By
From the Aaron Diamond AIDS Research Center and The Rockefeller University, New York, 10016
Recent studies have identified several coreceptors that are required for fusion and entry of Human Immunodeficiency Virus type 1 (HIV-1) into CD4+ cells. One of these receptors, CCR5, serves as a coreceptor for nonsyncytium inducing (NSI), macrophage-tropic strains of HIV-1, while another, fusin or CXCR-4, functions as a coreceptor for T cell line-adapted, syncytiuminducing (SI) strains. Using sequential primary isolates of HIV-1, we examined whether viruses using these coreceptors emerge in vivo and whether changes in coreceptor use are associated with disease progression. We found that isolates of HIV-1 from early in the course of infection predominantly used CCR5 for infection. However, in patients with disease progression, the virus expanded its coreceptor use to include CCR5, CCR3, CCR2b, and CXCR-4. Use of CXCR-4 as a coreceptor was only seen with primary viruses having an SI phenotype and was restricted by the env gene of the virus. The emergence of variants using this coreceptor was associated with a switch from NSI to SI phenotype, loss of sensitivity to chemokines, and decreasing CD4+ T cell counts. These results suggest that HIV-1 evolves during the course of infection to use an expanded range of coreceptors for infection, and that this adaptation is associated with progression to AIDS.
Primary isolates of HIV type 1 (HIV-1) show distinct
differences in their biological properties in vitro, including differences in replication kinetics, tropism, and syncytium-inducing capacity (1). Macrophage-tropic, nonsyncytium-inducing (NSI)1 viruses are most commonly
transmitted and predominate in the early stages of infection
(4). Syncytium-inducing (SI) isolates generally appear
later in infection (8, 9) and may acquire an expanded tropism in vitro, including the ability to replicate in CD4+ T
cell lines.
HIV-1 tropism is largely controlled by the env gene of
the virus (10) which encodes the surface (gp120) and
transmembrane (gp41) envelope glycoproteins. Although
not clearly defined, binding of gp120 to CD4 is believed to
trigger a series of conformational changes, leading to exposure of the NH2-terminal domain of gp41 and subsequent
fusion of the virus and host cell membranes (for review see
reference 15). Despite high affinity binding to gp120, expression of human CD4 on the surface of nonpermissive
cells is not sufficient to confer susceptibility to HIV-1 infection (16), indicating that additional factors are required for viral fusion and entry to take place.
Recent studies have identified several proteins that serve
as cofactors for HIV-1 fusion with CD4+ T cells (19).
These proteins are structurally related, G protein-coupled receptors characterized by seven transmembrane-spanning
regions (25). One receptor, termed fusin (23) or CXCR-4
(26, 27), has been shown to mediate entry of T cell line-
adapted (TCLA), SI strains of HIV-1 into CD4+ T cells,
but does not permit entry of NSI isolates. A second molecule, CCR5, has recently been identified as a coreceptor for macrophage-tropic, NSI strains of HIV-1 (20, 21, 24). CCR5 is a member of a family of receptors that bind While disease progression in many HIV-1-infected individuals is associated with a switch in viral phenotype from
NSI to SI, the ability of HIV-1 isolates to induce syncytium formation is defined largely on the basis of in vitro assays (29) with no clear in vivo correlate. We hypothesize
here that the molecular basis of the NSI to SI switch lies in
a change in coreceptor usage from CCR5 to CXCR-4.
Thus far, CXCR-4 has been shown to mediate entry of
predominantly TCLA strains of HIV-1 (23). However, it is
not yet clear whether primary strains of HIV-1 that use CXCR-4 emerge during the course of infection, and
whether use of this coreceptor relates to the switch from
NSI to SI phenotype.
Previously, we characterized in detail a series of sequential primary isolates of HIV-1 from three infected patients
(5). Two of these patients experienced a switch in viral
phenotype from NSI to SI followed by a marked drop in
CD4+ T cell counts and the onset of AIDS. The third patient remained clinically stable with only NSI isolates for
more than 10 yr after HIV-1 infection. Using sequential
isolates from all three patients, we examined the coreceptor
use and determined whether disease progression, marked
by a switch from NSI to SI phenotype, was associated with
the emergence of variants able to use CXCR-4 as a coreceptor, and whether this adaptation was associated with CD4+ T cell decline in vivo. We show here that HIV-1
uses primarily a single receptor, CCR5, in the early stages
of infection. However, the virus expands its coreceptor use
to include CCR5, CCR2b, CCR3, and CXCR-4 in patients with disease progression. The ability to use CXCR-4
as a coreceptor was only seen with viruses having an SI
phenotype and was restricted by the env gene of the virus. The emergence of these viruses was associated with a drop
in CD4+ T cell counts and clinical progression in the patients we studied.
Study Subjects.
Sequential isolates of HIV-1 were obtained
from two individuals, B and C, both of whom progressed rapidly
to AIDS after seroconversion. Each patient experienced a precipitous drop in CD4+ T cell counts that was preceded by an increase in viral load and a switch in viral phenotype from NSI to
SI (5). Sequential isolates were also evaluated from patient D, who
remained asymptomatic for more than a decade after HIV-1 infection (5). Isolates from this patient spanned a 5-yr period during
which time he was clinically stable with a low viral load and normal CD4+ T cell counts. All of the isolates from patient D had an
NSI phenotype (5).
Generation of Primary Isolates and Biological Clones.
Primary isolates of HIV-1 were obtained by a quantitative co-culture
method as previously described (5, 30). In brief, cryopreserved
patient PBMCs were thawed and the cells serially diluted fourfold
in replicates of 4-10 (from 1 × 106 down to ~103). The cells
were added to wells containing 2 × 106 PHA-stimulated normal
donor PBMCs and cultured in a final volume of 1.5 ml of RPMI
medium supplemented with 10% FCS, penicillin (100 U/ml),
streptomycin (100 µg/ml), and interleukin-2 (10 U/ml). The
culture supernatants were tested on days 7, 14, and 21 for the
presence of HIV-1 p24 antigen by a commercially available ELISA
assay (Abbott Labs., North Chicago, IL). A culture was considered positive if the p24 antigen level was >30 pg/ml. Virus
present in the first well of each dilution series was pooled from
the replicates and propagated by a single short-term (7-14 d) passage in PHA-activated normal donor PBMCs, while the biological clones from patient B were generated by similar propagation
of virus present in the last positive well of each series. The resulting stocks were titered on normal donor PBMCs and the 50% tissue culture infectious dose (TCID50) was determined by the
method of Reed and Muench (31). The syncytium-inducing
properties of each isolate were assessed as previously described (5,
30) using the MT-2 cell assay of Koot et al. (29).
Cell Lines.
Cells lines stably expressing CCR1, CCR2b,
CCR3, CCR4, CCR5, or CXCR-4 were generated as previously described (32). In brief, retroviral stocks were prepared using
the expression vector pBABE-puro and the appropriate coreceptor. Human osteosarcoma cells expressing human CD4 (HOS.CD4)
were infected with the pBABE-puro viruses, and 2 d later the
cells were selected in 1.0 µg/ml puromycin. When the cells became confluent (after 5-7 days), they were maintained in DMEM
containing puromycin, 10% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml).
Replication of Sequential HIV-1 Isolates.
To assess the coreceptor requirements of primary HIV-1 isolates and biologically
cloned isolates, HOS.CD4 (104/well) expressing either CCR1,
CCR2b, CCR3, CCR4, CCR5, or CXCR-4 were inoculated
with 103 TCID50 of each isolate in a final volume of 1.0 ml of
DMEM with added FCS, antibiotics, and 1.0 µg/ml puromycin.
The cells were incubated with virus for 24 h at 37°C, washed
three times, and samples of the culture supernatants were taken
on days 0, 4, 7, 10, and 14 for HIV-1 p24 antigen measurements.
Chemokine Blocking.
Biologically cloned isolates of HIV-1
were used to infect PHA-stimulated, normal PBMCs in the presence of increasing concentrations of recombinant human RANTES,
MIP-1 Single Cycle Infectivity Assays.
HIV-1 env expression vectors
were generated by direct DNA PCR amplification and cloning
using sequential PBMC samples from patient B as previously described (33). Env clones expressing full-length gp160/120 were
co-transfected with an env ( In initial experiments, sequential HIV-1
isolates from patients B, C, and D were used to infect
CD4+ cells expressing either CCR5, CCR3, CCR2b, or
CXCR-4. HIV-1 isolates obtained early in the course of
infection from all three patients replicated to low levels in
CD4+ cells expressing CCR5, but failed to replicate to detectable levels in cells expressing either CCR2b, CCR3, or
CXCR-4 (Table 1). A similar pattern of infection was seen
with all subsequent isolates from patient D. However, sequential isolates from patients B and C showed a shift in receptor use which coincided with a switch from NSI to SI
phenotype. While viruses isolated at early time points from
these patients initially used only CCR5 for infection, viruses isolated later in the course of disease infected cells expressing either CCR5 or CXCR-4. In addition, several
later isolates from patient C replicated in cells expressing
CCR2b and CCR3 (Table 1). These results suggest that
HIV-1 variants arise during the course of infection that use
an expanded range of coreceptors for infection of CD4+
cells. In particular, adaptation to use CXCR-4 as a coreceptor was associated with a switch from NSI to SI phenotype and a drop in CD4+ T cell counts.
Table 1.
Replication of Sequential Primary Isolates
-chemokines, including RANTES, MIP-1
, and -1
(28). Two
additional
-chemokine receptors, CCR3 and CCR2b,
have also been shown to function as coreceptors for some,
but not all, primary strains of HIV-1 (19, 22). Taken together, these findings suggest that HIV-1 may use a broad range of coreceptors for infection of CD4+ cells.
and -1
(R&D Sys., Inc., Minneapolis, MN). PBMCs
(2 × 106) were infected with 200 TCID50 of each virus in 1.0 ml
of medium. Chemokines were serially diluted twofold and added
at the time of infection, such that the final concentration of each
chemokine was 500 ng/ml in the highest well. Control cultures
were set up in quadruplicate and infected in the absence of added
chemokines. After overnight incubation at 37°C, the cells were
washed and resuspended in medium containing the appropriate
concentration of chemokines. HIV-1 p24 antigen was measured
in the culture supernatants on day 7 after infection and the
amount of chemokine blocking was calculated as the percent inhibition of HIV-1 p24 antigen production compared to controls.
) luciferase reporter vector, pNL43LucE
, to generate env-pseudotyped virions (34). Reporter viruses pseudotyped with either macrophage-tropic, NSI (HIV-1JRFL)
or TCLA SI (HIV-1HXB2) envelope glycoproteins were used as
controls. HOS.CD4 cells stably transfected with either CCR1,
CCR2b, CCR3, CCR4, CCR5, CXCR-4, or the retroviral expression vector (pBABE) were infected with each of the pseudotyped viruses (100 ng p24). Cells were lysed 4 d after infection
using 100 µl of luciferase lysis buffer (Promega Corp., Madison,
WI). The amount of luciferase activity in 20 µl of lysate was measured using commercially available reagents (Promega Corp.) in a
luminometer (Top Count; Packard Instrs., Meriden, CT).
Use of CCR5, CCR3, CCR2b, and CXCR-4 by Sequential HIV-1 Isolates.
Patient
Date
CD4 cells/mm3
Phenotype
p24 antigen*
CCR2b
CCR3
CCR5
CXCR-4
pg/ml
B
5/85
528
NSI
<30
<30
200
<30
11/86
677
NSI
<30
<30
26,200
<30
6/88
603
SI
<30
<30
29,500
400
11/88
117
SI
<30
<30
33,320
7,820
C
5/84
1,233
NSI
<30
<30
1,024
<30
1/85
1,038
NSI
<30
<30
72,400
<30
2/86
1,049
SI
<30
<30
84,800
1,666
7/86
485
SI
<30
<30
60,200
65,600
12/86
346
SI
16,240
12,700
31,000
43,100
5/87
141
SI
1,200
1,586
28,800
54,800
6/88
186
SI
<30
<30
30,200
19,460
11/89
38
SI
6,600
11,240
31,200
24,000
D
3/86
725
NSI
<30
<30
140
<30
6/87
690
NSI
<30
<30
181
<30
9/90
700
NSI
<30
<30
157
<30
3/91
750
NSI
<30
<30
155
<30
*
HIV-1 p24 antigen was measured in culture supernatants on days 0, 4, 7, 10, and 14 after infection. Values represent the peak p24 levels measured
on either day 10 or 14. A value of <30 pg/ml was considered negative.
The SI phenotype for each isolate was determined using MT-2 cells as previously described (5, 29).
Because primary HIV-1 isolates may consist of a mixture
of distinct variants with different phenotypes (3, 4), we
tested a panel of biologically cloned primary HIV-1 isolates
for their ability to infect CD4+ cells expressing CXCR-4
or the -chemokine receptors, CCR1, CCR2b, CCR3,
CCR4, and CCR5. These isolates were generated by limiting dilution and co-culture of PBMCs from patient B;
five had an NSI phenotype (2, 3-1, 3, 4-1, 5-2), and
two were SI (5, 5). As shown in Table 2, all five of the
NSI viruses replicated in cells expressing CCR5. They did
not replicate to detectable levels in cells expressing the
other coreceptors, but replicated efficiently in control cultures of PHA-stimulated normal donor PBMCs. Similarly,
the two SI viruses replicated well in control PBMC cultures. However, their coreceptor use was restricted to cells
expressing CXCR-4; no replication was detected in cells expressing CCR1, CCR2b, CCR3, CCR4, or CCR5. It
is not clear why some of the NSI biological clones replicated to lower levels in cells expressing CCR5 compared
with others (Table 2; clones 2-5 and 5-2); however, these
isolates may be characteristic of viruses with a "slow/low"
phenotype (3), or alternatively, may be restricted at a postentry step in HOS.CD4 cells expressing CCR5.
|
To determine whether coreceptor use was associated
with sensitivity to chemokine blocking, we tested the biologically-cloned viruses for blocking by either RANTES,
MIP-1, -1
, or a combination of all three chemokines.
As shown in Fig. 1, the NSI viruses (2, 3-1, 3, 5-2)
were inhibited in a dose-dependent manner by addition of
RANTES, MIP-1
, and -1
, either alone or in combination with an IC90 of 75-119 ng/ml. In contrast, the two SI
isolates (5, 5) were only partially blocked, even at the
highest chemokine concentration (500 ng/ml). These results suggest that there is a loss of sensitivity to the blocking
effects of
-chemokines that is associated with acquisition
of the ability to use CXCR-4 as a coreceptor for infection.
An NSI isolate from late stage infection (5-2) remained sensitive to chemokine blocking consistent with its use of the
CCR5 coreceptor (Table 2).
Taken together, our results suggest that HIV-1 predominantly uses a single coreceptor, CCR5, in the early stages of infection. Variants using CXCR-4 appeared later and were associated with a loss of sensitivity to chemokine blocking in vitro. The presence later in infection of an isolate using CCR5 as a coreceptor (5-2), is consistent with the persistence of macrophage-tropic, NSI variants throughout the course of infection (4).
Env-mediated Interaction.Fusion of HIV-1 with CD4+ T cells is mediated by interaction of the viral envelope glycoproteins with coreceptors expressed on the cell surface (15). Considerable variation in the env gene of HIV-1 occurs throughout the course of infection and is associated with changes in the tropism and SI properties of the virus. It follows that these changes may reflect the efficiency with which different viral envelope glycoproteins interact with specific coreceptors.
To test this hypothesis, we generated full-length env
clones by direct DNA PCR amplification from sequential
PBMC samples from patient B (33). Env clones 9-12 and
13-14 were derived from early NSI viruses (Table 1; 5/85
and 11/86, respectively), whereas env clone 15-1 was derived from an SI isolate (Table 1; 6/88). HIV-1 virions
containing a luciferase reporter gene were pseudotyped
with the cloned envelope glycoproteins and used in single
cycle infectivity assays to evaluate viral entry into CD4+ cells
expressing each of the -chemokine receptors or CXCR-4. As shown in Fig. 2, luciferase activity was detected in cells expressing CCR5, CCR3, and CCR2b after infection with
control viruses pseudotyped with envelope glycoproteins
from a macrophage-tropic, NSI strain of HIV-1 (HIV-1JRFL)
(Fig. 2 A). In contrast, a TCLA, SI virus (HIV-1HXB2) was
only able to infect cells expressing CXCR-4. Virions pseudotyped with envelope glycoproteins from two of the primary env clones from patient B (9, 13) infected cells
expressing CCR5, and to a lesser extent, CCR2b and
CCR3, while a third clone (15-1) only infected cells expressing CXCR-4. Another primary HIV-1 env clone derived from a long-term survivor (DH) used CCR5 and
CCR3 for infection. Sequencing of the V3 loop of env
clones 9-12, 13-14, and DH revealed the presence of uncharged amino acids at positions 11 and 28 (Fig. 2 B), characteristic of NSI viruses, while clone 15-1 had a positively
charged residue at position 11, characteristic of SI viruses
(35, 36).
In this study, we examined coreceptor use by sequential primary isolates of HIV-1 obtained throughout the course of infection. Our results indicate that disease progression is associated with the appearance of viral variants able to use an expanded range of coreceptors for infection of CD4+ cells. HIV-1 isolates initially used a single coreceptor, CCR5, for infection of CD4+ cells in vitro. Later in the course of disease, the virus expanded its coreceptor use to include CCR5, CCR3, CCR2b, and CXCR-4. In particular, the appearance of viruses using CXCR-4 as a coreceptor was associated with a decline in CD4+ T cell counts and clinical progression to AIDS. In contrast, only viruses capable of using CCR5 could be isolated from a third patient, who remained asymptomatic for over 10 yr after HIV-1 infection with a low viral burden and normal CD4+ T cell counts (5). These results indicate that the expanded coreceptor use is not simply the result of increasing env diversity over time, but instead suggest that there may be a selective advantage for viruses that can use coreceptors in addition to CCR5.
Recent studies have identified CCR5 as an essential determinant of sexual transmission (32). CD4+ T-cells from two multiply exposed/uninfected individuals were shown to resist infection with macrophage-tropic, NSI strains of HIV-1 (32, 37, 38) due to a 32-base pair deletion in the gene encoding CCR5 (32). Our results indicate that viruses using CCR5 persist during asymptomatic infection, and in one patient, were present as long as 7 yr after seroconversion. These viruses are typical of many primary NSI isolates in that they exhibit a dual tropism for both CD4+ T lymphocytes and macrophages. Both cells types are known to express CCR5 and may be targets for these viruses in vivo. The persistence of viruses using CCR5 suggests they may escape immune surveillance mechanisms, possibly by sequestration in long-lived cell populations such as macrophages or dendritic cells.
Selective pressure exerted by either specific or nonspecific immune mechanisms may lead to the evolution of HIV-1
variants with distinct coreceptor requirements. Because
CCR5 binds several -chemokines, including RANTES,
MIP-1
, and -1
, selection may favor variants that can replicate in the presence of high concentrations of these factors.
We identified several variants of HIV-1 that were resistant
to the blocking effects of exogenously added
-chemokines. This resistance was associated with loss of the ability to use CCR5 for infection. This suggests that HIV-1 may evolve
in vivo to use alternative coreceptors, such as CXCR-4, in
response to selection pressure exerted by the production of
-chemokines. Given the high rate of HIV-1 turnover in
vivo (39, 40) and the small number of amino acid changes
necessary to alter the virus phenotype (35, 36), it is not yet
clear why these viruses do not emerge earlier. However, they
may be more sensitive to neutralization by HIV-1-specific
antibodies due to changes in the envelope glycoproteins, or
alternatively, they may be blocked by the presence of stromal cell-derived factor 1, which was recently identified as
the ligand for CXCR-4 (26, 27).
Relatively subtle changes in the viral genome can influence both phenotype and coreceptor use. As few as two amino acid changes in the V3 loop of env have been shown to affect the ability of primary isolates to induce syncytium formation in vitro (35, 36). Moreover, substitutions at two amino acid positions in V3 were sufficient to overcome the resistant phenotype of CD4+ T cells from two exposed/ uninfected individuals to infection with SI strains of HIV-1 (38). This is presumably due to a change in coreceptor use from CCR5, which is defective in these individuals (32), to CXCR-4 or another functional coreceptor. Indeed, recent reports have shown that use of both CCR3 and CCR5 is dependent on the sequence of the V3 loop (19).
By cloning sequential env genes from an HIV-1 infected individual, we have shown that coreceptor use is controlled by the env gene of the virus, and have identified primary HIV-1 variants with distinct coreceptor requirements. Previously, we found amino acid substitutions in the V3 loop of these variants that were characteristic of an NSI to SI switch in phenotype (37), and have now shown that the corresponding env clones display a switch in coreceptor use from CCR5 to CXCR-4. Although the exact mechanism for this switch is unclear, it is possible that mutations in V3 may directly affect interaction of the virus with specific sites on the coreceptor molecule or may indirectly influence receptor binding by altering the global conformation of gp120.
The appearance of HIV-1 variants able to use CXCR-4 as a coreceptor was associated with an increase in viral load and a marked drop in CD4+ T cell counts in the patients we studied. It is not clear if these viruses are directly cytopathic for CD4+ T cells in vivo. The most straightforward explanation is that the SI phenotype reflects adaptation of HIV-1 to use CXCR-4 expressed on CD4+ target cells in vivo. Because the tissue distribution of CXCR-4 is much broader than CCR5, this may allow the virus access to a wider range of potential target cells, or alternatively, may permit fusion with more permissive target cells, thereby facilitating HIV-1 replication and spread. Further studies will be necessary to discriminate between these possibilities and delineate the pathogenic mechanisms associated with CD4+ T cell depletion in vivo and the role of HIV-1 coreceptor use in this process.
Address correspondence to Ruth I. Connor, Aaron Diamond AIDS Research Center and The Rockefeller University, 455 First Ave., 7th Floor, New York, NY 10016.
Received for publication 7 October 1996
1Abbreviations used in this paper: HOS.CD4, human osteosarcoma cells expressing human CD4; NSI, nonsyncytium-inducing; SI, syncytium-inducing; TCID50, 50% tissue culture infectious dose; TCLA, T cell line-adapted.We would like to thank Cladd Stevens and Pablo Rubinstein for providing PBMC samples and clinical information from patients B, C, and D; E. Fenamore for technical assistance, and Simon Monard for FACS® analysis.
This work was supported by grants and contracts (CA72149, AI36057, and AI45218) from the National Institutes of Health, from the American Foundation for AIDS Research in memory of Florence Brecher, and from the Aaron Diamond Foundation.
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