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Journal of Virology, August 2000, p. 6769-6776, Vol. 74, No. 15
Aaron Diamond AIDS Research Center, The
Rockefeller University, New York, New York 10021-6399
Received 13 March 2000/Accepted 1 May 2000
We examined the role of asparagine-linked glycosylation of the V2
loop of the human immunodeficiency virus (HIV) SF162 envelope on viral
replication potential and neutralization susceptibility. We report that
the asparagines located at the amino- and carboxy-terminal sites
(at positions 154 and 195, respectively), as well as within the V2 loop
of the SF162 envelope (at position 186), are glycosylated during in
vitro replication of this virus in human peripheral blood mononuclear
cells. Our studies indicate that glycosylation of the V2 loop, in
particular at its base, facilitates the interaction of the HIV envelope
with the CD4 and CCR5 receptor molecules present on the surface of
target cells and affects viral replication kinetics in a cell
type-dependent manner. In cells expressing high numbers of receptor
molecules on their surfaces, the SF162-derived V2 loop-deglycosylated
mutant viruses replicate as efficiently as the parental SF162 virus,
while in cells expressing small numbers of receptor molecules, the
mutant viruses replicate with markedly reduced efficiency. In addition
to expanding the viral tropism, V2 loop glycosylation at the three
sites examined prevents neutralization by anti-CD4 binding
site antibodies. In contrast, glycosylation at the amino- and
carboxy-terminal sites of the V2 loop but not within the loop itself
offers protection against anti-V3 loop antibodies. Thus, the
epitopes masked by the sugar molecules present on the three
glycosylation sites examined are not identical but overlap.
Approximately 50% of the mass of
the human and simian immunodeficiency virus (HIV and SIV, respectively)
envelope glycoproteins is attributed to N- and O-linked sugar molecules
(2, 28). Envelope glycosylation affects not only the
intracellular transport of these viral proteins, but also their
structure, and therefore influences the viral phenotype (6, 10,
15, 20, 23, 29, 31, 39, 45, 64).
Moreover, envelope glycosylation plays an important role in the
interaction of these viruses with the immune system of the infected
host by reducing the recognition of specific epitopes by T
lymphocytes (3) and by decreasing epitope immunogenicity (44). During infection of macaques by SIV and simian/human
immunodeficiency virus (SHIV), the emergence of
neutralization-resistant viruses in the blood circulation of the
infected host, which usually precedes or coincides with the development
of disease, is associated with changes in the envelope glycosylation
pattern (5, 36-38, 47). This resistance is partially due to
the repositioning of glycosylation sites within the viral envelope,
which results in the "masking" of neutralization epitopes
(1, 41, 44, 47-49).
Removal of glycosylation sites located in the first hypervariable
region (V1 loop) of the SIVmac239 envelope enhances the exposure of as yet unknown neutralization epitopes and increases their immunogenicity (44). During infection of macaques with mutant SIVmac239 viruses lacking asparagine-linked
glycosylation sites in the V1 loop, high titers of antibodies that
recognize the newly exposed epitopes are generated. Important for
vaccine development is the observation that sera collected from
macaques infected with such partially deglycosylated
SIVmac239 mutant viruses have a higher neutralization
potential against the parental fully glycosylated SIVmac239
virus than sera collected from macaques infected with the parental
virus itself (44).
The above observations strongly suggest that investigation of the
mechanisms by which HIV or SIV envelope glycosylation affects the viral
phenotype will not only provide us with information regarding the
biology of this pathogen, but may also assist us in the development of
more effective anti-HIV (or anti-SIV) envelope-based immunogens.
We previously reported that on the background of the
neutralization-resistant HIV-1 isolate SF162, the elimination of 30 amino acids (from T160 to Y189) from the central region of the second hypervariable envelope region (V2 loop) renders the virus (termed SF162 Elimination by mutagenesis of potential N-linked glycosylation
sites.
Elimination of the potential glycosylation sites was
performed as previously described (43, 44) by replacing the
asparagines (N) with glutamines (Q) at positions 154, 186, and 195 of
the SF612 envelope, using the QuickChange site-directed mutagenesis kit
(Stratagene). We designate the glycosylation mutant (GM) envelopes as
follows: N 154 replaced by Q is GM154; N 186 replaced by Q is GM186;
and N 195 replaced by Q is GM195.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
V2 Loop Glycosylation of the Human Immunodeficiency Virus Type 1 SF162 Envelope Facilitates Interaction of This Protein with CD4 and
CCR5 Receptors and Protects the Virus from Neutralization by
Anti-V3 Loop and Anti-CD4 Binding Site Antibodies
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
V2) highly susceptible to neutralization by antibodies present in sera from HIV-infected patients (54). It appears that on the background of the SF162 virus, this partial V2 loop deletion increases the exposure of neutralization epitopes whose structure is conserved among heterologous HIV-1 isolates. The above deletion results in the loss of one potential asparagine-linked glycosylation site located in the V2 loop (at position 186 of the SF162 envelope). Therefore, it is possible that the switch in viral phenotype observed upon V2 loop deletion is not due to the elimination of the 30 amino
acids per se, but rather to the elimination of the sugar moieties
present on the asparagine at position 186. In the studies presented
here, we investigated the role of this particular potential glycosylation site, as well as the roles of all other potential N-linked glycosylation sites present in the V2 loop of the SF162 envelope, in viral replication, tropism, and susceptibility to antibody-mediated neutralization. This investigation expands our understanding of the relationship between HIV envelope structure and
viral phenotype.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Generation of infectious virions with a partially deglycosylated
V2 loop.
293T cells were cotransfected with pUC DNA vectors
expressing the 5' and 3' (containing the parental SF162 or the
mutagenized envelope gene) halves of the viral genome using
Lipofectamine, as previously described (54). At 72 h
posttransfection, the cell supernatants were collected, subjected to
centrifugation (10 min at room temperature at 2,000 rpm), filtered
through 0.45-µm-pore membranes, and used to inoculate human
peripheral blood mononuclear cells (PBMC) that had been stimulated for
3 days with 3 µg of phytohemagglutinin (PHA; Sigma) per ml. Following
a 3-h incubation at 37°C, the viral inoculum was removed, and the
cells were cultured in RPMI medium supplemented with 10% fetal bovine
serum (FBS) (BioWhittaker), penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (2 mM) and interleukin-2 (IL-2; 20 U/ml)
(Hoffmann-La Roche, Inc.). Virus production was monitored by
determining the concentration of p24 antigen in the cell supernatant
every 3 to 4 days using an in-house p24 detection enzyme-linked
immunosorbent assay (ELISA). Supernatants with high p24 content were
collected, filtered, and stored as 0.5-ml aliquots at 
80°C. These
viral stocks were titrated in PBMC and used for all subsequent experiments.
Electrophoretic mobility analysis of virion-associated gp120 proteins. Intact infectious viral particles produced in PBMC were first separated from free gp120 molecules by centrifugation at 13,500 × g for 2 h at 4°C. The viral pellet was resuspended in 40 µl of lysis buffer (50 mM Tris-HCl, 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol) and subjected to boiling for 5 min. The denatured viral proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 5% gels, then transferred to Immobilon P membranes (Millipore), and incubated overnight at 4°C with goat-anti-SF2 gp120 serum antibodies (Chiron; 1:1,000 dilution) and for 2 h at room temperature with protein G-horseradish peroxidase (Bio-Rad; 1:1,000 dilution). Visualization of the gp120 envelope molecules was performed with the use of enhanced chemiluminescence reagents (Amersham).
Infectivity assays. (i) PBMC and macrophages. PHA-activated human PBMC were generated as previously described (9) from the blood of HIV-negative donors (New York Blood Center) and cultured in the above-mentioned RPMI medium. Macrophages were prepared by the plastic adherence method as previously described (9) and cultured in RPMI medium supplemented with 5% homologous human serum, 10% FBS, penicillin, streptomycin, and glutamine. PBMC (2 × 106) and macrophages (105 per well of a 12-well plate) were inoculated in triplicate with 200 or 400 50% tissue culture infective doses (TCID50), respectively, of each isolate for 3 h at 37°C. The viral inoculum was removed, and the cells were cultured in the appropriate medium (2 ml for PBMC and 1 ml for macrophages). The p24 antigen concentration in the cell supernatants was determined every 3 to 4 days, during which time the medium was replaced. The replication of all isolates (two clones for each mutant) was tested simultaneously. Experiments were repeated at least three times using target cells from different donors to eliminate potential donor-specific effects on viral infectivity.
(ii) HeLa cell lines. Infection of HeLa cells expressing various numbers of CD4 and CCR5 receptor molecules on their surface by the various viruses was analyzed as previously described (25, 42). Briefly, 2.5 × 105 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mM) were added per well of a 12-well plate (Corning). The next day, the medium was aspirated and the cells were inoculated with 500 TCID50 of each isolate and incubated for 3 h at 37°C. Triplicate wells for each isolate were used. The inoculum was removed, and fresh DMEM was added (1 ml per well). After 72 h, the medium was aspirated and the cells were fixed for 10 min with acetone-methanol (1:1) at room temperature. Thereafter, the cells were incubated (1 h at room temperature) with serum (1:150 dilution) from HIV-positive patients, then with a sheep anti-human immunoglobulin G (IgG)-horseradish peroxidase-coupled antibody (Amersham; 1:100 dilution), and finally with a diaminobenzidine peroxidase substrate solution (Sigma). The number of foci of infected cells present in each well was determined.
Neutralization assays. The neutralization assays were performed as previously reported (54, 58). Briefly, each virus (100 TCID50 in 50 µl of the above RPMI medium) was mixed with an equal volume of serially diluted heat-inactivated HIV-positive serum or monoclonal antibodies (MAbs) for 1 h at 37°C. For each serum or MAb dilution, triplicate wells of a U-bottomed 96-well plate were used. PBMC (4 × 105 in 0.1 ml of medium) were added to each well. Virus was also incubated with cells in the absence of HIV-positive serum or MAb. These wells served as positive controls (0% neutralization). Approximately 24 h later, half the cell supernatant of each well (containing virus and serum or MAbs) was replaced with fresh medium. This procedure was repeated twice. The percent neutralization in the presence of each serum or MAb dilution was determined as previously described (58). Neutralization experiments were performed two to three times using pooled PBMC from two donors in order to eliminate potential effects on viral infectivity that may be associated with the cells of a particular donor.
Determination of the relative envelope content of wild-type and mutant virions. We used well-established ELISAs to determine envelope content. Briefly, intact infectious virions from the wild-type and mutant viruses produced in PBMC were separated from free gp120 molecules by centrifugation as mentioned above. The virion pellet was lysed with Tris-buffered saline (TBS)-2% Empigen detergent by an overnight incubation at room temperature. Equal volumes (200 µl per well) from the viral lysates were added to ELISA wells coated with either 6203 (5 µg/ml) or 6205 (5 µg/ml) sheep antibodies (International Enzymes, Inc.), and twofold serial dilutions were made in TBS-2% Empigen. The first polyclonal antibody recognizes various regions of the HIV-1 p24gag protein, while the second recognizes the carboxy-terminal 15 amino acids of the HIV-1 gp120 subunit. The relative amount of the captured viral proteins was determined as previously described (55, 58). The ratio of optical density at 490 nm signals from the 6205 and 6203 wells reports on the relative gp120 to p24 protein ratio in these virions.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Glycosylation of asparagines at positions 154, 186, and 195 of the
SF162 envelope.
The positions of the three potential
asparagine-linked glycosylation sites in the V2 loop of the SF162 gp120
envelope subunit are shown in Fig. 1. It
is expected that the individual elimination by mutagenesis of a
utilized N-linked glycosylation site on the HIV-1 gp120 will reduce the
molecular size of this protein by approximately 2.5 kDa
(24). This in turn will increase the relative electrophoretic mobility of the mutated envelope gp120 protein compared
to the fully glycosylated parental protein. In contrast, if the site is
not utilized, no change in electrophoretic mobility will be recorded.
To examine whether the asparagines at the positions of interest
are glycosylated during the in vitro replication of SF162 in human
PBMC, we compared the electrophoretic mobility of
virion-associated gp120 molecules from the GM154, GM186, and GM195 virions to that of gp120 molecules derived from the parental SF162 virus (Fig. 2). The gp120 molecules
derived from all three mutant viruses migrated faster than those
derived from the SF162 virus. This indicates that the asparagines at
all three positions are normally glycosylated when SF162 is propagated
in human PBMC in vitro.
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Viral replication in PBMC and macrophages.
To examine whether
envelope glycosylation at positions 154, 186, and 195 of the SF162
envelope affects the replication potential of this virus, we compared
the replication kinetics of the three glycosylation mutant viruses to
that of the parental SF162 virus using as target cells PHA-activated
human PBMC and primary macrophages (Fig.
3). All three mutant viruses replicated
to titers as high as those of the parental SF162 virus in PBMC, with
only slight delays in their replication kinetics (Fig. 3A). Although
minor variations in the relative replication kinetics of the three
mutant isolates were recorded from experiment to experiment,
the GM154 virus consistently replicated with slower kinetics than
the GM186 and GM195 viruses.
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Infection of HeLa cell lines expressing various numbers of CD4 and CCR5 molecules on their surface. Differentiated human macrophages express fewer CD4 and CCR5 molecules than activated PBMC on their surfaces (16, 35, 63). The decreased replication ability of GM154 and GM195 viruses in macrophages could be due to a less efficient utilization of the CD4 and/or CCR5 molecules by their partially deglycosylated gp120 envelope molecules. To examine this, we compared the efficiency with which the various isolates infect HeLa cell lines expressing various amounts of CD4 or CCR5 molecules on their surfaces (25, 42).
Our results (Table 1) are in agreement with previous reports indicating that the infectious potential of SF162 is dependent on the presence of both CD4 and CCR5 molecules on the surface of target cells (8, 42). The sole presence of CD4 molecules (at both low and high numbers; cells HI-R and HI-J, respectively) on the target membranes in the absence of CCR5 molecules does not allow efficient entry. However, this may be dependent on the viral genetic background, because it has been shown that on the background of the ADA virus, deglycosylation of the V2 loop at positions very similar to those examined here alters the viral tropism such that the virus infects CCR5-expressing cells in a CD4-independent fashion (23). When CD4 numbers are kept low, an increase in the number of CCR5 molecules (compare the results obtained with HI-R cells to those obtained with the RC-12 cells) results in a 10-fold increase in entry of SF162 and GM186, but only a 2-fold increase in entry of GM154 and GM195. When the CCR5 numbers are kept low, an increase in the number of CD4 molecules (compare the results obtained with the RC-12 cells to those obtained with the JC-10 cells) results in an increase in entry of SF162 and GM186 by 13- and 12-fold, respectively, that of GM154 by 5-fold, and that of GM195 by 7.5-fold. When the CD4 numbers are high and the number of CCR5 molecules increases (compare the results obtained with the JC-10 cells to those obtained with the JC-53 cells), no further increase in the infectivity of SF162 and GM186 takes place, but in contrast a 3-fold increase in the case of GM186 and a 4.5-fold increase in the case of GM195 is recorded.
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Neutralizations.
Several reports have highlighted the
important role of envelope glycosylation on the susceptibility of HIV
and SIV to antibody-mediated neutralization (1, 5, 36, 44,
47-49). To evaluate whether glycosylation of the asparagines
located at positions 154, 186, and 195 of the V2 loop of the SF162
envelope offers protection against antibody-mediated neutralization, we
compared the neutralization susceptibility of the GM154, GM186, and
GM195 mutant viruses to that of the parental SF162 virus using sera
collected from HIV-infected patients (Table
2). These studies also allowed us to
compare the relative protective role of each of these three
glycosylation sites.
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Relative envelope content of wild-type and mutant viruses.
The
observed differences in the replication potential as well as the
susceptibility to neutralization between the SF162 virus and the three
mutant viruses could be due to less efficient incorporation of the
modified envelope proteins into the virion membrane. It is therefore
possible that the mutant viruses express fewer envelope molecules on
their surface. Our results indicate, however, that the relative ratio
of gp120 to p24 proteins in the SF162 and mutant virions is similar
(Fig. 4), suggesting that similar numbers
of gp120 molecules are present on the surface of all the viruses examined.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the studies presented here, we evaluated the role of asparagine-linked glycosylation sites located in the V2 loop of the HIV-1 envelope on the replication potential and neutralization susceptibility of the R5-using, macrophage-tropic, neutralization-resistant HIV-1 SF162 isolate (7, 8, 51, 52, 58). Three asparagine-linked glycosylation sites exist in this region of the SF162 envelope (Fig. 1); one is located within the V2 loop itself (position 186), and the other two are located at the amino and carboxy terminus of the base of this loop (positions 154 and 195, respectively).
During the in vitro replication of the SF162 virus in human PBMC, all three positions are glycosylated (Fig. 2). However, elimination of any one of these positions does not abrogate the ability of the virus to replicate in these cells (Fig. 3). These results are in agreement with those indicating that the pattern of HIV, SIV, or SHIV envelope glycosylation does not significantly affect viral replication ability in PBMC (5, 43, 47). In connection with the role of envelope glycosylation in viral tropism, previous studies have suggested that changes in envelope glycosylation could expand the tropism of the virus (10, 46). However, these studies were primarily conducted with established T-cell lines as target cells, and it is unclear whether an expansion in replication in established T-cell lines would have any relevance in vivo. Our current studies indicate that V2 loop glycosylation profoundly affects the ability of HIV to replicate in primary macrophages. In particular, glycosylation at the base of the V2 loop (at both the amino- and carboxy-terminal sides) appears to be crucial for efficient replication in these cells (Fig. 3).
The fact that the effect of the introduced modifications on viral replication is more pronounced when the target cells are macrophages than PBMC is most likely explained by the smaller number of CD4 and CCR5 receptor molecules on the surface of the former than the latter cells (16, 35, 63). Our studies conducted with HeLa cell lines (Table 1) expressing different numbers of CD4 and CCR5 molecules on their surfaces (42) suggest that the introduced modifications affect envelope interaction with both the CD4 and CCR5 receptor molecules on the surface of target cells. While glycosylation at position 186 appears to play a more important role during the interaction of the envelope with CD4 than with CCR5, glycosylation at positions 154 and 195 appears to play a role during both envelope-CD4 and envelope-CCR5 interaction (Table 1). Alternatively, the more efficient replication of the mutant viruses in PBMC than in macrophages could be related to potential differences in the oligomerization, glycosylation, and/or CD4 association of CCR5 molecules in these two cell types (17, 27). In this case, the fact that the SF162 virus replicates efficiently in both activated PBMC and macrophages, while the mutant viruses replicate less efficiently in macrophages, would suggest that the partially deglycosylated envelopes but not the wild-type envelope interacts less efficiently with the CCR5 molecules present on the surface of macrophages than those present on the surface of activated PBMC. Future studies will address this possibility.
We previously reported that CD4 binding to the SF162 gp120 envelope subunit is influenced by an envelope region comprising the V2 loop (56). Our current studies suggest that in addition to the amino acid sequence of the V2 loop, the extent of glycosylation of this region also influences the binding of HIV to the CD4 receptor. In addition to affecting envelope-CD4 binding, V2 loop glycosylation may affect the type and/or the extent of CD4-mediated envelope conformational changes (55) that are required for envelope-CCR5 binding. This may explain the observation that deglycosylation of the base of the V2 loop reduces the efficiency of interaction of virion-associated envelope molecules with CCR5 molecules present on the surface of target cells. Alternatively, the sugar molecules present on the V2 loop of SF162 interact directly with CCR5 molecules on the target cell surface. A third possible explanation for our observations could be that V2 loop deglycosylation alters the conformation of currently unknown envelope epitopes that interact with the CCR5 molecules.
As fully V2 loop-glycosylated envelopes appear to require less CCR5 for cell entry than partially deglycosylated envelopes (Table 1), our studies suggest that glycosylation of the V2 loop of the HIV envelope may offer CCR5-using HIV-1 isolates an initial advantage over CCR5-using isolates with a partially deglycosylated V2 loop in establishing infection in a new host by permitting a more efficient interaction of their envelope with the surface of naive CD4+ T lymphocytes, which express small numbers of CCR5 molecules.
In addition to allowing HIV to infect macrophages, glycosylation of the
V2 loop offers protection from antibody-mediated neutralization (Tables
1 and 2). Removal of any one of the three asparagine-linked glycosylation sites examined renders the virus more susceptible to
neutralization by antibodies produced during natural HIV infection. The
recorded differences in neutralization susceptibility are not due to
differences in the numbers of virion-associated envelope molecules
between SF162 and the mutant viruses (Fig. 4). Most likely, V2 loop
deglycosylation increases the exposure of neutralization epitopes.
We previously reported that the elimination of 30 amino acids (from
T160 to Y189), including the asparagine at position 186, from the
central region of the V2 loop from the SF162 isolate renders the virus,
termed SF162V2, highly susceptible to neutralization by the same
serum antibodies examined here (54). The studies conducted
here reveal that the elimination of the glycosylation site at position
186 from the SF162 background renders the virus (GM186) more
susceptible to neutralization than the parental SF162 virus. Even so,
the GM186 virus is severalfold less susceptible to neutralization than
the SF162V2 virus with the sera tested (see reference
54 for comparison). Therefore, although the
carbohydrate side chains present at position 186 of the SF162 envelope
mask neutralization epitopes and contribute to the
neutralization-resistant phenotype of this virus, this masking is not
as extensive as that offered by the 30 amino acids of the V2 loop
themselves. These amino acids may either mask additional epitopes
or more efficiently mask the same epitopes as those masked by the
sugar molecules present on the asparagine at position 186.
With the exception of serum B, which neutralized the GM154 and GM186 mutant viruses to the same extent as the parental SF162 virus, all the other HIV-positive human sera tested neutralized all three mutant viruses more efficiently than the SF162 virus. The fact that the three mutant viruses are not equally susceptible to neutralization by any given HIV-positive serum (Table 2) suggests that the sugar molecules present at positions 154, 186, and 195 of the SF162 envelope occlude neutralizing epitopes that are not identical but most likely overlap. It appears that glycosylation of the two asparagines located at the base of the V2 loop provides better protection from antibody-mediated neutralization to the SF162 isolate than glycosylation of the asparagine located within the V2 loop itself (position 186). Regardless, since the sera tested here were collected from patients infected with viruses heterologous to SF162, the epitopes that become exposed upon V2 loop deglycosylation of the SF162 envelope have common structural features among heterologous primary isolates. The fact that a given mutant virus is differentially susceptible to neutralization by the various HIV-positive sera tested suggests that the titers of antibodies recognizing the epitopes that are normally occluded by V2 loop glycosylation differ among the sera tested. Overall, the above results suggest that during HIV (or SIV) infection, even though the anti-envelope antibody responses evolve to neutralize the virus more efficiently (4, 12, 13, 32, 33), the virus itself can easily adapt and escape neutralization by altering the pattern of envelope glycosylation of the V2 loop (and most likely other regions) without affecting its replication ability in activated PBMC.
The sugar molecules present at the glycosylation sites examined appear to occlude neutralization epitopes located outside the V2 loop itself but within the CD4 binding site and the V3 loop (Table 3). These results are in agreement with conclusions from numerous studies reporting on a structural and functional interaction between the V1V2 region, the CD4 binding site, and the V3 loop of the HIV envelope (18, 21, 30, 34, 57, 66-69), as well as with results obtained following elucidation of the crystal structure of the gp120 core, which suggest that the V1V2 region of the HIV envelope folds over elements of the CD4 binding site and the V3 loop (26, 66-69). The three glycosylation sites examined, however, do not contribute equally to protection from neutralization by anti-CD4 binding site and anti-V3 loop antibodies. Glycosylation at the amino-terminal side (GM154) of the base of the V2 loop is more effective in preventing the binding of antibodies to these two envelope regions than glycosylation of the carboxy-terminal side (GM195). Also, in contrast to the protection from neutralization by anti-V3 loop MAbs offered by the glycosylation of the base of the V2 loop, glycosylation within the loop itself (GM186) does not appear to offer protection by such antibodies.
We previously reported that several V3 loop epitopes, including that recognized by MAb 391-95D, are occluded within the functional, virion-associated envelope (55). Our current studies indicate that this occlusion may be due (at least partially) to the sugar molecules present at the base of the V2 loop. Several groups, including ours, have reported that the V2 loop is implicated in determining viral tropism, either directly or in collaboration with the V3 loop (21, 22, 40, 61), which may interact with elements of the CCR5 (or CXCR4) coreceptor (11, 53, 70). Our present results suggest that V2 loop glycosylation, by masking specific V3 loop epitopes, can indirectly affect the interaction of the V3 loop with the coreceptor molecules and thus influence viral tropism.
In contrast to the observations made with the anti-CD4 binding site and anti-V3 loop MAbs, glycosylation of the three sites examined does not increase susceptibility to neutralization by MAbs 17b and 48d. These two MAbs recognize overlapping conformational epitopes that are CD4 induced and involved in envelope-chemokine receptor binding (60, 62, 65). Since elimination of the two glycosylation sites at the base of the V2 loop results in a less efficient interaction of the HIV envelope with the CCR5 chemokine receptor molecules on the surface of susceptible cells (Table 1), the sugar molecules present at these sites may be affecting an envelope-CCR5 binding step which follows that involving the epitopes recognized by MAbs 17b and 48d.
In summary, our current studies indicate that glycosylation of the V2 loop of HIV not only expands its cellular tropism by allowing it to more efficiently replicate in primary macrophages, but also protects the virus from neutralization by cross-reactive neutralizing antibodies. It would be interesting to compare the immunogenicity of envelope proteins derived from the GM154, GM186, and GM195 viruses to that of the SF162 envelope to determine whether the mutant envelopes elicit the generation of higher titers of neutralizing antibodies not only against SF162, but also against heterologous HIV-1 isolates.
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ACKNOWLEDGMENTS |
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This work was supported partially by a Jonathan S. Canno Research Fund award from AMFAR (02572-23-RGV) and by the NIH (AI 44309-01).
We thank C. Cheng-Mayer for many helpful comments and suggestions. We acknowledge D. Kabat for providing the HeLa cell lines and S. Zolla-Pazner, M. Gorny, J. Robinson, and D. Burton for providing the MAbs used in this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: Aaron Diamond AIDS Research Center, 455 First Avenue, 7th floor, New York, NY 10016. Phone: (212) 448-5000. Fax: (212) 448-5159. E-mail: lstamata{at}adarc.org.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, August 2000, p. 6769-6776, Vol. 74, No. 15
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(2004). Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology
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(2004). The V1/V2 Domain of gp120 Is a Global Regulator of the Sensitivity of Primary Human Immunodeficiency Virus Type 1 Isolates to Neutralization by Antibodies Commonly Induced upon Infection. J. Virol.
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Yang, Z.-y., Chakrabarti, B. K., Xu, L., Welcher, B., Kong, W.-p., Leung, K., Panet, A., Mascola, J. R., Nabel, G. J.
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McCaffrey, R. A., Saunders, C., Hensel, M., Stamatatos, L.
(2004). N-Linked Glycosylation of the V3 Loop and the Immunologically Silent Face of gp120 Protects Human Immunodeficiency Virus Type 1 SF162 from Neutralization by Anti-gp120 and Anti-gp41 Antibodies. J. Virol.
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Derdeyn, C. A., Decker, J. M., Bibollet-Ruche, F., Mokili, J. L., Muldoon, M., Denham, S. A., Heil, M. L., Kasolo, F., Musonda, R., Hahn, B. H., Shaw, G. M., Korber, B. T., Allen, S., Hunter, E.
(2004). Envelope-Constrained Neutralization-Sensitive HIV-1 After Heterosexual Transmission. Science
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Nabatov, A. A., Pollakis, G., Linnemann, T., Kliphius, A., Chalaby, M. I. M., Paxton, W. A.
(2004). Intrapatient Alterations in the Human Immunodeficiency Virus Type 1 gp120 V1V2 and V3 Regions Differentially Modulate Coreceptor Usage, Virus Inhibition by CC/CXC Chemokines, Soluble CD4, and the b12 and 2G12 Monoclonal Antibodies. J. Virol.
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Poumbourios, P., Maerz, A. L., Drummer, H. E.
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Srivastava, I. K., Stamatatos, L., Kan, E., Vajdy, M., Lian, Y., Hilt, S., Martin, L., Vita, C., Zhu, P., Roux, K. H., Vojtech, L., C. Montefiori, D., Donnelly, J., Ulmer, J. B., Barnett, S. W.
(2003). Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate. J. Virol.
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Labrijn, A. F., Poignard, P., Raja, A., Zwick, M. B., Delgado, K., Franti, M., Binley, J., Vivona, V., Grundner, C., Huang, C.-C., Venturi, M., Petropoulos, C. J., Wrin, T., Dimitrov, D. S., Robinson, J., Kwong, P. D., Wyatt, R. T., Sodroski, J., Burton, D. R.
(2003). Access of Antibody Molecules to the Conserved Coreceptor Binding Site on Glycoprotein gp120 Is Sterically Restricted on Primary Human Immunodeficiency Virus Type 1. J. Virol.
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Kitrinos, K. M., Hoffman, N. G., Nelson, J. A. E., Swanstrom, R.
(2003). Turnover of env Variable Region 1 and 2 Genotypes in Subjects with Late-Stage Human Immunodeficiency Virus Type 1 Infection. J. Virol.
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Srivastava, I. K., VanDorsten, K., Vojtech, L., Barnett, S. W., Stamatatos, L.
(2003). Changes in the Immunogenic Properties of Soluble gp140 Human Immunodeficiency Virus Envelope Constructs upon Partial Deletion of the Second Hypervariable Region. J. Virol.
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Lue, J., Hsu, M., Yang, D., Marx, P., Chen, Z., Cheng-Mayer, C.
(2002). Addition of a Single gp120 Glycan Confers Increased Binding to Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin and Neutralization Escape to Human Immunodeficiency Virus Type 1. J. Virol.
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Binley, J. M., Sanders, R. W., Master, A., Cayanan, C. S., Wiley, C. L., Schiffner, L., Travis, B., Kuhmann, S., Burton, D. R., Hu, S.-L., Olson, W. C., Moore, J. P.
(2002). Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins. J. Virol.
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Zhang, P. F., Bouma, P., Park, E. J., Margolick, J. B., Robinson, J. E., Zolla-Pazner, S., Flora, M. N., Quinnan, G. V. Jr
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Ogert, R. A., Lee, M. K., Ross, W., Buckler-White, A., Martin, M. A., Cho, M. W.
(2001). N-Linked Glycosylation Sites Adjacent to and within the V1/V2 and the V3 Loops of Dualtropic Human Immunodeficiency Virus Type 1 Isolate DH12 gp120 Affect Coreceptor Usage and Cellular Tropism. J. Virol.
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(2001). Equine Infectious Anemia Virus Genomic Evolution in Progressor and Nonprogressor Ponies. J. Virol.
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(2001). Differential CD4/CCR5 Utilization, gp120 Conformation, and Neutralization Sensitivity between Envelopes from a Microglia-Adapted Human Immunodeficiency Virus Type 1 and Its Parental Isolate. J. Virol.
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Means, R. E., Matthews, T., Hoxie, J. A., Malim, M. H., Kodama, T., Desrosiers, R. C.
(2001). Ability of the V3 Loop of Simian Immunodeficiency Virus To Serve as a Target for Antibody-Mediated Neutralization: Correlation of Neutralization Sensitivity, Growth in Macrophages, and Decreased Dependence on CD4. J. Virol.
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Pollakis, G., Kang, S., Kliphuis, A., Chalaby, M. I. M., Goudsmit, J., Paxton, W. A.
(2001). N-Linked Glycosylation of the HIV Type-1 gp120 Envelope Glycoprotein as a Major Determinant of CCR5 and CXCR4 Coreceptor Utilization. J. Biol. Chem.
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