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Journal of Virology, November 2000, p. 10229-10235, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Heterogeneous Spectrum of Coreceptor Usage among Variants
within a Dualtropic Human Immunodeficiency
Virus Type 1 Primary-Isolate Quasispecies
Anjali
Singh and
Ronald G.
Collman*
Department of Medicine, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received 28 June 2000/Accepted 1 August 2000
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ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) variants that use the
coreceptor CCR5 for entry (R5; macrophage tropic) predominate in early infection, while variants that use CXCR4 emerge during disease
progression. Some late-stage variants use CXCR4 alone (X4; T-cell
tropic), while others use both CXCR4 and CCR5 (R5X4; dualtropic).
It has been proposed that dualtropic R5X4 strains are intermediates in
the evolution from R5 to X4, and we hypothesized that a dualtropic
primary-isolate quasispecies might contain variants that represent
the spectrum of coreceptor use in vivo. We generated a panel of 35 functional full-length env clones from the primary-isolate quasispecies of a dualtropic prototype strain, HIV-1
89.6PI. Thirty of the functional env clones
(86%) were R5X4, four (11%) were R5, and one (3%) was predominantly
X4. V3 to V5 sequences did not reveal clustering by coreceptor usage,
and no specific sequence motif or V3 charge pattern corresponded to
coreceptor utilization. Complete sequencing of seven functionally
divergent Env proteins revealed 
98.7% homology and conservation of
structurally important domains. Chimeras between the R5X4 89.6 prototype and an R5 variant indicated that multiple regions contributed
to the use of CXCR4, while chimeras with the X4 variant implicated a
single residue in V4 in CCR5 use. These results confirm, at the
molecular level, both that dualtropic variants are a predominant
component of late-stage syncytium-inducing isolates and that variants
restricted to each coreceptor coexist with dualtropic species in vivo.
Coreceptor-restricted minority variants may reflect residual R5 species
from earlier in disease as well as emerging X4 variants.
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TEXT |
Human immunodeficiency virus type 1 (HIV-1) entry requires interaction of the viral envelope glycoprotein
with cellular CD4 and a seven-transmembrane G protein-coupled chemokine
receptor (reviewed in reference 1). Differential use
of distinct chemokine receptors by HIV-1 isolates has largely explained
differences in viral tropism and cytopathogenicity. While several
chemokine receptors and orphan receptors can mediate entry in vitro,
the principal HIV-1 coreceptors are the 
-chemokine receptor CCR5 and
the 
-chemokine receptor CXCR4. Non-syncytium-inducing (NSI) HIV-1
variants that use CCR5 (R5; macrophage [M] tropic) play a crucial
role in sexual, blood-borne, and vertical transmission and are the
predominant viral population immediately after seroconversion and
during asymptomatic infection (26, 37). Syncytium-inducing (SI) variants that use CXCR4 evolve in about 50% of individuals with
AIDS, coincident with CD4+ T-cell decline, and the
emergence of SI variants is strongly associated with accelerated
disease (32). Recent studies with SCID mice and lymphoid
tissue ex vivo have suggested that the emergence of CXCR4 use may be
responsible for, rather than simply a consequence of, enhanced immune
destruction (13, 22).
SI variants may use CXCR4 alone (X4; T-cell-line [T] tropic) or in
addition to CCR5 (R5X4; dualtropic). While the dominance of R5 strains
early in infection and the emergence of CXCR4-using variants later in
disease are widely recognized, several important questions regarding
the relationship between R5, R5X4, and X4 variants in vivo remain
incompletely resolved. (i) Since new infections are initiated by
M-tropic NSI R5 strains, and not by X4 or R5X4 strains even when these
variants are present in late-stage transmitters, does this reflect the
universal persistence of R5 variants alongside the CXCR4-using SI
strains as they emerge late in disease (26, 34)? (ii)
Although several recent studies document the ability of late-stage
primary-isolate swarms to use both CCR5 and CXCR4 (10, 25),
does this reflect mainly the coexistence of both X4 and R5 variants
within the swarm or mainly dualtropic R5X4 variants? (iii) Similarly,
while many late-stage SI strains are clearly R5X4 (8, 28),
single-coreceptor X4 isolates are well described (2, 33) but
their place in viral evolution is not clear. It may be that R5X4
strains represent intermediates in the transition from R5 to X4
(11), but it is not known whether X4 variants would
eventually emerge from R5X4 populations in vivo given sufficient time.
Thus, the spectrum of individual variants that coexist within the
late-stage SI quasispecies is an important question that may offer
insights into aspects of pathogenesis.
HIV-1 89.6PI is a dualtropic primary isolate from the blood
of an individual with AIDS (8). An infectious molecular
clone derived from this primary isolate provided the first indication that both macrophage tropism and the T-cell-line-tropic SI phenotype could be a feature of a single virus and not the result of multiple variants within a primary-isolate quasispecies. These characteristics have since been linked to the use of CCR5 and CXCR4, respectively, and
this well-characterized infectious clone is widely used as an R5X4
prototype. We hypothesized that the 89.6PI quasispecies might contain R5 variants reflecting earlier stages of infection and X4
variants related to disease progression in vivo. Identifying and
analyzing the spectrum of quasispecies within this isolate may thus
provide insight into the steps involved in the phenotypic transition
from R5 to R5X4 and possibly to X4 that occurs during HIV-1
pathogenesis. To address the relationship between R5, R5X4, and X4
variants within a dualtropic viral swarm, we evaluated the coreceptor
usage pattern of a panel of related full-length 2.5-kb envelopes that
we cloned from the original 89.6 primary-isolate quasispecies.
Construction of a panel of related env genes from an
R5X4 primary isolate.
We used high-fidelity PCR to make 50 full-length env clones from the dualtropic
89.6PI viral swarm (8). We selected this primary
isolate because the infectious molecular clone derived from it is
widely employed as an R5X4 prototype in studies of tropism and
coreceptor use as well as in both in vitro and in vivo studies of
pathogenesis (11, 14, 16). The panel of related
env clones was generated from the same cellular DNA pool from which the original 89.6 infectious clone was isolated by lambda
phage cloning (8). To ensure that each clone represented a
distinct proviral molecule, rather than potentially multiple amplification products of the same template, we used separate aliquots
of template DNA in independent PCRs, and only one env clone
from each amplification reaction was utilized. Amplification was
carried out with primers 5'-AGA AAG AGA AGA AGA CAG TGG CAA TGA-3' and
5'-TAG CCC TTC CAG TCC CCC CTT TTC TTT TAA-3' using rTth-XL
polymerase (Perkin-Elmer, Foster City, Calif.). Reaction mixtures were
heated to 95°C for 1 min, followed by 35 cycles at 94°C for 1 min,
54°C for 5 min, and 72°C for 7 min, and a final 10-min extension at
72°C. Amplification products were treated with Pfu
polymerase (Stratagene, La Jolla, Calif.) to generate blunt ends,
purified, and ligated into pCR-Blunt (Invitrogen, Carlsbad, Calif.)
downstream of the T7 promoter. Clones carrying properly sized and
oriented 2.5-kb env inserts were identified by restriction
analysis. For clarity, the uncloned primary isolate is referred to as
89.6PI, the env gene from the 9.7-kb infectious molecular clone is referred to as 89.6, and each clone is designated by
a number from 1 to 50.
Coreceptor fusion patterns of 89.6-related env clones.
env clones were tested for fusion with CCR5 and CXCR4 in a
cell-cell fusion assay that relies on a T7 polymerase-expressing recombinant vaccinia virus and a T7-driven luciferase reporter system,
as described previously (11, 29). Within each experiment, controls included effector cells lacking envelope and target cells with
CD4 but no chemokine receptor, and 10-fold enhancement of fusion
relative to controls was considered positive.
Thirty-five of the 50 clones (70%) encoded Env proteins that were
functional for fusion with at least one major coreceptor (Table
1). The others failed to achieve
10-fold-greater luciferase expression compared to control cells
without Env when mixed with CD4 and at least one coreceptor and also
failed to achieve 10-fold-greater luciferase expression when mixed
with cells expressing CD4 and one coreceptor than when mixed with cells
expressing CD4 alone. For most env clones that did not
achieve this threshold, there was little luciferase expression or none
at all. Those clones were considered nonfunctional and were not
investigated further. None of the Env proteins fused with a coreceptor
independent of CD4 or used CD4 in the absence of a coreceptor (data not
shown).
The majority (86%) of functional envelopes from the 89.6
PI
swarm used both CCR5 and CXCR4 for fusion and so were R5X4, like
the
prototype 89.6 molecular clone (Table
1). For most, the levels
of
fusion with the two coreceptors were similar. In addition,
we found a
minority of variants that were restricted to one individual
coreceptor.
Four of the functional genes (11%) used CCR5 but not
CXCR4 (clones 10, 13, 14, and 23). Only one variant (3%) was selective
for CXCR4 (clone
22). Fusion mediated by the functionally divergent
Env proteins, along
with one representative R5X4 Env (clone 2),
is shown in Fig.
1A. These results demonstrate the
presence of
viral species with distinct coreceptor usage within the
dualtropic
89.6
PI quasispecies.
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FIG. 1.
Fusion and infection mediated by functionally divergent
env clones within the 89.6PI quasispecies. (A)
Cloned env genes were analyzed for CCR5 and CXCR4-mediated
fusion in a cell-cell fusion assay, along with the R5 and X4 prototypes
JRFL and 3B, respectively. The env gene designated 89.6 is
derived from the full-length infectious clone described previously
(8, 11). Only data for the five variants with distinct R5 or
X4 patterns and one representative R5X4 env variant are
shown in this graph. Each envelope-coreceptor combination was tested a
minimum of three times. (B) Pseudotype virions generated with one R5
and one X4 env variant were used to confirm coreceptor
selectivity in infection. To generate pseudotype virions,
env genes were cotransfected with an
env-defective HIV-1 plasmid that carries the luciferase
reporter gene in place of nef. U87 cells were transfected
with CD4 and the indicated chemokine receptor, infected with equal
amounts of each pseudotype virus based on p24 antigen content, and
lysed 3 days later for measurement of luciferase expression. Pseudotype
infections were tested in three independent experiments.
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Env-mediated pseudotype virion infections.
We next determined
whether the clones' coreceptor specificity in cell-cell fusion
accurately reflected Env-coreceptor-mediated infection, using
luciferase reporter virus pseudotypes (9). Two functionally
distinct env genes, clones 14 and 22, were subcloned under
the control of the cytomegalovirus (CMV) promoter into pCDNA3 (Invitrogen) and cotransfected into 293T cells along with the NL4-3
backbone plasmid (pNL-luc-E
R) that bears a
defective env gene and the luciferase reporter gene in place
of nef (9) (kindly provided by N. Landau). Two days later, the supernatant was harvested, clarified by centrifugation, and quantified by p24 antigen content. U87 cells (2 × 105 cells per well in 24-well plates) were transfected with
CD4, with or without a coreceptor, and infected the following day with pseudotypes by using 20 ng of p24 antigen, in the presence
of Polybrene (5 µg/ml). Luciferase expression was quantified
in cell lysates 3 days later. In parallel, pseudotype virions were made with the R5X4 89.6 env, as well as the R5 JRFL and X4 3B prototypes.
As shown in Fig.
1B, pseudotype virions carrying the three
89.6
PI-derived variants differed in coreceptor-mediated
tropism.
Virions carrying
env variant 14 infected cells
expressing CD4
and CCR5 but not CXCR4, like JRFL. In contrast, virions
carrying
env variant 22 resembled 3B and infected cells
expressing CXCR4
but not CCR5. As expected, virions carrying the
prototype 89.6
Env infected cells expressing CD4 with either CCR5
or CXCR4. Thus,
Env-mediated infection showed the same selective
pattern of coreceptor
utilization as did fusion and confirmed the
presence of variants
restricted to CCR5 or CXCR4 for
infection.
Phylogenetic and V3 sequence analysis of related env
clones.
To determine the genetic relatedness among the 36 distinct
envelope genes derived from the 89.6PI viral swarm (35 functional clones generated here plus 89.6), we sequenced the V3-to-V5
region of each and compared their predicted amino acid and nucleic acid sequences (Fig. 2). Among all the
env variants, the overall nucleic acid homology was
97.5%, and 20 distinct sequences were identified. Fourteen sequences
were represented by one clone each, two sequences were each represented
by two clones, two sequences by three clones each, one sequence by four
clones, and one V3-to-V5 sequence was shared by eight of the
env variants including the 89.6 prototype. Since each
clone was generated independently from separate aliquots of template
DNA, they represent independent variants within the quasispecies
despite shared V3-to-V5 sequences.
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FIG. 2.
Genetic relatedness of all 36 functional env
clones from the 89.6PI swarm, determined on the basis of
the nucleotide sequences across a ~600-bp V3-to-V5 region. The
dendrogram was generated by using the Clustal method. Clones restricted
to CCR5 or CXCR4 are indicated by R5 or X4 to the right of the clone
number, while the rest of the env clones, for which no
coreceptor usage designation is given, were R5X4.
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All
env clones with identical V3-to-V5 sequences had the
same coreceptor use patterns. However, the R5
env clones as
a group
did not form a cluster when analyzed by V3-to-V5 amino acid or
nucleic acid sequence (Fig.
2), or when the analysis was restricted
to
V3 alone (data not shown). In fact, only 6
env clones
differed
from 89.6 within V3. The R5 variants 13 and 14 had a
non-charge-altering
I327M substitution, two R5X4 variants had a
non-charge-altering
T305A substitution, and one R5X4 variant had a
R300G substitution
which lowered the overall charge from +7 in 89.6 to
+6 but did
not alter coreceptor use. One additional
env
clone (clone 37)
differed from 89.6 at 6 sites in V3 (SIH instead of
RLS at residues
308 to 310, and TGD instead of RRN at residues 320 to
322), which
resulted in a considerably lower +3 V3 charge but also did
not
affect the R5X4 phenotype. Thus, the V3 sequence and charge
appeared
to have little role in regulating differential coreceptor
choice
among variants in this
panel.
Genetic analysis of functionally distinct envelope clones.
To
further address the genetic basis for coreceptor heterogeneity among
the related env clones, we obtained full-length 2.5-kb sequences for the functionally divergent subset (X4 clone 22 and R5
clones 10, 13, 14, and 23) and one representative R5X4 variant (clone
2), and compared them with the 89.6 prototype (Fig.
3). During this analysis we identified
one error in the published sequence of the 89.6 molecular clone Env
(Ala instead of Arg at amino acid 542 in gp41), which has been
corrected in GenBank.
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FIG. 3.
Amino acid alignment of functionally diverse
env gene sequences. Full-length sequences were determined
for the four R5 env clones (clones 10, 13, 14, and 23), one
X4 env clone (clone 22), and one representative R5X4
env clone derived from this pool (clone 2). Sequences were
aligned with that of the original R5X4 89.6 clone. R5 clones 13 and 14 had identical Env sequences.
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The R5X4 variant analyzed (clone 2) was identical to 89.6 in V3 to V5
but differed in several amino acids outside this region
(Fig.
3), while
two R5 clones had identical full-length Env sequences
(clones 13 and
14). Overall, there was
98.7% amino acid homology
among
fully sequenced Env proteins. All amino acids implicated
in direct CD4
binding were conserved among the seven variants
(
19), and 15 of 18 residues implicated in CCR5 binding were
conserved as well
(
23). The 7 Env proteins analyzed shared 29
of 30 potential
N-linked glycosylation sites. The exception was
one potential site at
N138 that was lacking in the R5X4 variant
2 and the R5 variant 23 but
was present in the others, and so
was not linked to coreceptor
specificity. Unexpectedly, one of
the R5
env clones (clone
23) had a single extra cysteine in gp120
(Y434C), confirmed by repeated
sequencing. Since this Env supports
fusion, the mechanism by which this
presumably unpaired cysteine
residue allows proper gp120 expression and
function is
uncertain.
Compared to 89.6, differences in gp120 sequence among the R5 clones
were most prominent in the signal peptide and V regions,
as expected
(Fig.
3). Only one V3 difference was found among the
functionally
divergent Env proteins (R5
env clones 13 and 14),
and that
was a single substitution (I330M) that did not affect
the strong
positive 89.6 V3 charge. Importantly, there was no
sequence motif
common to the R5 variants compared with the R5X4
env
variants that might suggest a common regulator of X4 utilization.
The
one X4 clone (clone 22) revealed only a single-amino-acid
difference in
gp120 compared with 89.6, in V4 (N410S), suggesting
that this residue
may contribute to CCR5 utilization in the background
of this
CXCR4
use.
Unexpectedly, sequencing of the portion corresponding to gp41 showed
that all of the
env variants were quite similar to one
another but differed from 89.6 in several locations (Fig.
3).
All six
gp41 variants shared three amino acid differences compared
with 89.6, and five of the six shared an additional three distinct
residues. This
suggests that the 89.6
env gene is the most divergent
among
the variants cloned from the primary-isolate swarm. Since
the gp41
differences did not correlate with R5, R5X4, or X4 patterns,
however,
they are not likely to be responsible for coreceptor
choice among these
variants.
Chimeric envelopes and molecular determinants of coreceptor
use.
Since sequences did not show any clear linkage to coreceptor
use, in order to begin to address the molecular regulation of individual coreceptor usage among related variants of a dualtropic swarm, we generated two pairs of reciprocal recombinants. We selected one R5 clone (clone 14) and one X4 clone (clone 22) and exchanged env domains with the 89.6 R5X4 env by using
overlap extension PCR (Fig. 4A). Three
overlapping fragments were amplified from each env clone
using rTth-XL polymerase. A ~1,000-bp 5' fragment was
amplified with the same upstream primer used for PCR cloning and
downstream primer 5'-CTT ATT ATG TTT CTT CTT GCA TAA-3'; a ~550-bp
V3-to-V5 fragment was amplified using upstream primer 5'-TTA TGC AAG
AAG AAA CAT AAT AAG-3' and downstream primer 5'-GCC CTG GTG GGT GCT ACT
CCT ATT-3'; and a ~1,400-bp fragment encompassing the 3' portion of
gp120 and gp41 was amplified with upstream primer 5'-AAT AGG AGT AGC
ACC CAC CAG GGC-3' and the downstream primer used for PCR cloning of
env. Reaction mixtures were heated to 95°C for 1 min,
followed by 25 cycles at 94°C for 30 s, 48°C for 30 s,
and 72°C for 1 min, with a final extension of 72°C for 10 min.
After gel purification, the 5' and middle fragments were mixed to
generate chimeric combinations and reamplified using the 5' outer
primer and 3' middle fragment primer. The process was repeated using
the purified products of this reaction and the 3' fragment amplified
with the 5' and 3' outer primers. The final amplification products were
treated with Pfu polymerase and ligated downstream of the T7
promoter into pCR-Blunt. Clones carrying properly sized and oriented
envelope inserts were identified by restriction analysis and verified
by complete sequencing.
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FIG. 4.
Coreceptor usage of chimeras generated between related
env genes with distinct phenotypes. (A) Construction of
recombinant env genes. Chimeras were made between the R5X4
89.6 env molecular clone and the R5 variant 14, and between
89.6 and the X4 variant 22. Overlap extension PCR was used to exchange
the indicated regions, which introduced the amino acid changes noted.
(B) Coreceptor selectivity of chimeric env variants. The
env chimeras were tested for CCR5 and CXCR4 utilization
based on cell-cell fusion.
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One pair of recombinants exchanged all four gp120 residues differing
between 89.6 and the R5 variant 14, except for one amino
acid in the
signal peptide (Fig.
4A). Introduction of these amino
acids from 89.6 into the R5 clone 14 (chimera 14-89-14) conferred
on it the ability to
use CXCR4 in addition to CCR5 (Fig.
4B).
However, replacing these
residues in 89.6 with sequences from
the R5 clone 14 (89-14-89) did not
abrogate the ability of 89.6
to use both CXCR4 and CCR5. When the
chimeric
env clones were
subcloned into CMV-driven vectors
and used to make pseudotype
virions, identical results were seen for
coreceptor-mediated infection
(data not shown). These results indicate
that these gp120 residues
are partially responsible for regulating
CXCR4 usage among these
related variants, but that other determinants
contribute as well.
Since the only other differences between 89.6 and
clone 14 are
in the signal peptide and in gp41, this suggests that
coreceptor
selectivity among these related species is complex and
dependent
on cooperation among multiple domains, including regions not
generally
considered typical tropism
determinants.
We then tested the pair of chimeras made between 89.6 and X4 variant
22. Since the only gp120 difference between variant 22
and 89.6 is a
single change in V4 (N407S), we used the same overlap
extension primer
pair described above, which resulted in exchange
of this residue only
(Fig.
4A). Replacing Ser 407 of the X4
env variant 22 with
the 89.6 Asn residue (22-89-22) resulted in an
env chimera
that fused with both CCR5 and CXCR4 (Fig.
4B), implicating
this residue
in the regulation of CCR5 use. However, changing
the 89.6 sequence to
that of clone 22 (89-22-89) reduced, but
did not completely block, the
use of CCR5. Pseudotype virions
generated with the chimeric
env variants showed that the same
pattern was true for
infection (data not shown). Thus, this V4
residue is responsible in
part for regulating CCR5 use in these
related variants. However, other
regions of Env, presumably in
gp41, appear also to contribute to or to
modulate the coreceptor
specificity encoded by
gp120.
Biological significance of heterogeneous coreceptor use within the
89.6PI swarm.
In this study we addressed the
biological and molecular relationship among R5, R5X4, and X4 variants
within a dualtropic primary-isolate quasispecies. This is important
because the relationship between R5, R5X4, and X4 HIV-1 variants in
vivo is not well understood, yet the evolution from R5 to R5X4, and
possibly to X4, is a major determinant of pathogenesis. Our results
show that at the molecular level (i) R5 variants persist alongside
X4-using strains, implicating a source for R5 viruses in
person-to-person transmission by advanced patients, and (ii)
CXCR4-restricted X4 variants may ultimately emerge from the R5X4 pool,
indicating that R5X4 variants may be evolutionary intermediates rather
than the ultimate end result of evolution in vivo. Furthermore, (iii)
the heterogeneous biological spectrum was independent of Env sequence
and charge patterns traditionally recognized as tropism and coreceptor
determinants, indicating that biological analysis, and not the sequence
alone, is necessary to understand these relationships in vivo.
Most of the functional
env genes in this panel of
genetically related clones efficiently used both CCR5 and CXCR4. In
addition
to the R5X4 majority, however, we also found a few variants
that
were selective for an individual coreceptor. The functional
diversity
of low-frequency minority variants within dualtropic
quasispecies
has not been studied previously, and we chose to analyze
this
primary isolate because the R5X4 prototype infectious molecular
clone and the
env gene derived from it are widely used for
in
vitro, ex vivo, and in vivo studies (
6,
11,
14,
16).
The
role played by R5X4 dualtropic variants in HIV-1 pathogenesis
still
requires clarification. Several groups have suggested that
most SI
primary isolates are dualtropic and use both R5 and X4
(
10,
25,
28,
34). However, this has been based mainly
on dual coreceptor
use by uncloned viral swarms, or on a limited
number of biologically
cloned isolates. The predominance of R5X4
env clones that we
found within this late-stage SI isolate demonstrates
at the molecular
level that it is indeed composed mainly of dualtropic
R5X4 variants and
is not predominantly a mix of M-tropic R5 and
T-tropic X4
variants.
Coexisting with the R5X4 majority, a small group of
env
clones within the swarm used only CCR5. R5 variants predominate early
in infection, and the emergence of CXCR4 utilization occurs as
a late
event. Thus, the genetically related R5 variants within
the 89.6 quasispecies may reflect variants from earlier in the
R5-to-R5X4
phenotypic evolution. While it is possible that R5
species would
eventually be replaced completely by variants competent
for CXCR4 use,
our findings suggest that single-coreceptor R5
variants persist
alongside R5X4. The persistence of CCR5-restricted
minority variants
goes along with the observation that this is
the virus subtype involved
in HIV-1 transmission, even from late-stage
individuals who harbor
mainly R5X4 or even X4
species.
We also found one
env clone that was restricted to CXCR4. In
contrast to the widely recognized transition from R5 to R5X4,
the
relationship between R5X4 and X4 variants in vivo is less
certain. The
discovery that many late-stage SI strains are R5X4
has led some to
suggest that R5X4 species are the selectively
advantaged phenotype and
the end result of evolution in vivo.
On the other hand, X4 primary
isolates are well described (
2,
33), and it has been
hypothesized that R5X4 variants are transitional
species in the
evolution from R5 to X4 (
11). Since the selective
forces
driving phenotypic evolution in vivo are poorly understood,
it is not
known whether X4 variants would eventually emerge in
most individuals
given sufficient time. The presence of X4 species
within this swarm,
albeit at low frequency, is consistent with
such a pattern and could
reflect early evidence of X4 emergence.
The high degree of genetic
similarity among the
env variants does
not allow for formal
evolutionary analysis that could definitively
show a temporal
relationship between the R5, R5X4, and X4 variants,
however. Of note,
we recently analyzed the
env variants contained
within
primary-isolate viral swarms obtained from blood of three
late-stage
AIDS patients, and we found that all of them harbored
X4 minority
variants, even though only one of the three primary
isolates had a SI
phenotype (
29). Thus, it is possible that
viral evolution in
vivo at the molecular level may precede the
appearance of phenotypic
features evident in bulk
populations.
Among the 36 functional envelope genes derived from the
89.6
PI swarm, there was a high degree of homology in the
V3-to-V5
region. However, no common sequence pattern, which might
suggest
either that R5 viruses were more closely related or that they
shared common determinants of coreceptor selectivity, distinguished
the
R5 from the R5X4 variants. Furthermore, the V3 region, surprisingly,
did not appear to regulate the coreceptor specificity of these
related
species, since the V3 sequences of the R5
env variants
did
not differ from the highly charged V3 sequence of 89.6. This
is in
contrast to prototype NSI R5 strains that display a relatively
low V3
charge pattern (
5,
36) and differs from the central
role
identified for V3 in many other studies of tropism and coreceptor
choice (
4,
7,
20,
31,
35). In fact, only one V3 difference
was identified among all the functionally distinct Env proteins,
and
this non-charge-altering substitution was not the principal
coreceptor
determinant. Interestingly, a previous report analyzed
a set of
molecular clones derived contemporaneously from an infected
individual
and also found marked differences in cell tropism despite
identical V3
sequences (
15). Together, these results demonstrate
considerable flexibility in biological characteristics for any
given V3
sequences. Thus, while the V3 pattern may evolve over
time from a
low-charge pattern typical of NSI, M-tropic, R5 strains
to a
high-charge, SI, T-tropic X4 profile (
27), at any given
time
variants with a range of tropisms and other biological characteristics
may still be retained within the population represented by that
V3
pattern.
Since no correlation was found between coreceptor choice and V3
sequences, genetic mapping was done and revealed complex determinants
of coreceptor choice. While extensive studies have focused on
Env
determinants of tropism and coreceptor selectivity, this is,
to our
knowledge, the first study to address coreceptor determinants
among
naturally occurring, related yet functionally distinct variants
within
a quasispecies. In analyzing the X4 versus the R5X4 phenotype
(i.e.,
CCR5 use on the background of CXCR4 utilization), an important
although
not absolute role in CCR5 usage was suggested for a single
residue in
V4. In analyzing the R5 versus R5X4 phenotype (i.e.,
CXCR4 use on the
background of CCR5 utilization), sequences in
gp120 and determinants
elsewhere appeared to contribute to CXCR4
use. It is well recognized
that multiple Env regions can contribute
to tropism and coreceptor use
in addition to or independently
of V3, particularly V1/V2 and V4/V5
(
3,
18). Except for a
single residue in the signal peptide,
however, the only other
differences between 89.6 and the R5
env variant tested were in
gp41. This suggests that elements
in the transmembrane subunit
can, in conjunction with determinants in
gp120, modulate coreceptor
use. Determinants in gp41 have not
previously been reported to
specifically influence viral tropism and
coreceptor use, and most
of the gp41 sequence differences were shared
by R5, R5X4, and
X4 variants, so it is not yet apparent how they
contribute to
regulation of coreceptor specificity. Nevertheless, these
data
support the idea that gp160 is a highly cooperative molecule where
specific amino acids may have very different effects depending
on the
context and the remainder of the
protein.
In previous studies we analyzed recombinants between 89.6 and unrelated
R5 and X4 prototype strains, and there also we found
that multiple Env
regions contributed to both cell tropism and
coreceptor choice
(
17,
30). The present data are consistent
with those results
and confirm that determinants of coreceptor
use in 89.6 and its related
variants differ from those identified
in prototype R5 and X4 strains.
The ability of multiple regions
to enable dual tropism or coreceptor
use suggests that for this
strain the dual phenotype is "dominant"
and that members of this
swarm may be rather plastic in their ability
to use the major
coreceptors. Genetic analysis has typically considered
structural
elements that confer specific coreceptor use. Alternatively,
it
is possible that the Env core is intrinsically able to interact
with
both coreceptors, and that structural determinants which
regulate
coreceptor choice interfere with the efficient use of
one or the other.
Thus, the 89.6 Env may possess a structure that
is inherently less
susceptible to downregulatory influences of
other domains. In addition
to CCR5 and CXCR4, 89.6 can use an
unusually large range of other
seven-transmembrane domain receptors
for fusion (
6,
11,
12,
24), which may also be consistent
with a highly plastic and
adaptable coreceptor utilization
capacity.
In vivo, aspects of pathogenesis that are linked to differences in
coreceptor use include the propensity of CCR5-using strains
to
participate in person-to-person transmission and
macrophage-dependent
sequelae such as neurological or pulmonary
disease, and the enhanced
ability of CXCR4-using strains to deplete
CD4
+ T cells. A limitation of this study is that it is not
known what
level of coreceptor use in vitro corresponds with coreceptor
use
and biologically related properties in vivo, and there may be
differences between coreceptor function in vitro and in vivo
(
14).
In addition, coreceptor use in vitro may vary
depending on the
cell types in which it is tested, expression level,
and whether
fusion or infection is used as the readout (
12,
21,
24).
Our studies with pseudotype viruses confirm that the
coreceptor
specificity observed in fusion also reflects coreceptor use
for
infection in vitro. It will be useful to compare the pathogenic
consequences of these viral variants using recombinant viruses
incorporating these
env genes in studies of pathogenesis ex
vivo
or in vivo with animal models. In addition, we anticipate that
this panel of genetically related but functionally distinct
env variants generated from the 89.6
PI
dualtropic prototype strain
will provide a valuable tool with which to
better understand the
structural basis for coreceptor choice of
naturally occurring
viruses that emerge in
vivo.
| |
ACKNOWLEDGMENTS |
We thank D. Williams for expert technical assistance, N. Landau for
pNL-luc-ER, and B. Hahn, S. Isaacs, D. Kolson, and R. Doms for valuable advice and discussions.
This work was supported by NIH grants HL 58004 and AI 35502.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Pennsylvania School of Medicine, 807 Abramson Building, 34th and Civic Center Blvd., Philadelphia, PA 19104-4399. Phone: (215) 898-0913. Fax:
(215) 573-4446. E-mail: collmanr{at}mail.med.upenn.edu.
| |
REFERENCES |
| 1.
|
Berger, E. A.,
P. M. Murphy, and J. M. Farber.
1999.
Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease.
Annu. Rev. Immunol.
17:657-700[CrossRef][Medline].
|
| 2.
|
Björndal, Å.,
H. K. Deng,
M. Jansson,
J. R. Fiore,
C. Colognesi,
A. Karlsson,
J. Albert,
G. Scarlatti,
D. R. Littman, and E. M. Fenyö.
1997.
Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.
J. Virol.
71:7478-7487[Abstract].
|
| 3.
|
Carrillo, A., and L. Ratner.
1996.
Cooperative effects of the human immunodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells.
J. Virol.
70:1310-1316[Abstract].
|
| 4.
|
Chan, S. Y.,
R. F. Speck,
C. Power,
S. L. Gaffen,
B. Chesebro, and M. A. Goldsmith.
1999.
V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1.
J. Virol.
73:2350-2358[Abstract/Free Full Text].
|
| 5.
|
Chesebro, B.,
K. Wehrly,
J. Nishio, and S. Perryman.
1992.
Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.
J. Virol.
66:6547-6554[Abstract/Free Full Text].
|
| 6.
|
Choe, H.,
M. Farzan,
Y. Sun,
N. Sullivan,
R. Rollins,
P. D. Ponath,
L. Wu,
C. R. Mackay,
G. LaRosa,
W. Newman,
N. Gerard,
C. Gerard, and J. Sodroski.
1996.
The -chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell
85:1135-1148[CrossRef][Medline].
|
| 7.
|
Cocchi, F.,
A. L. DeVico,
A. Garzino-Demo,
A. Cara,
R. C. Gallo, and P. Lusso.
1996.
The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection.
Nat. Med.
2:1244-1247[CrossRef][Medline].
|
| 8.
|
Collman, R.,
J. W. Balliet,
S. A. Gregory,
H. Friedman,
D. L. Kolson,
N. Nathanson, and A. Srinivasan.
1992.
An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.
J. Virol.
66:7517-7521[Abstract/Free Full Text].
|
| 9.
|
Connor, R. I.,
B. K. Chen,
S. Choe, and N. R. Landau.
1995.
Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes.
Virology
206:935-944[CrossRef][Medline].
|
| 10.
|
Connor, R. I.,
K. E. Sheridan,
D. Ceradini,
S. Choe, and N. R. Landau.
1997.
Change in coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185:621-628[Abstract/Free Full Text].
|
| 11.
|
Doranz, B. J.,
J. Rucker,
Y. Yi,
R. J. Smyth,
M. Samson,
S. C. Peiper,
M. Parmentier,
R. G. Collman, and R. W. Doms.
1996.
A dual-tropic HIV-1 isolate that uses fusin and the -chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell
85:1149-1158[CrossRef][Medline].
|
| 12.
|
Edinger, A. L.,
T. L. Hoffman,
M. Sharron,
B. Lee,
Y. Yi,
W. Choe,
D. L. Kolson,
B. Mitrovic,
Y. Zhou,
D. Faulds,
R. G. Collman,
J. Hesselgesser,
R. Horuk, and R. W. Doms.
1998.
An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus.
J. Virol.
72:7934-7940[Abstract/Free Full Text].
|
| 13.
|
Glushakova, S.,
J. C. Grivel,
W. Fitzgerald,
A. Sylwester,
J. Zimmerberg, and L. B. Margolis.
1998.
Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency.
Nat. Med.
4:346-349[CrossRef][Medline].
|
| 14.
|
Glushakova, S.,
Y. J. Yi,
J. C. Grivel,
A. Singh,
D. Schols,
E. De Clercq,
R. G. Collman, and L. Margolis.
1999.
Preferential coreceptor utilization and cytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo.
J. Clin. Investig.
104:R7-R11.
|
| 15.
|
Groenink, M.,
A. C. Andeweg,
R. A. M. Fouchier,
S. Broersen,
R. C. M. Van der Jagt,
H. Schuitemaker,
R. E. Y. de Goede,
M. L. Bosch,
H. G. Huisman, and M. Tersmette.
1992.
Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain.
J. Virol.
66:6175-6180[Abstract/Free Full Text].
|
| 16.
|
Karlsson, G. B.,
M. Halloran,
J. Li,
I. W. Park,
R. Gomila,
K. A. Reimann,
M. K. Axthelm,
S. A. Iliff,
N. L. Letvin, and J. Sodroski.
1997.
Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys.
J. Virol.
71:4218-4225[Abstract].
|
| 17.
|
Kim, F. M.,
D. L. Kolson,
J. W. Balliet,
A. Srinivasan, and R. G. Collman.
1995.
V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.
J. Virol.
69:1755-1761[Abstract].
|
| 18.
|
Koito, A.,
G. Harrowe,
J. A. Levy, and C. Cheng-Mayer.
1994.
Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization.
J. Virol.
68:2253-2259[Abstract/Free Full Text].
|
| 19.
|
Kwong, P. D.,
R. Wyatt,
J. Robinson,
R. W. Sweet,
J. Sodroski, and W. A. Hendrickson.
1998.
Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.
Nature
393:648-659[CrossRef][Medline].
|
| 20.
|
O'Brien, W. A.,
Y. Koyanagi,
A. Namazie,
J.-Q. Zhao,
A. Diagne,
K. Idler,
J. A. Zack, and I. S. Y. Chen.
1990.
HIV-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the CD4-binding domain.
Nature
348:69-73[CrossRef][Medline].
|
| 21.
|
Ohagen, A.,
L. Li,
A. Rosenzweig, and D. Gabuzda.
2000.
Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus.
AIDS Res. Hum. Retrovir.
16:27-35[CrossRef][Medline].
|
| 22.
|
Picchio, G. R.,
R. J. Gulizia,
K. Wehrly,
B. Chesebro, and D. E. Mosier.
1998.
The cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in SCID mice reconstituted with human peripheral blood leukocytes.
J. Virol.
72:2002-2009[Abstract/Free Full Text].
|
| 23.
|
Rizzuto, C. D.,
R. Wyatt,
N. Hernández-Ramos,
Y. Sun,
P. D. Kwong,
W. A. Hendrickson, and J. Sodroski.
1998.
A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding.
Science
280:1949-1953[Abstract/Free Full Text].
|
| 24.
|
Rucker, J.,
A. L. Edinger,
M. Sharron,
M. Samson,
B. Lee,
J. F. Berson,
Y. Yi,
B. Margulies,
R. G. Collman,
B. J. Doranz,
M. Parmentier, and R. W. Doms.
1997.
Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses.
J. Virol.
71:8999-9007[Abstract].
|
| 25.
|
Scarlatti, G.,
E. Tresoldi,
A. Bjorndal,
R. Fredriksson,
C. Colognesi,
H. K. Deng,
M. S. Malnati,
A. Plebani,
A. G. Siccardi,
D. R. Littman,
E. M. Fenyo, and P. Lusso.
1997.
In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.
Nat. Med.
3:1259-1265[CrossRef][Medline].
|
| 26.
|
Schuitemaker, H.,
N. A. Kootstra,
R. E. Y. de Goede,
F. de Wolf,
F. Miedema, and M. Tersmette.
1991.
Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture.
J. Virol.
65:356-363[Abstract/Free Full Text].
|
| 27.
|
Shankarappa, R.,
P. Gupta,
G. H. Learn, Jr.,
A. G. Rodrigo,
C. R. Rinaldo, Jr.,
M. C. Gorry,
J. I. Mullins,
P. L. Nara, and G. D. Ehrlich.
1998.
Evolution of human immunodeficiency virus type 1 envelope sequences in infected individuals with differing disease progression profiles.
Virology
241:251-259[CrossRef][Medline].
|
| 28.
|
Simmons, G.,
D. Wilkinson,
J. D. Reeves,
M. T. Dittmar,
S. Beddows,
J. Weber,
G. Carnegie,
U. Desselberger,
P. W. Gray,
R. A. Weiss, and P. R. Clapham.
1996.
Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.
J. Virol.
70:8355-8360[Abstract].
|
| 29.
|
Singh, A.,
G. Besson,
A. Mobasher, and R. G. Collman.
1999.
Patterns of chemokine receptor fusion cofactor utilization by human immunodeficiency virus type 1 variants from the lungs and blood.
J. Virol.
73:6680-6690[Abstract/Free Full Text].
|
| 30.
|
Smyth, R. J.,
Y. Yi,
A. Singh, and R. G. Collman.
1998.
Determinants of entry cofactor utilization and tropism in a dualtropic human immunodeficiency virus type 1 primary isolate.
J. Virol.
72:4478-4484[Abstract/Free Full Text].
|
| 31.
|
Speck, R. F.,
K. Wehrly,
E. J. Platt,
R. E. Atchison,
I. F. Charo,
D. Kabat,
B. Chesebro, and M. A. Goldsmith.
1997.
Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop.
J. Virol.
71:7136-7139[Abstract].
|
| 32.
|
Tersmette, M.,
R. A. Gruters,
F. de Wolf,
R. E. Y. de Goede,
J. M. A. Lange,
P. T. A. Schellekens,
J. Goudsmit,
H. G. Huisman, and F. Miedema.
1989.
Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates.
J. Virol.
63:2118-2125[Abstract/Free Full Text].
|
| 33.
|
Tscherning, C.,
A. Alaeus,
R. Fredriksson,
Å. Björndal,
H. K. Deng,
D. R. Littman,
E. M. Fenyö, and J. Albert.
1998.
Differences in chemokine coreceptor usage between genetic subtypes of HIV-1.
Virology
241:181-188[CrossRef][Medline].
|
| 34.
|
Valentin, A.,
J. Albert,
E. M. Fenyo, and B. Asjo.
1994.
Dual tropism for macrophages and lymphocytes is a common feature of primary human immunodeficiency virus type 1 and 2 isolates.
J. Virol.
68:6684-6689[Abstract/Free Full Text].
|
| 35.
|
Wang, W. K.,
T. Dudek,
M. Essex, and T. H. Lee.
1999.
Hypervariable region 3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization: therapeutic and prophylactic implications.
Proc. Natl. Acad. Sci. USA
96:4558-4562[Abstract/Free Full Text].
|
| 36.
|
Zhong, P.,
M. Peeters,
W. Janssens,
K. Fransen,
L. Heyndrickx,
G. Vanham,
B. Willems,
P. Piot, and G. Van der Groen.
1995.
Correlation between genetic and biological properties of biologically cloned HIV type 1 viruses representing subtypes A, B, and D.
AIDS Res. Hum. Retrovir.
11:239-248[Medline].
|
| 37.
|
Zhu, T.,
H. Mo,
N. Wang,
D. S. Nam,
Y. Cao,
R. A. Koup, and D. D. Ho.
1993.
Genotypic and phenotypic characterization of HIV-1 in patients with primary infection.
Science
261:1179-1181.
|
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