelleted for 5 min at 1,500 × g, and
postnuclear supernatant was incubated with either sheep anti-gp120
antiserum (AIDS Research Reagents Program, catalog number 288) or
rabbit anti-calnexin antiserum (Stressgene) and protein A-Sepharose
beads (Sigma) for 3 h. Immunoprecipitated samples were washed 3 times in their respective lysis buffers, separated by 7.5% SDS-PAGE,
and analyzed by autoradiography.
Immunoblotting--
Virion-associated viral proteins were
prepared from cell culture supernatants by centrifugation at 3,000 × g for 30 min in a Sorvall RT 6000B centrifuge followed by
filtration of the supernatants through a 0.2-µm membrane. Virus
particle-containing supernatants were concentrated by centrifugation
through a 30% sucrose cushion at 100,000 × g for 90 min in a Sorvall Ultra80. Viral pellets were resuspended in PBS.
Cell-associated viral proteins were analyzed by direct lysis of cells
(1 × 105) in 1 × loading dye (0.08 M Tris, pH 6.8, 2.0% SDS, 10% glycerol, 0.1 M
dithiothreitol, and 0.2% bromphenol blue), samples were boiled for 10 min, and proteins were separated by SDS-PAGE. Membranes were probed
with HIV-1-positive human serum (1:200) or sheep anti-serum against gp120 (1:500). Blots were developed using an alkaline phosphatase reaction.
PNGase F Treatment--
Cell-associated viral proteins were
lysed in the presence of 1% Triton, 10 mM Tris (pH 7.6),
300 mM NaCl, 50 mM
-mercaptoethanol, and
protease inhibitors. Postnuclear supernatants were then boiled for 5 min and treated with PNGase F (Genzyme) for 18 h at 37 °C in
the presence of 1% Nonidet P-40. Samples were boiled for 10 min in
SDS-PAGE loading dye and subjected to immunoblotting.
MAGI Assay--
Viral supernatants from COS-7 transfected cells
were pelleted through 30% sucrose as described above and resuspended
in PBS. Virions were normalized by reverse transcriptase activity as
described previously (31), and 10,000 cpm equivalent of virus was used to initiate an infection. Infections were performed in triplicate using
HeLa CD4-LTR-
-gal cells as targets. Assays were performed as
described previously (34).
 |
RESULTS |
The HIV-1 Env protein is synthesized as a 160-kDa glycoprotein
(gp160). The cleavage of gp160 to gp120 and gp41 indicates the
trafficking of Env through the Golgi stacks. The mature gp120 molecule
arises as a cleavage product of gp160 initially at ~80 min
post-translation as demonstrated in a pulse-chase analysis of H9
infected cells (Fig. 1A).
Additionally, a band of faster mobility than gp160, which reacted with
the anti-Env antisera, was observed in the 0 min chase lane (as
indicated with an asterisk; Fig. 1A). Because
this band was chased out at later time points, we reasoned that it
might be a translational/translocational intermediate of gp160. In
steady-state labeling of H9 infected cell lines, the presence of the
Env precursor (gp160) and the mature cleaved form of gp120 was evident
(Fig. 1B). Upon treatment of gp160 and gp120 with PNGase F
to remove the N-linked sugars, these molecules migrated more
rapidly, consistent with molecular mass of ~100 and 60 kDa,
respectively. A light band between gp120 and gp160 was detected (Fig.
1B, left panel). This protein band is likely to
be a translationally arrested product because its size dropped to ~90
kDa after treatment with PNGase F (Fig. 1B, right
panel).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Pulse-chase analysis of HIV-1 Env and
analysis of N-linked glycosylation. A,
H9 infected cells were pulse-labeled with [35S]cysteine
for 10 min and chased for up to 240 min. Cell lysates were analyzed by
immunoprecipitation with an antiserum to HIV-1 Env at the time
intervals indicated. Lanes containing uninfected cell lysates are
indicated as mock. Translation/translocation intermediate
is indicated by an asterisk. B, H9
infected cells were steady-state-labeled with
[35S]cysteine for 2 h. Cell lysates were
additionally subjected to treatment with PNGase F followed by
immunoprecipitation with an antiserum to HIV-1 Env.
|
|
We next examined the biosynthesis of gp160 during the time of protein
translation. HIV-1-infected cells were pulse-labeled for 2 min and
chased for up to 10 min to examine the translational intermediate
products. Surprisingly, distinct bands, which were immunoprecipitated
with an antiserum against gp160 but of lower molecular weight, were
observed (Fig. 2A). These
molecules were evident during very brief labeling periods and could be
chased out during the time period examined. To examine whether these molecules were translational/translocational intermediates, CHX was
added during the pulse period to stall translation, which resulted in
the sustained presence of these lower molecular weight Env (gp160)
intermediates (Fig. 2B). We additionally analyzed Env-related polypeptides, which were either treated with CHX or left
untreated during their biosynthesis, and ran them side-by-side on
SDS-polyacrylamide gels (Fig. 2C). With the addition of
deoxy-D-glucose and sodium azide to the tissue culture
system to deplete ATP (33), we observed a similar pausing of the Env
protein during translation (Fig. 2D). Disruption of
disulfide bond formation by the addition of DTT to the chase
media of infected cells (35) failed to significantly impede
translation of Env (Fig. 2E). Because translation and ER translocation are thought to be coupled in mammalian cells, it remained
difficult to make a distinction between these two steps. These data
demonstrate that the synthesis of the Env glycoprotein does not proceed
at a steady rate but rather is marked by periods of
translational/translocational pausing.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 2.
Analysis of translation/translocation pausing
in the biosynthesis of HIV-1 Env. H9 infected cells were
pulse-labeled with [35S]cysteine for 2 min, then
incubated with either CHX or DTT or depleted of ATP by the addition of
2-deoxy-D-glucose and sodium azide, and chased for up to 10 min. Cell lysates were analyzed by immunoprecipitation with an
antiserum to HIV-1 Env at the time intervals indicated. Following the
2-min pulse labeling H9 infected cells were either untreated
(A), treated with 0.5 mM CHX (B and
C), depleted of ATP by the addition of prewarmed
glucose-free media supplemented with 20 mM
2-deoxy-D-glucose and 10 mM sodium azide
(D), or treated with 5 mM DTT
(E). C, uninfected and infected H9 cell lysates,
which were either treated with 0.5 mM CHX or remained
untreated during a 10-min chase period, were run side-by-side and
analyzed by SDS-PAGE. Translation/translocation intermediates are
indicated by asterisks.
|
|
We next wanted to test the effect of DTT addition on the stability and
cleavage of Env in infected cells. In a pulse-chase analysis, the
addition of DTT to the chase media interfered with the cleavage of
gp160 into its gp120 form (Fig.
3B). When steady-state labeling experiments were performed in the presence of DTT, not only
were the higher molecular weight Env-related complexes disrupted (as
indicated by arrowheads in Fig. 3C), but the
overall abundance of gp160 was diminished. These data are consistent
with the notion that misfolding of Env causes it to be subjected to the
quality control mechanisms within the ER, leading to its ER retention and removal by decay pathways.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of in vivo DTT
addition on HIV-1 Env stability and processing. A and
B, H9 infected cells were pulse-labeled with
[35S]cysteine for 10 min and chased for up to 120 min.
Cell lysates were analyzed by immunoprecipitation with an antiserum to
HIV-1 Env at the time intervals indicated. Following the 10-min pulse
labeling H9 infected cells were either untreated (A)
or treated with 5 mM DTT (B). C, H9
infected cells were steady-state-labeled with
[35S]cysteine for 2 h in the presence or absence of
5 mM DTT. Cell lysates were immunoprecipitated with
antiserum against Env and either reduced with -mercaptoethanol
( -ME) or left untreated prior to SDS-PAGE analysis.
Immunoprecipitated Env-related complexes are indicated by
arrowheads.
|
|
One of the mechanisms by which functional maturation of glycoproteins
proceeds is through their association with resident ER chaperones. One
such molecule, calnexin, has been shown to retain misfolded molecules
within the ER in part via recognition of the
-terminal glucose of
N-linked glycoproteins (25). Again, using pulse-chase
analysis with antisera against Env (Fig.
4A) and calnexin (Fig.
4B), an association between gp160 and calnexin was shown to
have a t1/2 of 10 min after the initiation of
Env synthesis (Fig. 4B). To examine what role the
-terminal glucose had on the association of Env with
calnexin, cells were treated with an
-glucosidase inhibitor, CST,
prior to radiolabeling. Mock-infected and HXB2-infected cells were
pulse-labeled and -chased for 60 min followed by immunoprecipitation
with antiserum to Env and CN. Here we observed no significant
disruption of the association of gp160 and calnexin with CST treatment
(Fig. 5A). Treatment of
infected cells with CST caused Env to have a slower mobility by
SDS-PAGE (indicated by arrowheads). As a control, infected cells that were labeled with [35S]cysteine for 10 min in
the presence of DTT caused the association between gp160 and CN to be
significantly disrupted (Fig. 5B, lanes 6 and
8). In contrast, the treatment of HIV-1-infected cells with CST did not appear to impact the interaction between Env and CN (Fig.
5B, lanes 5 and 7). Besides the
effects that CST treatment has on the glucose trimming of Env, we
addressed whether it had any additional impact on the formation and
infectivity of virions. When CST was added in increasing
concentrations, a mobility shift in Env was evident for the
cell-associated molecules (Fig.
6A, indicated by
arrows), however no significant differences were observed
for the Gag-related proteins or for reverse transcriptase within the
cell (Fig. 6B). Env was present in similar quantities within
cell-free virions irrespective of whether viral producer cells were
grown in CST (Fig. 6C), indicating that CST has little influence on the trafficking or retention of Env through the secretory pathway. In addition, virion-associated Gag, Integrase, reverse transcriptase, and gp41 were not significantly altered by CST treatment
(Fig. 6D) nor was the infectivity of CST-treated virions impeded in a MAGI assay (Fig. 6E).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Association of HIV-1 Env with calnexin.
H9 infected cells were pulse-labeled with [35S]cysteine
for 2 min and chased for up to 30 min. Cell lysates were analyzed by
immunoprecipitation with an antiserum to HIV-1 Env
( -Env) (A) and calnexin
( -CN) (B) at the time intervals
indicated.
|
|

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of CST and DTT treatment on HIV-1 Env
association with calnexin. A, H9 mock-infected or
HXB2-infected cells were treated with 200 µM CST 45 min
prior to pulse labeling with [35S]cysteine for 10 min and
chased for 60 min in the presence of CST. B, H9 infected
cells were labeled for 10 min with [35S]cysteine either
in the absence of modifying chemicals or in the presence of 200 mM CST, 5 mM DTT, or a combination of both.
Cell lysates were analyzed by immunoprecipitation with an antiserum to
HIV-1 Env ( -Env) and calnexin
( -CN). Untreated gp160 are indicated by
arrowheads, and CST treated gp160 are indicated by
double-arrowheads.
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of CST treatment on HIV-1 virion
infectivity. COS-7 cells transfected with HXB2 were treated with
0.2 µM, 0.4 µM, 0.6 µM CST or
left untreated at the time of transfection. Three days
post-transfection, cell lysates (A and B) and
cell-free virions (C and D) were examined by
immunoblotting with antiserum against Env
( -Env) (A and C) or
HIV-1-positive patient serum ( -HIV-1)
(B and D). E, cell-free virions were
used to infect MAGI cells. Infected cells were scored as
-galactosidase-positive 48 h postinfection; experiments
were performed in triplicate. Untreated gp120 are indicated by
arrowheads, and CST-treated gp120 are indicated by
double arrowheads. Untreated gp160 are indicated by arrows,
and CST-treated gp160 are indicated by double arrows.
|
|
 |
DISCUSSION |
Evidence has emerged suggesting that the nascent chain polypeptide
may be exposed at times to the cytosolic side of the ER membrane during
cotranslational translocation (36). In fact, nascent polypeptides
containing specific amino acid sequences, termed pause-transfer
sequences, have been demonstrated to induce temporary pauses in
translocation that coincide with nascent chain accessibility to the
cytosolic side of the ER membrane (11, 12). These observations for
translational/translocational pausing from an in vitro
system have also been supported by evidence in tissue culture cells
with apolipoprotein B (37). In this report we examined the biosynthesis
of the Env protein in HIV-1-infected T-cells during the time of
translation/translocation into the ER. We find several pause-transfer
points as part of normal ER translocation, experiments which have not
been reported for a viral protein previously (Fig. 2). This
temporary pausing and restarting of cotranslational translocation was,
as expected, dependent on the ribosomal passage over the env
mRNA because CHX treatment impeded further
translation/translocation. Additionally, restarting of the paused
intermediates was dependent on ATP, which might suggest the involvement
of the ER translocation components rather than strictly the
translational machinery. It has previously been noted that ER lumenal
proteins, which use ATP as an energy source, may regulate the
translocation of nascent chain proteins into the ER (7-9). However,
demonstration of cytosolic Hsp70 binding to nascent chains of
apolipoprotein B in an ATP-dependent manner suggests the
additional involvement of non-ER lumenal components in cotranslational
translocation (38). Although the existence of pause-transfer sites may
be dependent on the amino acid sequences within the nascent chain
polypeptide itself (11), the restarting of these pausing events may
require additional associating molecules. The varied rate of
translation/translocation of HIV-1 Env highlights an additional level
of regulation. By pausing during translation/translocation of the
nascent chain polypeptide, the addition of sugars and
chaperone-assisted folding may occur in a regional manner to ensure the
production of functional Env. Our data suggest that the restarting of
Env pause sites during its biosynthesis appears to be dependent on translation as well as ATP and not the formation of disulfide bonds.
The ER houses several molecule chaperones such as BiP,
protein-disulfide isomerase, 94-kDa glucose-regulated protein,
calnexin, calreticulin, and ERp57, which serve to prevent the
misfolding of newly synthesized glycoproteins (25). To this end,
misfolded proteins tend to be retrieved from further transport through
the secretory pathway until properly folded. This understanding has raised the notion that the ER acts as a cellular quality control compartment (25). Retention of misfolded molecules within the ER
usually leads to their degradation (26), a process dependent on the
cytosolic 26 S proteasome (27). ER misfolding pathways may be studied
by the introduction of mutations into glycoproteins and by the addition
of chemicals, such as DTT, which disrupt the disulfide bonding of these
molecules (35). The use of DTT to treat HIV-1-infected T-cells had
little effect on the restarting of Env translational/translocational
pause intermediates (Fig. 2E), however it did result in a
failure of gp160 to reach the Golgi compartment as evidenced by the
absence of the cleaved gp120 form of Env (Fig. 3). DTT addition
in vivo consequently leads to a marked reduction in the
overall levels of Env (Fig. 3C, lane 2), which
was independent of its translation/translocation (Fig. 3B).
ER resident chaperones are thought to associate with newly synthesized
glycoproteins to regulate their folding and retain misfolded molecules
within the ER. Because the addition of DTT disrupted the association
between Env and calnexin (Fig. 5B), it suggests that other
chaperones may dictate the retention of misfolded Env within the ER.
For example, BiP has been shown to bind newly synthesized glycoproteins
in a sequential manner prior to calnexin (39).
It has been shown that treatment of cells with CST, an
-glucosidase
inhibitor, can disrupt the association of calnexin with various viral
glycoproteins because calnexin is thought to recognize the trimmed
monoglucosylated form of these glycoproteins (22, 25, 39, 40). However,
the association of HIV-1 Env with calnexin after treatment with CST
appeared to be unaltered (Fig. 5). This is consistent with a previous
report that demonstrated that CST treatment failed to abolish the
association of gp160 with calnexin (41). These studies suggest that
calnexin does not recognize HIV-1 Env only through its interaction with
the monoglucosylated core glycans. In a recent report, it was
demonstrated that calnexin recognized the polypeptide portion of
unfolded molecules in vitro, suggesting that it may not be
acting solely as a lectin-associating chaperone (24). Although
association of calnexin and Env in infected T-cells may not be
dependent on the glucosylation state of Env, we cannot rule out the
possibility of subtle interaction differences between CST-treated and
untreated samples. Additionally it could be argued that Env association
with calnexin may be through a trimeric complex with ERp57 and that
this thiol-dependent reductase plays a more critical role
in this particular association (21, 22). It remains possible that
calnexin binds the unfolded state of Env and plays a role in preventing
the aggregation of misfolded molecules. This remains intriguing
considering the association of translation/translocation intermediates
of gp160 with calnexin in the earliest chase times (Fig. 4B,
2 and 5 min). Although the inhibition of
terminal glucose trimming demonstrated limited disruption in calnexin
association with Env, their inaction may still be critical for the
formation of functionally active Env.
Previous studies have examined the effects of CST on HIV-1-infected
cells and have shown a reduction in the formation of syncytia and virus
infectivity (28, 29). These reports have either assayed the effect of
CST on viral replication in multiple rounds or have used cell culture
supernatants containing CST as a source of virus input. Because CST
does not specifically target viral glycoproteins, it remained plausible
that its use may confer sublethal cell toxicity that could alter other
molecules of cellular origin effecting virus cell-to-cell spread. In
the present study, we observed little to no effect in the inhibition of
infectivity of cell-free virions produced in the presence of CST (Fig.
6E). The differences in the results may be explained by the
fact that we tested cell-free virions in a one-round infectivity assay
rather than growing virally infected cells in the presence of
-glucosidase inhibitors over the course of several days.
Additionally, we removed all CST from the cell culture supernatants by
pelleting virus before infecting target cells, thus minimizing
the effects CST may have on these cells. In fact, we demonstrate that
CST caused a retardation in the mobility of both gp160 and gp120,
characteristic of the absence of glucose trimming of these molecules
(Fig. 6). Additionally, the treatment of HIV-1-infected cells with CST
has no significant impact on the virion incorporation of gp120 and gp41, suggesting that trafficking of Env through the secretory pathway
was unimpeded. These results are consistent with reports using DNJ and
NB-DNJ to inhibit glucosidase I and II in the production of Env, which
demonstrated no effect on CD4 binding (13, 30). However, treatment of
HIV-1-infected cells with NB-DNJ did display a post-CD4 binding entry
defect (30), which was not observed with CST treatment (Fig.
6E). These differences may be explained because NB-DNJ
inhibits ER glucosidases generally (42), potentially conferring a more
severe phenotype. Our data suggest that glucose trimming is not
essential for the initiation of cell-free virus infection nor does it
significantly influence the association of HIV-1 Env with calnexin.
 |
ACKNOWLEDGEMENTS |
We thank Philippe Depres, Ari Helenius, and
Vishwanath Lingappa for useful discussion and Carolyn Machamer and
Richard Markham for critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by a grant from the National
Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: 615 N. Wolfe St.,
Baltimore, MD 21205. Tel.: 410-955-3768; Fax: 410-614-8263; E-mail:
xfyu@jhsph.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008933200
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
Env, envelope;
ER, endoplasmic
reticulum;
BiP, immunoglobulin heavy chain binding protein;
CST, castanospermine;
DNJ, deoxynojirimycin;
NB-DNJ, N-butyldeoxynojirimycin;
PBS, phosphate-buffered saline;
CHX, cycloheximide;
DTT, dithiothreitol;
PAGE, polyacrylamide
gel electrophoresis;
PNGase, N-glycosidase F;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CN, calnexin.
 |
REFERENCES |
1.
|
Freed, E. O.
(1998)
Virology
251,
1-15[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Willey, R. L.,
Bonifacino, J. S.,
Potts, B. J.,
Martin, M. A.,
and Klausner, R. D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
9580-9584[Abstract]
|
3.
|
Hunter, E.,
and Swanstrom, R.
(1990)
Curr. Top. Microbiol. Immunol.
157,
187-253[Medline]
[Order article via Infotrieve]
|
4.
|
Earl, P. L.,
Moss, B.,
and Doms, R. W.
(1991)
J. Virol.
65,
2047-2055[Medline]
[Order article via Infotrieve]
|
5.
|
Freed, E. O.,
and Martin, M. A.
(1995)
J. Biol. Chem.
270,
23883-23886[Free Full Text]
|
6.
|
Matlack, K. E. S.,
Mothes, W.,
and Rapoport, T. A.
(1998)
Cell
92,
381-390[Medline]
[Order article via Infotrieve]
|
7.
|
Vogel, J.,
Misra, L. M.,
and Rose, M. D.
(1990)
J. Cell Biol.
110,
1885-1895[Abstract]
|
8.
|
Nguyen, T. H.,
Law, D. T. S.,
and Williams, D. B.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
1565-1569[Abstract]
|
9.
|
Sanders, S. L.,
Whitfield, K. M.,
Vogel, J. P.,
Rose, M. D.,
and Schekman, R. W.
(1992)
Cell
69,
353-365[Medline]
[Order article via Infotrieve]
|
10.
|
Nicchitta, C. V.,
and Blobel, G.
(1993)
Cell
73,
989-998[Medline]
[Order article via Infotrieve]
|
11.
|
Chuck, L. M.,
and Lingappa, V. R.
(1992)
Cell
68,
9-21[Medline]
[Order article via Infotrieve]
|
12.
|
Hegde, R. S.,
and Lingappa, V. R.
(1996)
Cell
85,
217-228[Medline]
[Order article via Infotrieve]
|
13.
|
Fennie, C.,
and Lasky, L. A.
(1989)
J. Virol.
63,
639-646[Medline]
[Order article via Infotrieve]
|
14.
|
Lee, W.-R., Yu, X.-F.,
Syu, W.-J.,
Essex, M.,
and Lee, T.-H.
(1992)
J. Virol.
66,
1799-1803[Abstract]
|
15.
|
Warrier, S. V.,
Pinter, A.,
Honnen, W. J.,
Girard, M.,
Muchmore, E.,
and Tilley, S. A.
(1994)
J. Virol.
68,
4636-4642[Abstract]
|
16.
|
Weissman, J. S.,
and Kim, P. S.
(1993)
Nature
365,
185-188[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Melnick, J.,
Dul, J. L.,
and Argon, Y.
(1994)
Nature
370,
373-375[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Degen, E.,
and Williams, D. B.
(1991)
J. Cell Biol.
112,
1099-1115[Abstract]
|
19.
|
Ou, W.-J.,
Cameron, P. H.,
Thomas, D. Y.,
and Bergeron, J. J. M.
(1993)
Nature
364,
771-776[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Ware, F. E.,
Vassilakos, A.,
Peterson, P. A.,
Jackson, M. R.,
Lehrman, M. A.,
and Williams, D. B.
(1995)
J. Biol. Chem.
270,
4697-4704[Abstract/Free Full Text]
|
21.
|
Oliver, J. D.,
van der Wal, F. J.,
Bulleid, N. J.,
and High, S.
(1997)
Science
275,
86-88[Abstract/Free Full Text]
|
22.
|
Molinari, M.,
and Helenius, A.
(1999)
Nature
402,
90-93[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Zapun, A.,
Petrescu, S. A.,
Rudd, P. M.,
Dwek, R. A.,
Thomas, D. Y.,
and Bergeron, J. J. M.
(1997)
Cell
88,
29-38[Medline]
[Order article via Infotrieve]
|
24.
|
Ihara, Y.,
Cohen-Doyle, M. F.,
Saito, Y.,
and Williams, D. B.
(1999)
Mol. Cell
4,
331-341[Medline]
[Order article via Infotrieve]
|
25.
|
Ellgaard, L.,
Molinari, M.,
and Helenius, A.
(1999)
Science
286,
1882-1888[Abstract/Free Full Text]
|
26.
|
Klausner, R. D.,
and Sitia, R.
(1990)
Cell
62,
611-614[Medline]
[Order article via Infotrieve]
|
27.
|
Hiller, M. M.,
Finger, A.,
Schweiger, M.,
and Wolf, D. H.
(1996)
Science
273,
1725-1728[Abstract/Free Full Text]
|
28.
|
Gruters, R. A.,
Neefjes, J. J.,
Tersmette, M.,
de Goede, R. E. Y.,
Tulp, A.,
Huisman, H. G.,
and Ploegh, H. L.
(1987)
Nature
330,
74-77[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Walker, B. D.,
Kowalski, M.,
Goh, W. C.,
Kozarsky, K.,
Krieger, M.,
Rosen, C.,
Rohrschneider, L.,
Haseltine, W. A.,
and Sodroski, J.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
8120-8124[Abstract]
|
30.
|
Fischer, P. B.,
Collin, M.,
Karlsson, G. B.,
James, W.,
Butters, T. D.,
Davis, S. J.,
Gordon, S.,
Dwek, R. A.,
and Platt, F. M.
(1995)
J. Virol.
69,
5791-5797[Abstract]
|
31.
|
Dettenhofer, M.,
and Yu, X. F.
(1999)
J. Virol.
73,
1460-1467[Abstract/Free Full Text]
|
32.
|
Braakman, I.,
Hoover-Litty, H.,
Wagner, K. R.,
and Helenius, A.
(1991)
J. Cell Biol.
114,
401-411[Abstract]
|
33.
|
Braakman, I.,
Helenius, J.,
and Helenius, A.
(1992)
Nature
357,
260-262
|
34.
|
Klimpton, J.,
and Emerman, M.
(1992)
J. Virol.
66,
2232-2239[Abstract]
|
35.
|
Braakman, I.,
Helenius, J.,
and Helenius, A.
(1992)
EMBO J.
11,
1717-1722[Abstract]
|
36.
|
Liao, S.,
Lin, J.,
Do, H.,
and Johnson, A. E.
(1997)
Cell
90,
31-41[Medline]
[Order article via Infotrieve]
|
37.
|
Thrift, R. N.,
Drisko, J.,
Dueland, S.,
Trawick, J. D.,
and Davis, R. N.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
9161-9165[Abstract]
|
38.
|
Zhou, M.,
Wu, X.,
Huang, L. S.,
and Ginsberg, H. N.
(1995)
J. Biol. Chem.
270,
25220-25224[Abstract/Free Full Text]
|
39.
|
Hammond, C.,
and Helenius, A.
(1994)
Science
266,
456-458[Medline]
[Order article via Infotrieve]
|
40.
|
Hammond, C.,
Braakman, I.,
and Helenius, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
913-917[Abstract]
|
41.
|
Otteken, A.,
and Moss, B.
(1996)
J. Biol. Chem.
271,
97-103[Abstract/Free Full Text]
|
42.
|
Saunier, B. R.,
Kilker, D. J.,
Tkacz, J. S.,
Quaroni, A.,
and Herscovics, A.
(1982)
J. Biol. Chem.
257,
14155-14161[Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.