Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype

Abstract

The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.

Full text

JOURNAL OF VIROLOGY,
0022-538X/97/$04.0010Oct. 1997, p. 7478–7487 Vol. 71, No. 10
Copyright © 1997, American Society for Microbiology
Coreceptor Usage of Primary Human Immunodeficiency Virus
Type 1 Isolates Varies According to Biological Phenotype
ÅSA BJO
¨RNDAL,
1
* HONGKUI DENG,
2
MARIANNE JANSSON,
1
JOSE
´R. FIORE,
1,3
CLAUDIA COLOGNESI,
4
ANDERS KARLSSON,
5
JAN ALBERT,
6
GABRIELLA SCARLATTI,
1,4
DAN R. LITTMAN,
2
AND EVA MARIA FENYO
¨
1
Microbiology and Tumorbiology Center, Karolinska Institute,
1
Swedish Institute for Infectious Disease Control,
6
and Venha¨lsan,
South Hospital,
5
Stockholm, Sweden; Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine,
New York University Medical Center, New York, New York
2
; and Unit of Immunobiology of HIV, DIBIT,
San Raffaele Scientific Institute, Milan,
4
and Clinic of Infectious Diseases,
University of Bari, Bari,
3
Italy
Received 13 February 1997/Accepted 30 June 1997
The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according
to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high,
syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability
to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C
chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the
principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4
glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have
tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from
primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype
of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary
HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low
viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine
receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used
CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to
inhibition by b-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host
range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient
patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with
multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using
CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original
population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry
the particular coreceptor required by the virus.
Primary human immunodeficiency virus (HIV) isolates can
be subdivided into two distinct groups according to their bio-
logical phenotype. Fast replication to high titers and syncytium
formation in peripheral blood mononuclear cells (PBMC) and
the capacity to infect and replicate in a broad range of T-
lymphoid and monocytoid cell lines often characterize viruses
isolated from immunodeficient patients (17, 37, 44). In con-
trast, slow replication to low titers and induction of small, if
any, syncytia in PBMC are characteristic of viruses isolated
from individuals with no or mild symptoms of HIV infection.
These viruses lack the capacity to infect and replicate in estab-
lished cell lines and thus do not induce syncytia in such cells.
The two distinct groups of primary HIV isolates were classified
as rapid/high or syncytium-inducing (SI) and slow/low or non-
syncytium-inducing (NSI) isolates, respectively (17, 37, 45).
HIV-1 has been genetically classified into a major (M) group
and a more distant outlier (O) group, and the M group has
been further subdivided into nine subtypes on the basis of
sequence diversity (29). Members of the same subtype differ by
less than 10%, and those of different subtypes differ by 15% or
more (25). Similarly to the phenotypes first described for
HIV-1 of genetic subtype B, distinct biological phenotypes of
subtypes A, D, E, F, and G have recently been identified (19,
21, 26, 43). All the available data suggest that the HIV-1
biological phenotype varies with the severity of HIV-1 infec-
tion across genetic subtypes (16).
Studies with HIV-1 of genetic subtype B have shown that
progression of the infection from the asymptomatic phase to
immunodeficiency is accompanied by a gradual increase in the
replicative capacity of the viruses isolated, culminating in ac-
quisition of the ability to induce syncytia in PBMC and repli-
cation in cell lines (5, 23, 38, 39, 45). The in vitro biological
phenotype of HIV-1 has therefore been recognized as a pre-
dictive marker for progression (15, 18, 24, 35). In agreement,
the early presence of rapid/high virus during seroconversion in
adults or shortly after birth in infants leads to a higher rate of
decline in CD4
1
T lymphocytes and early development of
AIDS in both groups (33).
Several chemokine receptors have recently been shown to be
necessary, along with CD4, for fusion of HIV-1 envelopes to
the plasma membrane of target cells (reviewed in reference
12). In response to chemokines, these molecules, which belong
to the family of seven transmembrane G-protein-coupled re-
ceptors, transduce signals that result in chemotaxis and, poten-
tially, developmental processes (reviewed in reference 31).
* Corresponding author. Mailing address: Microbiology and Tumor-
biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm,
Sweden. Phone: 46-8-728 6311. Fax: 46-8-33 13 99. E-mail: Asa.Bjorn
dal@mtc.ki.se.
7478

Since chemokines fall into two distinct groups based on the
properties of their primary sequence, the corresponding recep-
tors are named accordingly C-C or C-X-C receptors. Signifi-
cantly, envelope glycoproteins from T-cell-line-adapted (T-
tropic) viruses were found to utilize CXCR4 whereas envelope
glycoproteins from primary non-T-cell-line adapted (also
called macrophage-tropic or M-tropic) viruses utilized CCR5
and, in few cases, CCR3 (1–3, 6, 9–11, 14, 30). In line with this
coreceptor usage, the b-chemokines, of the C-C type, RAN-
TES, MIP-1a, and MIP-1b, have been shown to inhibit the
infectivity of HIV-1 isolates unable to replicate in established
cell lines (7, 22). These results have suggested that viruses with
different biological phenotypes differ in the choice of chemo-
kine receptor(s) (2, 6, 9, 11, 14). In the present study, we tested
the receptor specificity of a set of cloned envelope glycopro-
teins from primary HIV-1 isolates. To further analyze the
potential correlation of biological phenotype and stage of in-
fection with coreceptor usage, a panel of 36 HIV-1 primary
isolates from 30 individuals was tested for chemokine receptor
specificity by infecting derivatives of the U87 glioma cell line
(42) stably expressing CD4 and one of the chemokine recep-
tors CCR1, CCR2b, CCR3, CCR5, or CXCR4. The results
show that the previously established phenotypic differences
between HIV-1 isolates correspond to distinct coreceptor us-
age.
MATERIALS AND METHODS
Patients and virus isolates. Virus isolates 92RW020, 92UG037, 92BR020,
92TH014, 92BR025, 92UG021, and 92UG024 were obtained through the WHO
Network for HIV Isolation and Characterization (32, 43). Cloning and sequenc-
ing of envelopes have been described previously (19). Virus isolates A130, A136,
A145, and A196 from transmitting mothers and A245 from a nontransmitting
mother were characterized within the framework of studies on mother-to-child
transmission (34). Isolates V2, V8, and V9 were part of a study on sexual
transmission and were obtained during seroconversion (18). All other isolates
were from patients with progressive HIV-1 infection (17, 23, 39, 45).
Prior to these experiments, the primary HIV-1 isolates were passaged three to
five times in peripheral blood mononuclear cells (PBMC). Virus stocks were
prepared by infecting 5 310
6
phytohemagglutinin P (PHA-P; Pharmacia, Upp-
sala, Sweden)-stimulated PBMC from two blood donors with cell culture super-
natant containing 4 ng of HIV-1 p24 antigen per ml determined by an in-house
HIV-1 p24 antigen enzyme-linked immunosorbent assay (36). PBMC cultures
were maintained in RPMI 1640 medium (Gibco/BRL, Paisley, United Kingdom)
with 10% fetal calf serum (Flow Laboratories, Costa Mesa, Calif.),5Uof
recombinant interleukin-2 (IL-2; Amersham, Little Chalfont, United Kingdom)
per ml, 2 mg of Polybrene (Sigma, St. Louis, Mo.) per ml, and antibiotics (IL-2
medium). On day 7, the cultures were screened for HIV-1 p24 antigen. Subse-
quently, culture supernatants were harvested, clarified by centrifugation, and
filtered (pore size, 0.45 mm), and aliquots were stored at 280°C until used.
Cell lines. U87.CD4 cell lines stably expressing CCR1, CCR2b, CCR3, CCR5,
or CXCR4 were established as described previously (9). Briefly, cDNAs encod-
ing the chemokine receptors were subcloned into pBABE-puro. Amphotropic
virus stocks were prepared by transfecting BING packaging cells with the result-
ing plasmids. Supernatants were collected 48 h later and used to infect U87.CD4
cells. After another 48 h, cells were selected in medium containing 1 mgof
puromycin per ml. The C-C-type chemokine receptors were expressed on the
surface of the U87.CD4 cells, as assessed by mobilization of intracellular free
Ca
21
in response to the appropriate chemokines. Expression of CXCR4 was
monitored by flow cytometry with a monoclonal antibody (13).
Cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco/
BRL) with high glucose and with the addition of 15% fetal calf serum, 1 mM
sodium pyruvate (Gibco/BRL), nonessential amino acids (Gibco/BRL), and an-
tibiotics. The cultures were grown in 25-cm
2
tissue culture flasks (Costar) and
split at a ratio of 1:3 twice a week by treatment with 5 mM EDTA (pH 8.0). For
coculture experiments, the cells were seeded in 12-well plates (Costar) at a
concentration of 10 310
5
cells per well in 4 ml of medium. For cell-free infection
with primary isolates, the cells were seeded in 24-well plates (Costar) at 5 310
5
cells per well in 2 ml of medium. The plates were further incubated at 37°C under
5% CO
2
until cultures reached half confluence (after 1 to 3 days); they were then
used for the different assays.
Infectivity assay with HIV-luciferase reporter virus. The infectivity assay with
HIV-luciferase reporter virus has been described previously (8). In brief,
U87.CD4 cells were seeded in 24-well tissue culture dishes (0.5 310
5
cells per
well) and were infected on the following day with luciferase reporter viruses (50
ng of p24 antigen). Reporter viruses pseudotyped by a panel of genetically
diverse envelopes were prepared by transfecting 293T cells with NL-luc-Env(2)
and the appropriate Env expression vector (10 mg each), quantitated as previ-
ously described and stored in aliquots at 280°C. Lysates (120 ml) were prepared
2 days postinfection, and the luciferase activity in 20-ml samples was measured
with commercial luciferase assay reagents (Promega) and a Wallac scintillation
counter.
Cocultivation of infected PBMC with the U87.CD4 cell lines. PBMC cultures
were infected with virus as described above. At 7 days postinfection, 5 310
5
to
8310
5
infected PBMC/well were added to 12-well plates with the U87.CD4 cell
lines. Noninfected PHA-P-stimulated PBMC and medium only were included as
controls. At 48 h after initiation of cocultures, the plates were washed by being
rinsed twice with phosphate-buffered saline (PBS), whereafter fresh medium was
added. On days 2, 4 and 7, a sample of culture supernatant was withdrawn and
stored at 220°C until tested for p24 antigen. Samples removed on day 2, before
the washing procedure, contained input virus and were used as controls for the
washing procedure. Samples from different time points within one experiment
were tested for p24 antigen in the same enzyme-linked immunosorbent assay.
Between days 3 and 5, wells with a confluent cell layer were treated with 5 mM
EDTA and split 1:3, and fresh medium was added. Cell cultures were monitored
under the microscope daily for 7 days for the presence of cytopathic effects.
Cell-free virus infection of U87.CD4 cell lines. Virus stocks were subjected to
titer determination on PHA-stimulated PBMC as previously described (40).
U87.CD4 cell lines were infected with 100 to 1,000 50% tissue culture infective
doses (TCID
50
) (corresponding to 2.5 to 30 ng of p24 antigen). Before infection,
the cells were rinsed once with PBS and virus was added in a 1-ml final volume.
At 24 h later, 1 ml of fresh medium was added. The cultures were rinsed twice
with PBS on day 2 and observed daily for cytopathic effects, and supernatants
were harvested on days 2 (before washing), 4, 6, 8, and 10 and tested for p24
antigen.
Serial passage of HIV-1 primary isolates in CCR3-, CCR5-, or CXCR4-ex-
pressing cells. Two primary isolates of the rapid/high phenotype with a broad
coreceptor usage (isolates 29 and 31) were selected for this experiments. Virus
stocks whose titers had been determined were used at 1,000 TCID
50
for cell-free
infection of glioma cells in 24-well tissue culture plates as described above. When
clear syncytium formation was observed (for CCR5- and CXCR4-expressing cells
on day 4 or 5 and for CCR3-expressing cells on day 7 or later), the cell culture
supernatants were harvested and centrifuged and 0.5-ml samples were used for
further passage. Four passages were carried out on U87.CD4-CCR5 and
U87.CD4-CXCR4 cells, and three passages were carried out on U87.CD4-CCR3
cells. Subsequently, passaged virus isolates were retested on all U87.CD4 cell
lines in parallel with the original virus stocks.
Chemokine inhibition assay. A chemokine inhibition assay with sequential
isolates with a phenotype switch was performed with the b-chemokines RAN-
TES, MIP-1a, and MIP-1bas described previously (22). For the six virus isolates
obtained from different genetic subtypes, chemokine inhibition was performed as
follows. In brief, PHA-P-stimulated PBMC (10
5
cells/well) from one blood donor
were infected with each isolate at three fivefold dilutions in a round-bottom
microtiter plate (Nunc, Roskilde, Denmark). The infection was performed over-
night at 37°C in the presence or absence of RANTES or MCP-1 (R & D Systems,
Minneapolis, Minn.) at 250- and 62-ng/ml concentrations in duplicates. Every
48 h, the cells were washed by centrifugation and chemokine was added in fresh
IL-2 medium. A TCID
50
assay of serial fivefold virus dilutions was performed in
parallel. Inhibition by b-chemokines was evaluated by measuring p24 antigen
production 7 to 12 days postinfection.
RESULTS
Envelopes of primary HIV-1 isolates cause segregation of
chemokine receptor usage according to the biological pheno-
type, and not the genetic subtype of the virus. A representative
panel of genetically diverse env genes was recently prepared
from 35 primary HIV-1 isolates collected at major epicenters
of the current AIDS pandemic (19). These genes were assessed
for biological activity in the context of HIV-1 virions, and 15 of
them were shown to encode fully functional envelope glyco-
proteins. We used these 15 env genes to generate HIV-lucif-
erase reporter virus and tested for chemokine receptor usage
on U87 cells stably expressing human CD4 and CCR1, CCR2b,
CCR3, CCR5, or CXCR4 (9). The results, shown in Table 1,
indicate that CCR5 and CXCR4 are the main coreceptors for
HIV-1 subtypes A to E and G. The receptor usage of these
isolates was closely associated with their biological phenotype
but not their genotype: NSI isolates used CCR5, whereas SI
isolates used CXCR4. One isolate (92HT593), previously clas-
sified as NSI, was able to use both CCR5 and CXCR4 as
coreceptor. In addition, we tested the chemokine receptor
usage and sensitivity to the b-chemokine RANTES of selected
VOL. 71, 1997 CORECEPTOR USAGE OF HIV-1 ISOLATES 7479

primary isolates (Table 2). With one exception, coreceptor
usage of primary isolates correlated with that of the corre-
sponding envelopes. In the exceptional case, the virus isolate
(92UG024) used CCR3 in addition to CXCR4, used by its
cloned envelope. Receptor usage of the primary isolates
correlated with sensitivity to RANTES, in that 92UG037,
92TH014, and 92BR020, using the CCR5 receptor, were in-
hibited whereas 92UG024 and 92UG021, using CXCR4, were
resistant. None of the isolates were inhibited by MCP-1 (data
not shown).
Chemokine receptor usage of primary HIV-1 isolates from
patients with HIV-1 infections of different severities. (i) Cocul-
tivation of infected PBMC with the U87.CD4 cell lines. The
next question was whether chemokine receptor usage of pri-
mary HIV-1 isolates would correlate with the severity of HIV-1
infection. For this purpose, a panel of 20 HIV-1 primary iso-
lates from 19 individuals was tested for chemokine receptor
usage. The 20 isolates used in these experiments showed dif-
ferences in their biological phenotype according to the clinical
condition of the patient at the time of virus isolation (Table 3).
Patients from whom rapid/high (SI) virus was isolated had
invariably low (#230 310
6
cells/liter) CD4
1
T-lymphocyte
counts, whereas patients with slow/low (NSI) virus had CD4
1
counts in a higher range ($280 310
6
cells/liter). In the first
series of experiments, we found that productively infected
PBMC were able to infect and induce syncytia in U87.CD4
cells expressing particular chemokine receptors. The pattern of
syncytium formation segregated according to the biological
phenotype of the virus isolate. Beginning 24 h after coculture,
syncytia were evident in CCR5-expressing cells infected with
TABLE 1. Coreceptor usage by envelopes of different genetic subtypes of HIV-1
Envelope
a
Genetic
subtype Biological
phenotype
b
Luciferase activity (10
3
cps) in U87.CD4 cell lines expressing chemokine receptor
c
:
CCR1 CCR2b CCR3 CCR5 CXCR4
92RW020.5 A NA 0.1 0.1 0.1 2,896.7 0.1
92UG037.8 A NSI 0.2 0.3 0.1 151.1 0.1
92US715.6 B NSI 0.1 0.1 0.3 375.9 0.2
92HT593.1 B NSI 0.1 0.1 0.3 120.8 922.6
92HT599.24 B SI 0.2 0.5 0.4 0.1 432.3
92BR020.4 B NSI 0.3 0.7 0.5 3,324.7 0.3
92TH014.12 B NSI 0.1 0.1 1.9 927.2 0.3
91US005.11 B NSI 0.6 0.1 0.1 1,787.7 0.4
92BR025.9 C NSI 0.4 0.1 0.1 6,848.1 0.1
93MW965.26 C NSI 0.1 0.1 0.4 128.8 0.1
92UG021.16 D SI 0.1 0.3 0.1 0.1 221.8
92UG024.2 D SI 0.1 0.1 0.7 0.1 38.4
93TH966.8 E NSI 0.4 0.2 0.1 1,495.1 0.1
93TH976.17 E NA 0.1 0.1 0.1 2,808.3 0.1
92UG975.10 G NSI 0.4 0.1 0.1 6,761.8 0.1
HXB2
d
B 1.7 0.3 1 0.2 952.9
ADA
e
B 4 3.5 1.1 521.7 0.4
VSV-g
f
691.4 540.2 432.2 165.9 170.5
a
HIV-1 envelopes were derived from cloned virus isolates as previously described (19).
b
The biological phenotype of all primary isolates listed was determined through syncytium induction and replication in MT-2 cells (23). NA, not assayed. In addition,
isolates obtained through the WHO Network for HIV Isolation and Characterization were tested on CEM and U937 clone 2 cells and were classified as slow/low (NSI)
or rapid/high (SI) (32).
c
U87.CD4 cells expressing chemokine receptors were incubated with HIV-luc pseudotyped by the above panel of envelopes, and luciferase activity was measured
as described previously (8). The values represent the mean value from duplicate experiments.
d
HIV-1 clone derived from the primary isolate HIV-1
IIIB
, previously described as prototype T-cell-line tropic.
e
HIV-1 primary isolate, prototype non-T-cell-line tropic (M tropic).
f
Envelope glycoprotein from vesicular stomatitis virus (VSV) was used as a control.
TABLE 2. Coreceptor usage and chemokine sensitivity of primary HIV-1 isolates of different genetic subtypes
a
Virus
isolate Genetic
subtype Biological
phenotype
b
% Inhibition by RANTES
in PBMC cultures
c
:Syncytium induction and p24 antigen production in U87.CD4 cell
lines expressing chemokine receptor
d
:
250 ng/ml 62 ng/ml CCR1 CCR2b CCR3 CCR5 CXCR4
92RW020 A NA NA NA 2 2 2 111 2
92UG037 A NSI 96 96 2 2 2 111 2
92BR020 B NSI 100 54 2 2 2 111 2
92TH014 B NSI 96 74 2 2 2 111 2
92UG021 D SI 0 0 2 2 2 2 111
92UG024 D SI 7 10 2211
e
2 111
a
From the WHO Network for HIV Isolation and Characterization. All patients were asymptomatic at the time of virus isolation.
b
See Table 1, footnote b.
c
Percent inhibition was evaluated at 10 to 52 TCID
50
; NA, not assayed.
d
Cells were infected with 1,000 TCID
50
. Syncytium formation was evaluated on day 7 postinfection: 2, no syncytia (,0.2 ng of p24 per ml); 11, large syncytia
detected in every field (1 to 2 ng/ml); 111, large syncytia cover the entire well (.2 ng/ml). None of the isolates replicated or induced syncytia in the U87.CD4 parental
cell line.
e
p24 antigen values increased on days 4 to 7 postinfection; syncytium formation appeared on day 9.
7480 BJO
¨RNDAL ET AL. J. VIROL.

slow/low viruses and in CXCR4-expressing cells infected with
rapid/high viruses. In addition, most of the rapid/high isolates
were able to infect several cell lines, inducing syncytia in cells
transfected with CCR3, CCR5, and, in one case, CCR2b (Ta-
ble 3; Fig. 1). The cocultures were tested for p24 antigen
production on days 2 (before washing), 4, and 7. Cultures with
syncytia had increased levels of extracellular p24 antigen,
whereas cultures negative for syncytia had decreased levels
(data not shown). The results show that distinct chemokine
receptor usage corresponds to the phenotype of primary
HIV-1 isolates. Moreover, all primary isolates are able to in-
duce syncytia in U87 cells, provided that the cells express the
chemokine receptor used by the virus.
(ii) Cell-free infection of the U87.CD4 cell lines. Next, we
analyzed the virus dose necessary to achieve productive infec-
tion and syncytium induction in the U87.CD4 cell lines. Six
virus isolates were selected for these experiments, and 100 to
1,000 TCID
50
was used for infection. All viruses infected cells
and induced syncytia according to the previously established
pattern, provided that the virus dose was high enough, i.e.,
1,000 TCID
50
(Table 4). Variable results were obtained at
lower doses. In most cases, a TCID
50
of 100 was not sufficient
to result in a productive infection and syncytium induction
within 7 to 10 days. None of the virus isolates induced syncytia,
even at the highest dose, in CCR1-expressing cells (Fig. 1A). In
general, syncytia tended to be larger in CXCR4-expressing
cells (Fig. 1E) than in the other cell lines, with the smallest
syncytia being present in CCR2b-expressing cells (Fig. 1B).
Sequential isolates obtained from patients with progressive
HIV-1 disease differ in coreceptor usage and sensitivity to
b-chemokines. From a particularly interesting group of five
patients with high rates of CD4
1
T-lymphocyte decline, pairs
of virus isolates that had been obtained sequentially and had
distinct phenotypes were tested for receptor usage. Viruses
obtained from the same individual but differing in biological
phenotype (in these cases distinguished by replication and syn-
cytium induction in the MT-2 cell line) also differed in chemo-
kine receptor usage. MT-2 negative (NSI) isolates used CCR5,
while MT-2-tropic (SI) isolates used CXCR4 instead of or in
addition to CCR5 (Table 5). Interestingly, viruses capable of
using both receptors usually used CCR3 as well. Thus, the
viruses appear to evolve over time to use several receptors. The
results also show that b-chemokine receptor usage corre-
sponds strictly to sensitivity to b-chemokines, expressed as the
50% inhibitory concentration in nanograms per milliliter. A
mixture of RANTES, MIP-1a, and MIP-1bcould block the
infectivity for PBMC of viruses using CCR5 but not those using
CXCR4 (Table 5) (22).
TABLE 3. Cocultivation of U87.CD4 cell lines and PBMC infected with primary HIV-1 isolates obtained from patients
with HIV-1 infection of different severities
Virus
isolate
Patient clinical data Virus isolate
Clinical
status
a
No. of CD4
1
cells
(10
6
/liter) Replication
in cell lines
b
Biological
phenotype
Syncytium induction in U87.CD4 cell lines expressing
chemokine receptor
c
:
CCR1 CCR2b CCR3 CCR5 CXCR4
196A AS 183 1Rapid/high 2 2 11 111 111
245A AS 187 1Rapid/high 2 2 2 111 111
31 AIDS 20 1Rapid/high 2 2 111
d
111
d
111
26 AIDS 230 1Rapid/high 2211
e
2 111
24 PGL 147 1Rapid/high 2 2 2 2 111
25 AIDS 100 1Rapid/high 21
d
111 111 111
29 AIDS 90 1Rapid/high 2 2 111 111 111
V4 AIDS 89 1Rapid/high 2 2 1 111 111
V7 AIDS 65 1Rapid/high 2 2 2 2 111
6A PGL 370 2Slow/low 2 2 6 111 2
6B PGL 470 2Slow/low 2 2 2 111 2
8 PGL 804 2Slow/low 2 2 2 111 2
12 PGL 330 2Slow/low 2 2 2 111 2
130A AIDS 280 2Slow/low 2 2 2 111 2
145A AS 600 2Slow/low 2 2 2 111 2
136A AS 290 2Slow/low 2 2 2 111 2
V2 Seroconv. 565 2Slow/low 2 2 2 111 2
V6 PGL 465 2Slow/low 2 2 2 111 2
V8 Seroconv. 684 2Slow/low 2 2 2 111 2
V9 Seroconv. 382 2Slow/low 2 2 2 111 2
IIIB
f
2 2 2 2 111
BaL
g
2 2 2 111 2
a
At the time of virus isolation: Seroconv., sample obtained during seroconversion; AS, asymptomatic; PGL, persistent generalized lymphadenopathy.
b
All viruses were tested for replication in MT-2 cells: 2, no replication; 1, virus replication (p24 antigen-positive culture supernatant) and syncytium induction. In
addition, virus isolates 130A, 136A, 145A, 196A, and 245A were tested on Jurkat and U937 clone 2 cells (34); V2, V4, V6, V7, V8, and V9 were tested on C8166 and
HuT-78 cells (reference 18 and unpublished results); and 6A, 6B, 8, 12, 24, 25, 26, 29, and 31 were tested on Jurkat, CEM, H9, and U937 clone 16 cells (39, 45). Virus
isolates 6A and 6B were collected from the same patient 6 months apart.
c
Syncytium induction: 2, no syncytia (,0.2 ng of p24 per ml); 6, rare small syncytia (0.2 to 0.5 ng/ml); 1, small syncytia apparent in every field (0.5 to 1 ng/ml);
11, large syncytia detected in every field (1 to 2 ng/ml); 111, large syncytia covering the entire well (.2 ng/ml). Cultures were observed daily, and results obtained
on day 2 are presented, except where noted. CCR3-positive isolates were tested two to five times.
d
Results obtained on day 7.
e
Results obtained on day 4.
f
Laboratory strain, prototype T-cell-line adapted.
g
Laboratory strain, prototype macrophage-tropic.
VOL. 71, 1997 CORECEPTOR USAGE OF HIV-1 ISOLATES 7481

FIG. 1. Syncytium formation in U87.CD4 cell lines after direct infection with primary HIV-1 isolates. (A to E) Virus isolates 25 (A and B), 31 (C), 29 (D), and V7
(E). (F to K) Noninfected cells. The chemokine receptors used were CCR1 (A and F), CCR2b (B and G), CCR3 (C and H), CCR5 (D and I), and CXCR4 (E and
K).
7482 BJO
¨RNDAL ET AL. J. VIROL.

Serial passage in cell lines expressing individual chemokine
receptors reveals heterogeneity in HIV-1 isolates. The ability
of some isolates to use several different chemokine receptors
was further explored. Isolates 29 and 31, which used multiple
receptors, were selected for these experiments. Each of the
virus isolates was passaged four times in U87.CD4-CCR5 or
U87.CD4-CXCR4 cells or three times in U87.CD4-CCR3 cells
and retested for receptor usage (Table 6). In both cases, pas-
sage in CCR5-expressing cells selected for virus that was able
to replicate in CCR5-expressing cells but not in CXCR4-
or CCR3-expressing cells. Interestingly, viruses selected for
growth in cells expressing CXCR4 or CCR3 had a phenotype
identical to that of the original isolate, maintaining their ability
to use all three receptors. The results suggest that the primary
isolates used in these experiments contained heterogeneous
viral populations with regard to receptor requirements, with
one using CCR5 only and the other using CCR3, CCR5, or
CXCR4.
Replication kinetics of primary HIV-1 isolates using differ-
ent coreceptors. The results of serial passage in cell lines ex-
pressing individual chemokine receptors also suggested that
viruses using CCR5 only as the coreceptor may replicate more
efficiently than multitropic viruses in the U87.CD4 cells and
hence may outgrow the more promiscuous viruses in these
cells. In fact, while four passages (4 to 5 days each) were
carried out in the CCR5- or CXCR4-expressing cell lines, only
three passages (at least 7 days each) could be carried out in the
CCR3-expressing cells during the same period. Even so, one of
the viruses passaged was lost (isolate 31), due to slow replica-
tion during the period allotted.
This prompted us to study the replication kinetics of three
virus isolates differing in coreceptor requirements (Fig. 2).
Virus levels increased rapidly in cells expressing CCR5 (Fig. 2)
or CXCR4, as indicated by increasing p24 antigen values in
TABLE 4. Cell-free infection of U87.CD4 cell lines with
selected primary HIV-1 isolates
Virus
isolate TCID
50a
Syncytium induction and p24 antigen production
in U87.CD4 cell lines expressing
b
:
CCR1 CCR2b CCR3 CCR5 CXCR4
31 500 NA 2 11 111 111
24 100 22221
500 2 2 2 2 111
1,000 2 2 2 2 111
25 100 22211
c
111
500 26
c
1
c
111 111
1,000 (1)
c
6
c
11
c
111 111
29 100 226
c
11
c
11
c
500 2 2 11 111 111
1,000 2 2 11 111 111
V7 100 222211
c
500 2 2 2 2 111
1,000 2 2 2 2 111
8 100 22222
500 2 2 2 111 2
1,000 2 2 2 111 2
a
Corresponds to 2 to 30 ng of p24 antigen per ml. Results obtained with 500
or 1,000 TCID
50
represent the mean values from two independent experiments.
b
2, no syncytia (,0.2 ng of p24 per ml); 6, rare small syncytia detected (0.2
to 0.5 ng/ml); 1, small syncytia apparent in every field detected (0.5 to 1 ng/ml);
11, large syncytia detected in every field (1 to 2 ng/ml); 111, large syncytia
covering the entire well (.2 ng/ml); (1), no syncytia detected (0.5 to 1 ng/ml);
NA, not assayed. Cultures were observed daily, and results obtained on day 3 are
presented, except where noted. Viruses were retested two to five times with 1,000
TCID
50
.
c
Results obtained between days 7 and 10.
TABLE 5. Cell-free infection of U87.CD4 cell lines with pairs of primary HIV-1 isolates sequentially obtained from patients
with progressive HIV-1 infection
Patient Virus
isolate Clinical
status
a
CD4
1
T lymphocytes Time
c
(mo) MT-2
tropism
d
Chemokine
sensitivity
(ng/ml)
e
Syncytium induction and p24 antigen production in
U87.CD4 cell lines expressing
f
:
Cell count
(10
6
/liter) Rate of
decline
b
CCR1 CCR2b CCR3 CCR5 CXCR4
A J2195 AS 600 210 2 2 2 111 2
J4052 AS 260 211, 1 20 1.200 2211
g
111 111
B J562 AS 250 2,72 2 2 111 2
J975 AIDS 110 25, 8 8 1.200 2211
g
111 111
C J669 AS 320 245 2 2 2 111 2
J1629 AIDS 210 28, 5 15 1.200 2 2 2 2 111
D J1874 AS 360 216 2 2 2 111 2
J2337 AS 410 25, 5 6 1.200 2 2 2 2 111
E J2090 ARC 50 242 2 2 2 111 2
J2822 ARC* 20 26, 1 6 1.200 (1)
g
(1)
g
111
g
111 111
a
At the time of virus isolation: AS, asymptomatic; ARC, AIDS-related complex; ARC*, patient developed AIDS 4 months later.
b
The rate of CD4
1
T-lymphocyte decline (10
6
cells/liter/month) was calculated by linear regression from multiple determinations during the entire observation
period (40 to 100 months) (23). Patients B and C received zidovudine at the time of second isolation (virus isolates J975 and J1629, respectively); patient E received
zidovudine at both samplings.
c
Between the two samples indicated.
d
All viruses were tested for replication (p24 antigen production) and syncytium induction in MT-2 cells: 2, no syncytia/no p24; 1, syncytia/p24 detected.
e
Chemokine sensitivity as measured by the 50% inhibitory concentration of a mix of equal concentrations of chemokines MIP-1a, MIP-1b, and RANTES, starting
with 200 ng of each per ml as previously described (22).
f
For data on syncytium induction and p24 antigen production, see Table 4, footnote b. Cultures were observed daily, and results obtained on day 2 are presented,
except where noted. CCR3-positive isolates were retested twice. Except for J2822, none of the isolates replicated or induced syncytia in the U87.CD4 parental cell line.
g
Results obtained on day 4 or 5.
VOL. 71, 1997 CORECEPTOR USAGE OF HIV-1 ISOLATES 7483

cultures infected with viruses using these receptors (virus iso-
late J2822 from patient E and isolate J975 from patient B [Fig.
2A and B, respectively]). However, in CCR3-expressing cul-
tures, an increase in p24 antigen level could not be detected
before day 6. Replication of the J2822 virus in CCR2b-express-
ing cells showed a similar pattern. These observations may be
due to relatively low-level expression of CCR2b and CCR3 or
to their relatively poor function as viral receptors. Alterna-
tively, this result may reflect the relative levels of viruses with
different chemokine receptor specificities in mixed isolates.
Isolates J2822 (Fig. 2A) and 25 (data not shown) were par-
ticularly interesting because they were also able to infect the
parental U87.CD4 cell line. The viruses replicated in these
cells, as shown by extracellular p24 antigen production (illus-
trated in Fig. 2A by isolate J2822), but did not induce syncytia
in these cultures or in CCR1-expressing cells. However, both
isolates induced large syncytia if the cells infected expressed
CCR3, CCR5, or CXCR4 in addition to CD4 (Tables 3 to 5).
The two isolates differed in their capacity to induce syncytia in
CCR2b-expressing cultures, in that isolate 25 induced syncytia
while J2822 did not. These results suggest that in some patients
HIV-1 may evolve to use coreceptors, other than those de-
scribed above, that are expressed in U87 cells. Alternatively,
there may be rare viruses that require only CD4 for infection,
like those described by Shimizu et al. (35a).
DISCUSSION
Our results indicate that primary HIV-1 isolates with distinct
biological phenotypes utilize chemokine receptors for entry
into target cells according to a characteristic pattern. The abil-
ity of rapid/high (SI) viruses to infect established T-lymphoid
and monocytoid cell lines is determined by their capacity to use
the chemokine receptor CXCR4 as a coreceptor to CD4. This
is in line with previous studies (9, 14) showing that the pattern
of coreceptor usage depends on whether the envelope glyco-
protein is derived from T-cell-line-adapted or nonadapted
HIV-1 isolates. Furthermore, we show that among primary
HIV-1 isolates, CCR5 is the most commonly used coreceptor.
In general, such slow/low (NSI) viruses, unable to replicate or
induce syncytia in cell lines, have a narrow host range and use
CCR5 exclusively. In the present study, one slow/low virus (of
19 primary isolates tested), isolated from a patient with pro-
gressive HIV-1 infection (39), could use CCR3 in addition to
CCR5, as shown by syncytium formation and p24 antigen pro-
duction upon infection of cells expressing these coreceptors. In
contrast, rapid/high viruses were often characterized by a
broad host range and used CCR2b, CCR3, or CCR5 in addi-
tion to CXCR4. In a parallel study, Connor et al. made similar
observations. A study of sequential isolates from three patients
showed that dual-tropic viruses were associated with low CD4
counts in two of them (8a). In our study, this broadening of the
host range may be even greater than currently suspected, since
two viruses from AIDS patients were able to infect the parental
U87.CD4 cells, which lack any transfected chemokine recep-
tor. Although some viruses may evolve to infect cells through
CD4 alone, it is more likely that the U87.CD4 cells express a
novel coreceptor. Studies on infection of U87.CD4 with HIV-
luc pseudotyped with simian immunodeficiency virus (SIV)
envelope glycoproteins indicate that at least one additional
coreceptor, utilized by SIV, is expressed in these cells (9a).
FIG. 2. Kinetics of virus replication in different U87.CD4 cell lines following
infection with three selected virus isolates: patient E, isolate J2822 (A); patient
B, isolate J975 (B); and patient C, isolate J669 (C). Culture supernatants were
tested for p24 antigen at the times indicated. Symbols: j, CCR1; }, CCR2b; E,
CCR3; X, CCR5; ■, CXCR4; h, CD4 only.
TABLE 6. Passage of primary HIV-1 isolates on
chemokine receptor-expressing cells
Virus
isolate Cells used for
passage
Syncytium induction and p24
antigen production in U87.CD4
cell lines expressing
a
:
CCR3 CCR5 CXCR4
29 U87.CD4-CCR3 111 111 111
U87.CD4-CCR5 2 111 2
U87.CD4-CXCR4 111 111 111
31 U87.CD4-CCR3 NA NA NA
U87.CD4-CCR5 2 111 2
U87.CD4-CXCR4 111 111 111
a
See Table 2, footnote d. None of the isolates replicated or induced syncytia
in the parental U87.CD4 cells or cells expressing CCR1 or CCR2b.
7484 BJO
¨RNDAL ET AL. J. VIROL.

Since many chemokine receptors are expressed in the central
nervous system, it would not be surprising to find that addi-
tional relevant receptors are expressed in this glial cell line.
Our results also show that envelopes of primary HIV-1 iso-
lates segregate chemokine receptor usage according to the
biological phenotype and not the genetic subtype of the virus.
In a recent publication, Zhang et al. came to the same conclu-
sion (44). We infected the U87.CD4 cells, in the cases where
the primary virus isolate was available, and found that core-
ceptor usage agreed with the pattern established by using HIV-
luc pseudotyped with the corresponding envelope glyco-
protein. This clearly shows the role of the viral envelope
glycoprotein in determining the biological phenotype of the
virus. In one exceptional case (of 15 tested), the biological
phenotype and chemokine receptor usage of the primary iso-
late and the corresponding envelope clone were discordant,
indicating that a random selection of one clone from the het-
erogeneous population of a virus isolate may not necessarily
yield viruses representative of the original virus isolate (28).
The pattern of coreceptor usage corresponded strictly to
sensitivity to b-chemokines. The emerging general pattern is
that viruses using the CCR5 coreceptor are sensitive to RAN-
TES (Table 2) or to a mixture of RANTES, MIP-1a, and
MIP-1b(Table 5) whereas viruses using CXCR4 are resistant.
MCP-1, a chemokine not using CCR5 as the receptor, did not
affect replication of the six isolates tested (data not shown),
whereas isolates using the CCR5 receptor were shown to be
sensitive to RANTES (Table 2). Changes in chemokine recep-
tor usage of isolates obtained from the same patient over time
paralleled changes in sensitivity to b-chemokines. Thus, pro-
gressive HIV-1 disease not only is associated with a change in
the virus host range, enabling the virus to infect a different or
a wider range of cells, but also seems to be linked to resistance
to b-chemokines as well (22).
All primary isolates tested were able to induce syncytia pro-
vided that the cells expressed the particular coreceptor used by
the virus. It is interesting that even if two virus isolates (isolates
25 and J2822) were able to infect and replicate in the parental
U87.CD4 cells, syncytium induction was present only in cells
coexpressing CXCR4, CCR5, CCR3, or, in one case, CCR2b.
It is tempting to speculate that the presence of these chemo-
kine receptors not only predisposes cells to infection with
HIV-1 but also renders them more vulnerable to the cytopathic
effect of the virus. However, it is important to recognize that
the cell system used in these experiments is a model and that
the U87 cell line and the in vivo target cells for HIV may show
important differences. One such difference is evident when the
replication kinetics of the different viruses are compared.
While viruses appeared to replicate with similar kinetics in
U87.CD4-CCR5 and U87.CD4-CXCR4 cells, one of the main
distinctive properties of primary HIV-1 isolates was their
speed of replication in primary PBMC cultures. This difference
led to the previous designation of slow/low and rapid/high (45).
Choe et al. (6) have pointed out the importance of the levels of
cell surface expression of chemokine receptors for HIV-1 co-
receptor function. Cells that stably express CD4 and the core-
ceptor, like the U87 series, may have relatively large amounts
of these receptors, whereas lower CCR5 levels may be present
in PBMC. This may explain why distinct phenotypes of primary
HIV-1 isolates could be established in PBMC. If PBMC indeed
have lower CCR5 concentrations than the U87.CD4-CCR5
cell line, replication by viruses using this receptor may be slow
and not accompanied by syncytium induction. In line with this
reasoning, PBMC and U87 cells would differ in the relative
level of expression, but no intrinsic difference between the
CCR5 and CXCR4 receptors would exist. In support of this
model are the recent findings of Bleul et al. (4), showing that
CCR5 expression is very low on unstimulated T cells (resting
cells) and only weakly up-regulated by PHA-P compared to
CXCR4 levels. In fact, CCR5 levels increase only after several
days of IL-2 stimulation. Alternatively, a fundamental differ-
ence may exist between these receptors, and this may account
for why CXCR4 usage is associated either with a more virulent
phase of disease or, if such virus is transmitted, with an early
and rapid decline in the number of CD4
1
cells. Conceivably,
the CCR5 and CXCR4 receptors may set off different signal-
ling pathways in the in vivo target cells, even if this is not
evident in U87 cells.
In our study, viruses using the CCR3 receptor were able to
use CCR5 or CXCR4 as well. No virus able to use CCR3 as the
only coreceptor for CD4 was observed. One virus (isolate 6A)
could use CCR3 or CCR5, similarly to the ADA and YU2
envelopes reported by Choe et al. (6). In our material, this
phenotype was encountered once in 36 isolates and might
therefore represent a rare phenotype among primary isolates.
It remains to be seen if this is a true phenotype or if it reflects
a mixture of viruses. Conceivably, faster replication of viruses
utilizing CCR5 relative to the CCR3 coreceptor may result in
selection for viruses able to use CCR5, competing out viruses
using CCR3. In model experiments involving serial passage of
two isolates in U87.CD4 cells expressing different coreceptors,
we could show that, indeed, viruses using CCR5 only were
selected from a population of viruses using CCR3, CCR5, or
CXCR4. This indicates that viruses using CCR5 only as the
coreceptor have a selective advantage over viruses that use
several coreceptors. Recently, Simmons and colleagues com-
pared coreceptor usage of biological clones, obtained through
limiting dilution, with the parental virus isolates (35b). All the
viruses were dual-tropic, indicating that the original isolates
contained homogeneous populations. Different virus isolates
are expected to contain different mixtures of viral populations
with regard to receptor requirements; consequently, the prop-
erties of envelopes in the starting material will determine the
outcome of selection, and the results may vary for different
primary isolates. Again, we must remember that these results
were obtained in the U87 model system, which may differ
significantly from in vivo conditions. Nevertheless, we may
envisage that selective mechanisms which operate at the re-
ceptor level play an important role in HIV pathogenesis.
It is intriguing that, over time in the same patient, the re-
ceptor usage and resistance to the direct inhibitory effect of
b-chemokines change. Such changes may conceivably confer a
replicative advantage to HIV-1 in vivo. It is also possible that
the emergence of viruses with higher replicative capacity,
which use the CXCR4 coreceptor, is a mere reflection of the
collapse of the immune system, which finally allows uncon-
trolled replication. Broadening of the capacity to use several
coreceptors may allow HIV-1 to infect a wide variety of cells
and thereby accelerate immune deterioration. Since infection
with rapid/high virus leads to a rapid loss of CD4
1
lymphocytes
(18), it is tempting to speculate that the viral phenotype plays
a major role in driving the progression of HIV-1-related dis-
ease through multiple mechanisms. Viral RNA levels in plas-
ma have been shown to correlate with progression rates and
can predict the severity of HIV-1 infection (20, 27, 41). The
HIV-1 biological phenotype may be one of the determinants of
viral load, of increased replicative capacity, and of broader cell
tropism, which together may result in higher levels of viral
RNA in plasma. Sensitivity to b-chemokines, as shown here, as
well as humoral and cellular immune responses, may all con-
tribute to the control of virus replication and thereby slow the
disease process.
VOL. 71, 1997 CORECEPTOR USAGE OF HIV-1 ISOLATES 7485

ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Medical Re-
search Council, the Swedish National Board for Industrial and Tech-
nical Development, the European Comission (Concerted Action on
HIV Variability) (to E.M.F. and Å.B.), the National Institutes of
Health (to D.R.L.), the Istituto Superiore di Sanita`, VII and IX pro-
getto AIDS, grant 9405-02 (to G.S.), and the Aaron Diamond Foun-
dation (to H.K.D.). D.R.L. is an Investigator of the Howard Hughes
Medical Institute.
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