From the Department of Human Retrovirology, Academic Medical
Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Received for publication, October 26, 2000, and in revised form, December 18, 2000
The variable V1V2 and V3 regions of the human
immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (gp120) can
influence viral coreceptor usage. To substantiate this we generated
isogenic HIV-1 molecularly cloned viruses that were composed of the
HxB2 envelope backbone containing the V1V2 and V3 regions from viruses isolated from a patient progressing to disease. We show that the V3
amino acid charge per se had little influence on altering
the virus coreceptor phenotype. The V1V2 region and its
N-linked glycosylation degree were shown to confer CXCR4
usage and provide the virus with rapid replication kinetics. Loss of an
N-linked glycosylation site within the V3 region had a
major influence on the virus switching from the R5 to X4 phenotype in a
V3 charge-dependent manner. The loss of this V3
N-linked glycosylation site was also linked with the
broadening of the coreceptor repertoire to incorporate CCR3. By
comparing the amino acid sequences of primary HIV-1 isolates, we
identified a strong association between high V3 charge and the loss of
this V3 N-linked glycosylation site. These results demonstrate that the N-linked glycosylation pattern of the
HIV-1 envelope can strongly influence viral coreceptor utilization and the R5 to X4 switch.
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INTRODUCTION |
Many seven-transmembrane chemokine receptors, in conjunction with
the CD4 molecule, have been shown to facilitate entry of human
immunodeficiency virus type-1
(HIV-1)1 into the various
cell types that it infects (1-9). The CC-chemokine receptor CCR5 and
the CXC chemokine receptor CXCR4 are the most significant coreceptors
with regard to HIV-1 transmission and pathogenesis (10-15). Viruses
utilizing CCR5 (R5), also referred to as nonsyncytia-inducing or
macrophage tropic (M-tropic) viruses, are those preferentially
transmitted, whereas those using CXCR4 (X4), also termed
syncytia-inducing (SI) or T cell line tropic (T-tropic) viruses,
are those more associated with later stage disease and disease
progression (16-19). The full molecular events leading to alterations
in coreceptor activity or to receptor switching in vivo are
not fully understood.
The most striking association to date between viral envelope variation
and biologic phenotype has been the overall amino acid charge of the V3
region, with a higher positive charge associated with the SI phenotype
and utilization of the CXCR4 coreceptor (20-34). The V3 region is not
the sole determinant of biological phenotype or coreceptor utilization,
and other envelope regions, namely the V1 and V2, have been implicated
as important (32-41). The V4 and V5 regions, in conjunction with the
V1 and V3, have also been shown to influence coreceptor usage of a
dual-tropic R5X4 virus (42). Additionally, N-linked
carbohydrate moieties have been shown to have an influence on certain
biological properties of both HIV-1 and HIV-2 viruses (43-47), some of
which would be predicted to alter coreceptor binding and utilization
(44-47).
Several chemokine and chemokine receptor genotypes have been associated
with HIV-1 transmission and disease progression, strongly suggesting
that these factors play a significant role in controlling viral
replication in vivo (12), with their efficacy dependant on
the interaction of the virus with the relevant coreceptors (13-15,
48-52). The natural ligands for the CCR5 receptor, RANTES (regulated
on activation normal T cell expressed and secreted), macrophage
inflammatory protein (MIP)-1
, and MIP-1
, have been shown to
inhibit viral entry through either competing for the CCR5 receptor or
by down-regulating its surface expression (53-55). A virus with
altered coreceptor activity is therefore likely to be selected during
disease progression as a consequence of chemokine inhibition pressure.
Indeed many studies show that minor alterations within the V3 region of
the HIV-1 envelope can render a virus resistant to the blocking effects
of the CC-chemokines (54-56). A better knowledge of the molecular
events contributing to viral evolution, specifically the switch from
the R5 to X4 phenotype will be important in broadening our
understanding of HIV-1 pathogenesis as well as providing information
relevant to both HIV-1 therapy design and vaccine development.
To define the molecular events involved with the coreceptor switching
in vivo, we created and studied a panel of molecularly cloned viruses based upon the V1V2 and V3 envelope regions of viral
isolates derived from an HIV-1-infected individual who has progressed
to disease. The V1V2 region was shown to be significant in providing
the virus with R5X4 dual tropism but not in affecting the level of CCR5
utilization. Here we demonstrate that the loss of an
N-linked glycosylation event within the V3 region of the envelope was significant in determining strong CXCR4 utilization and in
the virus switching from the R5 to X4 phenotype. In addition, we have
compared the V3 sequences from a large number of primary isolates and
found a strong association between an increase in V3 charge and the
loss of the V3 N-linked glycosylation event, highlighting
its in vivo significance. These results demonstrate that the
N-linked glycosylation pattern of the gp120 envelope can
contribute to viral coreceptor utilization and switching.
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EXPERIMENTAL PROCEDURES |
Molecular Cloning and Sequencing of Viruses--
The initial
panel of molecularly cloned HIV-1 chimeric viruses were composed of the
LAI viral backbone carrying the HxB2 envelope where the V3 loop
was replaced with the corresponding region amplified from a patient
(ACH168) progressed in the disease course. These viruses, termed X, are
shown in Fig. 1A and were created as previously described
(22, 27). With the X viruses, we have replaced the V1V2 region of the
envelope glycoprotein with the corresponding region of a T-tropic
isolate, 168.10, originating from the same patient (ACH168) and from a
late time point in disease. The new set of viruses, termed X.10, of
which the V1 region is shown in Fig. 1B, were created as
follows. The BlueScirpt plasmid was modified for our requirements
through replacing the KpnI site with a NcoI site
by inserting the linker 5'-TTC CAT GGA AGT AC-3' at the
KpnI site, resulting in the pBSn plasmid. The
NcoI-BamHI fragment of the X virus HIV-1 envelope
(HxB2 nucleotides 5675-8475) was sub-cloned into the
NcoI-BamHI-cloning sites of the pBSn plasmid,
resulting in the pAG plasmid. The KpnI-StuI
fragment of the HXB2 envelope (nucleotides 6347-6832) was replaced by
the KpnI-StuI fragment of the 168.10 envelope,
resulting in the pAGX.10 plasmids. Finally, the envelope constructs
were transferred into the pLAI backbone by
NcoI-BamHI digestion and ligation, resulting in
the X.10 infectious molecular clone plasmids.
For all the mutagenesis reactions the QuikChange site-directed
mutagenesis kit (Stratagene) was used with the pAG plasmid series as
the starting material. All procedures were performed according to the
manufacturer's specifications. The modification of the
N-linked glycosylation site in the V1 loop was achieved with
the primers 5'-TGC CAC TAA TGG TAG CTG GGA AAA GAT GGA AAA AGG-3' and
5'-CCT TTT TCC ATC TTT TCC CAG CTA CCA TTA GTG GCA-3', which altered
the amino acid sequence of the V1 loop from NSTTNATIGSWE to
NSTTYATIGSWE. The modification of the N-linked
glycosylation site in the V3 loop was achieved with the primers 5'-GAC
ATT AAT TGT ACA AGA CCC AAC AAC AAT ATA AGA AAA AGG-3' and 5'-CCT TTT TCT TAT ATT GTT GTT GGG TCT TGT ACA ATT AAT GTC-3'. The amino acid
sequence of the V3 loop changed from CTRPNNNTRK to CTRPNNNIRK, and both amino acid sequences were found in primary isolates derived from individual ACH168. The construction of
all molecularly cloned viruses and the production of viral stocks were
monitored throughout using the technique of standard automated sequencing.
Generating Infectious HIV-1 Viral Stocks--
Infectious
molecularly cloned HIV-1 viral stocks were generated by transfection of
the relevant HIV-expressing plasmid into the human cervical carcinoma
cell line C33A. Transfections were performed with 10 µg of plasmid
DNA using the CaCl2 precipitation method. All plasmid DNA
used for transfection was prepared using Qiagen kits in accordance with
the manufacturer's instructions. The DNA precipitate was split between
two wells of C33A cells plated 24 h earlier at 1.5 × 106 cells/well in a 6-well tissue culture plate. Each
transfection was performed in a final volume of 8 ml of Dulbecco's
modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum. The following day, the
cells were washed with phosphate-buffered saline and given fresh
culture media, and the viral stocks were harvested on day 3 of culture
in multiple freezing vials after a 0.2 µM filtration step
(Millipore). All culture supernatants were assayed at the time of
freezing for levels of the viral p24 antigen with a standard p24
enzyme-linked immunosorbent assay. For all viruses produced, the levels
of p24 were within the range 105-106
pg/ml.
Determining Viral Coreceptor Utilization--
The coreceptor
utilization phenotype was determined by measuring viral replication on
the U87.CD4+ cell line expressing an array of different
chemokine receptors (CCR1, CCR2b, CCR3, CCR5, and CXCR4), a gift from
Dr. Dan Littman, Skirbill Institute, New York. These cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum plus the antibiotics puromycin (1 µg/ml) and
neomycin (300 µg/ml). Coreceptor utilization was determined by adding
200 µl of virus stock (between 105 and 106
p24 pg/ml) to 3.0 × 104 U87.CD4+ cells
(plated 20-24 h previously in a 96-well flat-bottomed culture plate)
expressing the specific receptor under analysis. Cells were infected
for 18 h before being washed twice with phosphate-buffered saline
and fed with 200 µl of fresh media. On day 10 of the culture, cells
were scored for syncytia formation, and the p24 levels in the culture
supernatants were determined using a standard enzyme-linked immunosorbent assay.
Determining Viral Infectivity and Replication Phenotype on
CD4+ Lymphocytes--
All viral stocks were assayed for
tissue culture infectious dose (TCID50) on
CD4+-enriched lymphocytes isolated from individuals who did
not carry the
32CCR5 allele, screened for by standard polymerase
chain reaction technique. PBMCs were isolated from fresh buffy
coats Central Laboratory Blood Bank, Amsterdam) by standard
Ficoll-Hypaque density centrifugation. PBMCs were frozen in multiple
vials at a high concentration and, when required, PBMCs were thawed and activated with 5 µg/ml phytohemagglutinin and cultured in RPMI medium containing 10% fetal calf serum, penicillin (100 units/ml), and
streptomycin (100 units/ml) with recombinant interleukin-2 (100 units/ml). On day 4 of culture, the cells underwent CD4+
enrichment by incubating with CD8 immunomagnetic beads (Dynal Oslo,
Norway) and separating out the CD8+ lymphocytes.
CD4+-enriched lymphocytes were plated at 2 × 105 cells/well in 96-well plates with 5-fold serial
dilutions of the virus. The cells were fed on day 7 with fresh media
and scored on day 14 for p24 levels, and the number of positive wells
were identified. This figure was used to determine the
TCID50 value for each virus. PBMCs from the same donor were
preferentially used throughout the study to minimize any potential
experimental errors resulting from variation between donor cells. Where
the same donor PBMC samples were not available, then cells behaving similarly in their replication capacity for R5 viruses were selected.
The replication kinetics of each virus was measured by infecting
CD4+-enriched lymphocytes from either CCR5+/+
or CCR5
/
individuals at 1,000-3,000
TCID50. The kinetics of virus replication were monitored by
measuring p24 antigen levels in the culture supernatants on days 4, 7, and 10 of infection. All experimental results with regard to
replication kinetics, unless otherwise stated, are representative of
three independently performed experiments.
Electrophoretic Mobility of Virion-associated gp120
Proteins--
We tested for the induced N-linked
glycosylation pattern variations by following the electrophoretic
mobility of virion-associated gp120 proteins. Infectious viral
particles were pelleted (18,000 × g for 2 h at
4 °C) from 0.5 ml of culture supernatant harvested from transfected
C33A cells. The virus pellet was resuspended in 40 µl of of lysis
buffer (50 mM Tris-HCl, 100 mM
2-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and
heated to 95 °C for 5 min. The denatured proteins underwent
SDS-polyacrylamide gel electrophoresis (PAGE) on 5% gels and
transferred to nitrocellulose membrane. After overnight incubation at
4 °C with rabbit-anti-gp120 serum followed by a 1-h incubation with
a goat anti-rabbit antibody conjugated to alkaline phosphatase, the
position of the gp120 glycoprotein on the molecular weight scale was
detected by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
color development.
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RESULTS |
Effect of V3 Charge on Coreceptor Utilization--
Our starting
material for this study was a panel of molecularly cloned chimeric
viruses that consisted of the HxB2 envelope in the LAI viral backbone.
The V3 region of the HxB2 envelope was replaced by the equivalent V3
region from a M-tropic viral isolate (168.1), obtained from a patient
early in the disease course (patient ACH168 of the Amsterdam Cohorts
Studies) (27). Subsequently, the V3 region of this virus was altered by
site-directed mutagenesis to resemble the amino acid alterations that
confer the positive charge changes seen in the V3 region of a late
stage disease, T-tropic viral isolate (168.10). Fig.
1A depicts the V3 amino acid
sequence differences between the early and late isolates (168.1 and
168.10) and the sequences of the sequentially altered V3 regions, which
confer differences in overall positive charge. An additional virus (G)
was generated that provided a further +3-charged V3 envelope for this
study.

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Fig. 1.
Schematic representation and coreceptor
utilization of the original viruses. A, amino acid
comparisons of the V3 sequences of the early 168.1 and late 168.10 isolates and the original panel of chimeric viruses (X) composed by
replacing the V3 region from donor ACH168 into the HxB2 envelope gp120
glycoprotein. Amino acid alterations are indicated, and the
potential N-linked glycosylation site is boxed.
MT-2 and SupT1 cells were transfected with plasmids expressing the
original viruses and syncytia formation determined. MT-2 transfections
were scored for syncytia formation on day 4 post-transfection (+,
syncytia; , no syncytia) and SupT1 on day 7 (++++, very high; +++,
high; ++, intermediate; +, low; , nil). Virus stocks generated by
transfection of C33A cells were tested for infection on
U87/CD4+ cells expressing different coreceptors and on
CD4+ lymphocytes isolated from CCR5+/+ and
CCR5 / individuals. Infection was monitored by measuring
p24 on day 10 of infection (++++ > 106 pg/ml; +++,
105-106 pg/ml; ++,
104-105 pg/ml; +,
103-104 pg/ml; and , <103
pg/ml). B, amino acid sequence comparison of the V1 regions
for the HxB2 virus and the early (168.1) and late (168.10) isolates
from donor ACH168. The additional N-linked glycosylation
site in the isolate 168.10 is boxed.
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Upon transfection of these viral plasmids into either MT-2 or Sup-T1
cells, the viral phenotype changed from that of M-tropic to T-tropic,
as determined by the appearance of syncytia (Fig. 1A). This
result suggested that as the positive charge of the V3 envelope
increased, the viruses began to utilize the CXCR4 receptor as a
coreceptor for viral entry. To test this hypothesis, we measured the
infectivity of viral isolates generated by the transfection of the
human C33A cell line on U87.CD4+ cells expressing either
the CCR5 or CXCR4 receptors. Surprisingly, we found that all viruses
were strong users of the CCR5 receptor, and none were capable of
utilizing CXCR4 (Fig. 1A). The full-length 168.10 viral
envelope cloned into the LAI backbone produced a virus that showed
strong CXCR4 usage but that could not utilize CCR5 (Fig.
1A). These results demonstrated that the V3 charge alterations, which could confer syncytia formation upon transfection of
MT-2 and Sup-T1 cell lines, could not predict successful CXCR4 utilization and thereby suggested that some other region or factors within the envelope were responsible.
Influence of V1V2 Region on Coreceptor Utilization--
Since we
were unable to confer CXCR4 utilization onto our virus panel by
altering V3 charge alone, we tested what effect the V1V2 region had on
viral coreceptor utilization. The differences in the V1V2 region
between the early isolate (168.1) and the late virus isolate (168.10)
as well as the HxB2 virus can be seen in Fig. 1B. Sequence
comparison revealed a strong homology between the V1V2 region of the
HxB2 and the 168.1 envelopes, whereas both differed from the 168.10 isolate, with the main difference being the addition of a potential
N-linked glycosylation site in the latter. We generated
a panel of viruses where we replaced the V1V2 region of our original
panel of viruses (termed X) with the V1V2 region of the late isolate,
168.10. We produced 10 new viruses that encompassed the array of
different V3 charges (+3 to +6) (termed X.10). The new viruses along
with the original (X) viruses were tested for replication on
U87.CD4+ cells expressing either CCR5 or CXCR4 and on
CD4+ lymphocytes isolated from individuals homozygous for
the
32 CCR5 allele (CCR5
/
) or from those who did not
carry this allele (CCR5+/+). In this experiment
non-diluted viral stocks were used to infect the
U87.CD4+ cells, and a 3,000 TCID50 was used to
infect the CD4+ lymphocytes. With the original X viruses
(top panel, Fig. 2), no
replication was seen on the CCR5
/
CD4+
lymphocytes, indicating no CXCR4 utilization. With some of the V3
higher charged viruses we did observe slight CXCR4 utilization but at
less than 0.1% of that seen for CCR5. Surprisingly, the X.10 viruses,
irrespective of V3 charge, all demonstrated replication on
U87.CD4+ cells expressing CXCR4 or CCR5 and also replicated
on CCR5
/
CD4+ lymphocytes (bottom
panel, Fig. 2). Interestingly, there was no reduction in the
replication of the X.10 viruses on the U87.CD4+ cells
expressing CCR5, and actually all showed higher levels of replication
opposed to the U87.CD4+ cells expressing the CXCR4
receptor. We therefore conclude that the V1V2 region from the late
isolate, 168.10, allows the virus to utilize CXCR4 while not
diminishing its ability to use CCR5.

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Fig. 2.
Replication of X and X.10 viruses. Virus
stocks of the X (without the V1V2 of the late disease isolate 168.10)
and X.10 (with the V1V2 of the late disease isolate 168.10) viruses
generated by transfection of C33A cells were tested for infection of
U87/CD4+ cells expressing either CCR5 or CXCR4 and on
CD4+ lymphocytes isolated from CCR5+/+ and
CCR5 / individuals. The U87 cell replication results are
shown with error bars based on the S.D. derived from three
separate data points included in the same experiment. For replication
on CD4+ lymphocytes, the results are depicted without error
bars but are representative of three separately performed
experiments.
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The replication kinetics of the X.10 viruses proved to be identical on
both CCR5+/+ and CCR5
/
CD4+
lymphocytes irrespective of the V3 charge. However, the viruses did
(closed circles, Fig. 3) show
faster rates of replication than the corresponding panel of X viruses
on CCR5+/+ lymphocytes (open circles, Fig. 3),
indicating that the 168.10 V1V2 region can provide HIV-1 with a faster
replicating phenotype irrespective of the charge in the V3 region.
Again, the original X viruses showed no replication on CD4+
lymphocytes from individuals lacking a functional CCR5 receptor.

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Fig. 3.
Kinetics of virus replication on
CD4+ lymphocytes. Replication of molecular cloned
viruses with the variant V3 regions inserted into the HxB2 envelope
backbone on CD4+-enriched lymphocytes from
CCR5+/+ and from CCR5 / individuals.
Open circles represent the original (X) panel of viruses,
without the 168.10 V1V2 region. Closed circles represent the
X.10 viruses, with the V1V2 region from the 168.10 isolate. The V3
charge and sequence are represented for each virus. The results shown
are representative of three separately performed experiments.
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Contribution of V1 and V3 N-Linked Glycosylation Events to
Coreceptor Utilization--
The panel of viruses with the 168.10 V1V2
region (X.10) were still capable of using CCR5 strongly, even with the
high V3 charges, whereas the 168.10 full envelope was not, suggesting
that other features within the envelope of the 168.10 isolate were
contributing to its solo CXCR4 utilization. We therefore became
interested in the V3 amino acid difference between the early 168.1 and
late 168.10 isolates that resulted in the loss of a potential
N-linked glycosylation site (Fig. 1A). We were
also interested in whether the additional N-linked
glycosylation site in the V1 region of the 168.10 envelope could
contribute to coreceptor utilization since this site has previously
been predicted to play a role in determining viral tropism (57). To
test the contribution made by these two N-linked
glycosylation events to viral coreceptor utilization and replication
phenotype, we generated a panel of viruses where these sites were
altered by site-directed mutagenesis (
gV1 or
gV3). We chose a
panel of viruses (G, N, RN, and RTQN together with the equivalent G.10,
N.10, RN.10, and RTQN.10 viruses) that covered the array of V3 charges
(+3 to +6).
Initially, to test whether the N-linked glycosylation sites
were indeed glycosylated or not, we pelleted the viruses and determined the gp120 envelope glycoprotein molecular weight by SDS-PAGE gel electrophoresis and Western blot analysis using a gp120-specific polyclonal antibody. In Fig. 4 we show
the result for the N series of viruses, where the envelopes lacking one
N-linked glycosylation site are seen to migrate faster.
Since we utilized molecular-cloned viruses altered by site-directed
mutagenesis, the variation in molecular size shown in Fig. 4 should
reflect the alterations to the N-linked glycosylation
pattern.

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Fig. 4.
Electrophoretic mobility of virion associated
gp120 envelope molecules during SDS-PAGE. Electrophoretic mobility
of virion-associated gp120 molecules during SDS-PAGE. The preparation
of virion-associated gp120 molecules from the chimeric RN, RN.10, and
glycosylation mutant viruses and SDS-PAGE were performed as described
under "Experimental Procedures." No attempt was made to correct for
differences in the gp120 content of the samples.
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The results for the replication of these viruses on
U87.CD4+ cells expressing CCR3, CCR5, and CXCR4 are shown
in Fig. 5. For the +3V3 virus (G.10),
there is no influence on coreceptor utilization with the presence or
absence of V1 glycosylation, but CXCR4 usage does appear to be much
weaker than for the CCR5 receptor. For the +4V3 virus (N.10), there is
a dramatic decrease in CXCR4 utilization (>99.5%) when the
N-linked glycosylation event in V1 is missing, and
furthermore, this virus also showed a reduced replication capacity on
CD4+ lymphocytes isolated from a CCR5
/
individual (data not shown). The N.10
gV1 virus also gained the ability to use the CCR3 receptor expressed on U87.CD4+
cells. Interestingly, the +5V3 and +6V3 virus, which were lacking the
V1 glycosylation event (RN.10
g V1 and RTQN.10
gV1), showed no
reduction in their ability to utilize CXCR4, with the RTQN.10
gV1 virus gaining CCR3 usage. These observations show that the degree of
glycosylation in the V1V2 region can be critical for coreceptor utilization, but in a context restricted by the charge found in the V3
region.

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Fig. 5.
Replication of the X, X.10 and the
glycosylation variant viruses. Virus stocks of the X, X.10, and
de-glycosylated (X.10/ gV1, X/ gV3, and X.10/ gV3) viruses in
either the V1 or V3 region were generated by transfection of C33A cells
and tested for infection of U87/CD4+ cells expressing
either CCR3, CCR5, or CXCR4. The V3 charge and amino acid substitutions
are indicated for each panel of viruses. The results are shown with
error bars based on the S.D. derived from three separate
data points included in the same experiment. Due to DNA cloning
difficulties, the RTQN.10 gV3 virus was not included in this
study.
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More dramatic effects were observed with the loss of the V3
N-linked glycosylation event, shown in Fig. 5. The +5V3
virus (RN
gV3) could replicate to equal levels on
U87.CD4+ cells expressing CCR5 or CXCR4 and had gained the
ability to use CCR3, whereas the original RN virus was only capable of
using CCR5. The +5V3 virus with the 168.10V1V2 region (RN.10
gV3)
showed high levels of replication on U87.CD4+ cells
expressing CXCR4 while at the same time showing a loss in the capacity
to utilize CCR5 (<2.0% of the RN.10 virus). Remarkably, the loss of
the V3 N-linked glycosylation site in the +6V3 virus (RTQN
gV3) resulted in the abolition of CCR5 usage, the maintenance of its CXCR4 usage, and a gain in the ability to use CCR3 as a functional receptor for replication (Fig. 5, lower right
panel). At the lower V3 charges (+3 and +4), no effect was seen
with the alteration of the V3 N-linked glycosylation site
(Fig. 5, top two panels). We can therefore conclude that for
our panel of viruses, the N-linked glycosylation pattern of
the V3 region plays a crucial role in directing coreceptor utilization
and coreceptor switching. In accordance, the replication kinetics of
these viruses (lacking the N-linked glycosylation site) on
CCR5
/
CD4+ lymphocytes demonstrate that the
loss in V3 N-linked glycosylation provides for a virus with
increased replication when the V3 charge is highest
(filled circles in lower panel,
Fig. 6A).

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Fig. 6.
Kinetics of viral replication and infectivity
profiles of de-glycosylated viruses. A, kinetics of
virus replication on CD4+ lymphocytes isolated from
CCR5+/+ or CCR5 / individuals (open
circles, X/ gV3; closed circles, X.10/ gV3). The
results shown are representative of three separately performed
experiments. B, percentage of TCID50
determination on CCR5 / CD4+ lymphocytes
opposed to CCR5+/+ CD4+ lymphocytes.
TCID50 determination is described under "Experimental
Procedures."
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We tested further the effect that the glycosylation modifications
within the gp120 envelope glycoprotein had on viral phenotype by
determining the TCID50 of each virus separately on
CCR5
/
and CCR5+/+ CD4+
lymphocytes and calculating this as a percentage (Fig. 6B).
We confirmed that as the charge of the V3 increased, the lack of V3
glycosylation (
gV3) significantly enhanced the viral infectivity of
CD4+ lymphocytes from CCR5
/
donors,
presumably reflecting an enhanced infectivity via the CXCR4 coreceptor.
We also observed that both the +5 and +6 viruses lacking the V1
N-linked glycosylation event had an increased infectivity profile on CCR5
/
CD4+ lymphocytes.
Frequency of the V3 N-Linked Glycosylation Pattern with Relation to
V3 Charge--
To address the biological relevance of our findings, we
compared the amino acid sequences from primary viral isolates taken from the Los Alamos data base and compared the frequency of
N-linked glycosylation events in the V1V2 and V3 regions
with the V3 amino acid charge. Analyzing the sequences of a large
number of viruses (n = 2,562) from different HIV-1
subtypes, we found no correlation between any N-linked
glycosylation events in the V1V2 regions and the charge of the V3 loop
(data not shown). We did, however, find a strong association between
high V3 charge and the lack of the N-linked glycosylation
event within the V3 loop for the different subtypes of HIV-1 (A, B, D,
and E), but not for subtype C, where the envelope charges remained
relatively low, and no de-glycosylation was observed (Fig.
7). This finding is in accordance with
the low frequency of SI viruses found in nature for subtype C viruses
compared with the high frequency encountered for subtypes D and E.

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Fig. 7.
Association between V3 charge and V3
de-glycosylation. The frequency of V3 de-glycosylation at
different overall V3 charges is given for the different HIV-1 subtypes
analyzed: A (n = 890), B (n = 393), C
(n = 531), D (n = 275), and E
(n = 473). Gray bars represent the frequency
of glycosylation, and the black bars represent the frequency
of de-glycosylation in the V3 region.
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DISCUSSION |
We have shown for our panel of molecularly cloned viruses that the
overall V3 charge is not the solo determinant of viral coreceptor
utilization or replication phenotype in vitro but does play
a significant role. With our viruses, the V3 charge is only important
with regard to these two parameters when placed within the correct V1V2
amino acid context and/or V1 and V3 glycosylation environments. This
result is somewhat surprising given the previous findings, where it was
demonstrated that the V3 charge was the minimal requirement to be
associated with the T-tropic phenotype (22, 27). We show that the V1V2
region has the capacity to significantly alter coreceptor utilization
and generate dual-tropic (R5X4) viruses as well as confer a
rapid-growing phenotype on CD4+ lymphocytes. The loss of an
N-linked glycosylation event within the V3 region, in
association with a high positive charge, can lead to the complete
switch of the virus from the R5 to X4 phenotype. The same
de-glycosylation event in the V3 region within or out of the context of
the V1V2 region is able to render a virus capable of utilizing the CCR3
coreceptor and, again, with a restriction based around the V3 charge.
These facts, combined, demonstrate that coreceptor utilization and
switching can result from an association between different events in
the V1V2 and V3 regions of the envelope and that N-linked
glycosylation patterns can be highly significant with regard to
coreceptor function.
The effect of the 168.10 V1V2 region was significant with respect to
CXCR4 usage irrespective of the V3 charge, whereas de-glycosylation of
the V3 region rendered the virus incapable of using CCR5 when the V3
charge was high. Since transmitted viruses are expected to have the
lowest V3 amino acid charges, it would be predicted that the
alterations in the V1V2 are more likely to be selected earlier in
infection for both CXCR4 utilization and faster replication kinetics
than the de-glycosylation event of the V3 region. The virus was
substantially weakened for CXCR4 utilization when the 168.10 V1V2
region was linked with a lower V3 charge, in conjunction with V3
de-glycosylation, thereby suggesting that the V3 de-glycosylation event
is only favorable for viral replication efficiency after the charge of
the V3 has risen. The broadening in the coreceptor repertoire of the
R5X4 dual-tropic chimeric viruses to include CCR3 by alterations in
N-linked glycosylation of the envelope suggests that the
viruses with the higher V3 region charges may be in a more structurally
open conformation.
We emphasize that we have studied in depth the V1V2 and V3 regions and
their interactions with the coreceptor of viral isolates established
from a single patient. In other individuals the equilibrium of these
interactions may be somewhat different. Nevertheless, our data
demonstrate that the V1V2 and V3 regions play a crucial role. A
previous study reports that the de-glycosylation of the V3, when
associated with a specific V3 backbone sequence, can have a negative
effect for CXCR4 utilization (58), and in our study the HxB2 envelope
lost its infectivity with the loss of the V3 region N-linked
glycosylation site (data not shown). This suggests that in certain
cases the de-glycosylation of the V3 can be detrimental for the virus
with respect to CXCR4 utilization, but it is worth noting that the +9V3
charge of HxB2 is unusually high and infrequently found in nature.
Our results are in agreement with the concept that the V1V2 region
interacts with the V3 region of the envelope to determine coreceptor
usage, and we show here that the N-linked glycosylation pattern can determine the effectiveness of this interaction toward coreceptor utilization. An interesting observation in this study has
been the finding that upon transfection of the original panel of
viruses (X, without the 168.10 V1V2) into either Sup-T1 or MT-2 cells,
the SI is seen in relation to the positive charge of the envelope, but
infectious virus capable of utilizing CXCR4 is not produced. However,
when the viruses were altered by removing the
N-glycosylation site in the V3 region, the virus was capable of using this coreceptor. This would indicate that as the charge of the
V3 region of the virus becomes more positive, the affinity of the
envelope for CXCR4 increases, but the V3 N-linked
glycosylation event in the V3 region inhibits the virus from being able
to utilize this receptor. The maintenance of the N-linked
glycosylation event also allows the virus at high charge to maintain
CCR5 utilization. This is pertinent given the recent finding that the
N-linked glycosylation pattern of the CXCR4 chemokine
receptor itself can influence viral coreceptor utilization patterns
(59). These results taken together strongly suggest that glycosylation
patterns, either envelope- or receptor-specific, are capable of
determining coreceptor utilization but not necessarily coreceptor
binding and that the interaction of the V1V2 and V3 regions is a
dynamic phenomenon in the course of the evolution of the virus.
The study of the kinetics of viral replication on
CD4+-enriched lymphocytes provides for an interesting
analysis. The addition of the late 168.10V1V2 region provides for
rapid-replicating viruses irrespective of the charge in the V3 region,
whereas alterations in V3 N-linked glycosylation results in
a virus with a slower rate of replication, although CXCR4 utilization
appears stronger. At higher V3 charges the N-linked
glycosylation of the V3 seems more significant in determining
replication kinetics than the V1V2 region.
A recent study has also suggested that an N-linked
glycosylation event in the V2 region has a significant effect on
CD4+/CCR5 receptor interactions and that this can influence
the neutralization potential of this virus by specific monoclonal
antibodies (60). Additionally, with simian immunodeficiency
virus it has been shown that de-glycosylation events within the
V1 region of the envelope result in a virus with increased antibody
neutralization potential (61). With HIV-1, the N-linked
glycosylation pattern has also been shown to have a varied effect on
the induction of neutralizing antibodies (44, 62-67). It is therefore
possible that the selection of viruses with different coreceptor
utilization patterns and replication phenotypes, based upon altered
envelope glycosylation events, may be aided by the escape from a
neutralizing antibody response. The emergence of CXCR4 viruses late in
disease and the concurrent rapid rise in viral loads seen in a
proportion of infected individuals may merely reflect viral escape from
a controlling neutralizing antibody response influenced by the
N-linked glycosylation pattern of the envelope. It is likely
that a combination of events encompassing altered viral replication
phenotype and immune evasion results in the rapid rise in viral load
and progression to disease. Alterations in glycosylation events
influencing coreceptor utilization will also allow the virus to escape
the controlling responses of the CC chemokines in vivo,
which are believed to play an effective role in inhibiting viral
replication and slowing disease progression (68). The strong
association observed between the V3 charge and the loss of the V3
N-linked glycosylation site for primary viral isolate
sequences suggests that the V3 de-glycosylation event may be worth
targeting to prevent the virus switching toward using the CXCR4 and
CCR3 coreceptors and the patient ultimately progressing to disease.
Subtype C envelopes have low V3 charges and a high frequency of V3
glycosylation, which is in support of reports describing a low
frequency of SI isolates among subtype C-infected individuals even when
the patients had low CD4+ cell counts and had progressed to
AIDS (69). In accordance, HIV-1 subtypes D and E demonstrate the
highest frequency of V3 de-glycosylation, even at the lower V3 charges,
and both these subtypes reportedly have a high frequency of SI viruses
among infected individuals (71, 72). It will be interesting to address why subtype C viruses keep a low charge in the V3 region, whereas rates
of disease progression do not appear to be different nor do the viral
loads seem to be lower than for the other subtypes (70). We can
speculate that the low V3 charge maintenance, hence CCR5-using
phenotype, in combination with high viral load may help explain why
subtype C viruses are spreading rapidly throughout the world.
Structural constraints in the envelope or biological constraints on the
virus may keep subtype C with the more optimal CCR5 phenotype for
efficient transmission. The elucidation of such mechanisms will be
important for the better understanding of HIV-1 disease pathogenesis.
We thank J. J. de Jong for providing the
original plasmid material and to A. Artsen for technical assistance,
Dr. H. Huisman for providing the anti-gp120 sera, and Dr. A. Trkola for
critical review of the manuscript.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M009779200
The abbreviations used are:
HIV-1, human
immunodeficiency virus type-1;
SI, syncytia-inducing;
M-tropic, macrophage tropic;
T-tropic, T cell line tropic;
PBMC, peripheral blood mononuclear cell;
TCID, tissue culture infectious
dose;
PAGE, polyacrylamide gel electrophoresis.
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