Copyright © 1998, American Society for Microbiology
A Broad Range of Chemokine Receptors Are Used by Primary
Isolates of Human Immunodeficiency Virus Type 2
as Coreceptors with CD4
A
´ INE M
CKNIGHT,
1* MATTHIAS T. DITTMAR,
1JOSE´ MONIZ-PERIERA,
2KOYA ARIYOSHI,
3JACQUELINE D. REEVES,
1SAM HIBBITTS,
1DENISE WHITBY,
1EMMA AARONS,
4AMANDA E. I. PROUDFOOT,
5HILTON WHITTLE,
3ANDPAUL R. CLAPHAM
1Section of Virology, Chester Beatty Laboratories, Institute of Cancer Research,
1and Department of GUM,
St. Mary’s Hospital,
4London, United Kingdom; Faculdade de Farmacia, Universidada de Lisboa,
Lisbon, Portugal
2; Medical Research Council Laboratories, Fajara, The Gambia
3; and Geneva
Biomedical Institute, Glaxo Wellcome Research and Development SA, Geneva, Switzerland
5Received 29 September 1997/Accepted 21 January 1998
Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2
re-quires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family
of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can
use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite
broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood
mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these
cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to
infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the
ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.
CD4 is the primary cell surface receptor for entry of human
immunodeficiency virus (HIV) and simian immunodeficiency
virus (SIV) into cells. Coreceptors for HIV and SIV have been
identified as G-protein-coupled receptors with seven
trans-membrane domains (reviewed in references 14, 42, 59, and 60).
Early evidence suggested that there may be more than one
coreceptor, reflecting the extended tropism of
syncytium-in-ducing (SI) HIV-1 strains for CD4-positive T-cell lines
com-pared to non-syncytium-inducing (NSI) strains, which infect
mainly primary macrophage and T-cell cultures. CXCR4 was
the first HIV type 1 (HIV-1) coreceptor to be defined (25) and
was shown to be the major coreceptor for T-cell-line-adapted
and primary SI HIV-1 isolates (21, 25, 40, 54, 61). CCR5 is the
main coreceptor for primary NSI HIV-1 isolates (3, 10, 18, 21,
22, 61). HIV-1 viral entry is inhibited in the presence of the
ligands to these chemokine receptors. Thus, RANTES,
MIP-1
a
, and MIP-1
b
, the ligands for CCR5, inhibit
macrophage-tropic isolates, while SDF-1, the specific ligand for CXCR4,
inhibits entry of T-cell-tropic isolates (5, 15, 34, 44).
CCR5 is important for transmission. Individuals with a
CCR5 gene deletion are largely protected from HIV-1
infec-tion (17, 31, 33, 37, 46, 50). CCR5-using strains are present
throughout the course of infection in an individual, while
CXCR4-using viruses often develop late in disease (16). Some
HIV-1 isolates have the ability to exploit other chemokine
re-ceptor molecules, such as CCR3, CCR2b, BOB, and BONZO
(4, 10, 19, 21, 24, 36), in addition to CCR5 and CXCR4. The
efficiency of these other coreceptors for HIV entry is
contro-versial.
Phylogenetic analysis has shown that HIV-1 clusters with
SIV isolated from chimpanzees (SIVcpz) and that HIV-2
clus-ters with SIV from sooty mangabeys (SIVsm) (7, 26, 27, 43).
HIV is likely to have originated as a result of cross-species
transmission of SIV from African apes or monkeys to humans
(53). Like HIV, SIV also requires a coreceptor in addition to
CD4 for viral entry, since several nonhuman cell types
express-ing human CD4 fail to support SIV viral entry (39). Chen et al.
reported that SIV isolates (SIVmac, SIVsmSL92a,
SIVsmLib-1, and SIVcpz GAB) could use human or rhesus monkey CCR5
for entry but not human CXCR4, CCR1, CCR2b, CCR3, or
CCR4 (8). SIV, however, could infect several cell lines (now
known to be CCR5 negative) that resist HIV-1 infection,
in-dicating that other coreceptors may be exploited by SIV (12,
32). Several groups have recently reported three likely
candi-dates for this unknown receptor(s). BOB/gpr15 (19, 24, 29) and
BONZO/STRL33 (4, 36) both assist entry of SIVmac and
HIV-2 as well as certain SI and NSI strains of HIV-1. Another
orphan receptor, GPR1, allows entry of SIVmac but not HIV-1
into CD4-positive cells (24).
Coreceptors used by the laboratory-adapted isolate HIV-2
ROD were recently studied by Bro¨n et al. (6). In cell-to-cell
fusion assays, ROD was particularly promiscuous and induced
fusion of CD4-positive cells expressing CCR1, CCR2b, CCR3,
CCR5, CXCR2, and CXCR4. In contrast, in cell-free
infectiv-ity assays, ROD used CCR3 and CXCR4 but not CCR5. A
variant of HIV-2, ROD/B, that efficiently infects some cell
types in the absence of CD4 (13) also used CXCR4 and CCR3
or an orphan receptor, V28, in the absence of CD4 (23, 48).
Re-cently, Sol et al. (56) showed that CCR3, CCR5, and CXCR4
were used by HIV-2 primary strains, while Heredia et al. (30)
reported that some primary isolates of HIV-2 could use CCR1,
CCR2b, and CCR5 as well as CXCR4 but not CCR3 or CCR4.
However, there was no indication of the relative efficiencies of
these infections. Deng et al. (19) also showed that some
pri-mary HIV-2 isolates used several coreceptors, including the
newly identified BOB/gpr15 and BONZO/STRL33.
Here we report on the coreceptor use of various primary
* Corresponding author. Mailing address: Section of Virology,
Chester Beatty Laboratories, Institute of Cancer Research, London
SW3 6JB, United Kingdom. Phone: 8133. Fax:
44-171-352-3299. E-mail: aine@icr.ac.uk.
4065
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isolates of HIV-2 isolated mainly from symptomatic patients
and cultured in peripheral blood mononuclear cells (PBMCs).
We find that these primary HIV-2 isolates can use a broad
range of coreceptors, including CCR1, CCR2b, CCR3, CCR4,
CCR5, and CXCR4. Not all human cell types that express an
appropriate coreceptor supported virus replication, indicating
the presence of other factors influencing viral tropism.
MATERIALS AND METHODS
Cells.U87/CD4 cells expressing chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 were a gift from Dan Littman and have been previously described (18). CCC/CD4 cells derived from cat kidney (CCC S1L2), RD/CD4 cells derived from human rhabdomyosarcoma, and HeLa/CD4 cells have been previously described (12, 39). SCL/CD4 cells are from human skin (9), and WI-38/CD4 cells are simian virus 40-transformed human lung fibroblasts (28). Human CD4 was stably expressed on these cell types by using an amphotropic retroviral vector (9). MT-2 (41), C8166 (11), Molt 4, H9, and Sup T1 cells are all human CD41T-cell lines and have been described elsewhere (13, 39). Peripheral
blood PBMCs were stimulated for 2 days with phytohemagglutinin (PHA) (0.5 mg/ml) and then cultured in RPMI plus 10% fetal calf serum (FCS) supple-mented with interleukin-2 (IL-2) (20 U/ml).
Chemokines and coreceptor ligands.A recombinant form of SDF-1 that retains the N-terminal methionine residue was used. This, MCP-1, and AOP-RANTES (55) were provided by Glaxo Wellcome. vMIP-II, a chemokine en-coded by the Kaposi’s sarcoma-associated herpesvirus (35), was provided by T. W. Schwartz, University of Copenhagen. AMD3100 is a bicyclam derivative that reacts with CXCR4 (51).
Isolation of virus and preparation of stocks.CBL-20, CBL-23, and V9 were isolated from individuals from The Gambia. CBL-20 and CBL-23 have been described before (52). CBL-20 and V9 were isolated from individuals with AIDS, whereas CBL-23 is from an individual with AIDS-related complex. prCBL-20 was propagated only in primary PBMC cultures, while CBL-20 was adapted for growth in T-cell lines. MIR was isolated from an AIDS patient who died in 1987. A-ND was from a Portuguese individual with progressive disease. ST was from a Senagalese asymptomatic individual (53). Viruses were prepared as follows. PBMCs from HIV-2-infected patients were cocultured with PHA-stimulated uninfected PBMCs in the presence of recombinant IL-2 (20 IU/ml). Supernatant from a day 28 culture was passaged into a culture of fresh uninfected PBMCs, which was monitored weekly for reverse transcriptase (RT) activity. When the culture became positive for RT activity (.50 pg/ml), fresh PBMCs were added and virus was allowed to grow for a further week before the supernatant was harvested. Stocks of ST were made from Molt 4-derived virus passaged into PBMCs. Virus aliquots were stored under liquid N2.
Plasmids.The chemokine receptors CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4 were subcloned into the vector pcDNA3.1 (Invitrogen) for trans-fection and transient expression in CCC/CD4 cells.
Transfection of CCC/CD4 cells.Cells were plated overnight in six-well trays at 23105cells per ml. For each well, 1mg of plasmid DNA in 100ml of FCS-free medium was added to a solution of 7ml of Lipofectamine (GIBCO BRL) (1 mg/ml) in 100ml of FCS-free medium and allowed to stand on the bench for 45 min. Cells were washed twice before addition of 0.8 ml of FCS-free medium followed by the plasmid and Lipofectamine mixture. Control transfections (mock) were done by using the vector pcDNA3.1 without insert. After 5 h of incubation, the medium was replaced, and after overnight incubation, the cells were set up in 24-well trays for infection the following day.
Syncytium induction assays.High-titer virus stocks were added to 0.5 ml of target cells (23105cells/ml for MT-2, C8166, Molt 4, and Sup T1 cells or 13 106cells/ml for PBMCs) in a 48-well tray. Cells were passaged twice weekly for 2 weeks and scored for syncytia as follows:2, no syncytia;1, at least one syncytium in cultured cells but fewer than 5% of nuclei in syncytia;11,.5% but ,19% of nuclei in syncytia,111, 20 to 40% of nuclei in syncytia; and1111, .40% of nuclei in syncytia. After 2 weeks, RT activity in the PBMC cultures was measured to assess the presence of virus.
Determination of RT activity. Virus production into the supernatant was assayed by measurement of RT activity by a sensitive nonradioactive method (Retrosys RT activity kit; Cavidi Tech, Uppsala, Sweden).
Virus infectivity and inhibition assays.Cells were plated at 53104per well in a 24-well plate or at 13104per well in a 48-well dish. A total of 0.1 ml (48-well dishes) or 0.2 ml (24-well plates) of 10-fold serial dilutions of virus was added to cells and incubated at 37°C for 1 h before washing in growth medium and reincubation for 3 to 5 days. Infected cells were detected by specific immuno-staining. For titration of virus on PBMCs, virus (50ml) was added to 105cells per well of a 96-well tray. For inhibition-of-infection studies with PBMCs, cells were first treated with SDF-1 at 400 or 1,600 ng/ml, with AOP-RANTES at 200 or 800 ng/ml, or with MCP-1 at 800 ng/ml for 20 min before addition of virus.
Immunostaining of HIV-infected cells.The immunostaining method has been described before (39). Briefly, HIV-infected cells were washed in serum-free phosphate-buffered-saline (PBS) and fixed in methanol-acetone (1:1) at220°C. After washing of the cells in PBS–1% FCS (PBS-FCS), HIV-2-positive serum
was added at 1/5,000 (in PBS-FCS) to detect virus antigen. After three washes in PBS-FCS, the cells were incubated for 1 h with mouse anti-human immunoglob-ulin G conjugated withb-galactosidase (Southern Biotech). After a further three washes in serum-free PBS, 0.5 mg of X-Gal (5-bromo-4-chloro-3-indolyl-b-D
-galactopyranoside) substrate (Novolabs) per ml in PBS containing 3 mM potas-sium ferricyanide, 3 mM potaspotas-sium ferrocyanide, and 1 mM magnepotas-sium chloride was added. Clusters of blue cells were counted as foci of infection (focus-forming units [FFU]) to estimate the virus titer.
PCR amplification of CCR5 genes.Oligonucleotide primers spanning the CCR5 32-bp deletion (D32) were used to amplify DNA fragments from PBMC cultures (37). This procedure produces a PCR DNA fragment of 183 bp from wild-type CCR5 DNA and a fragment of 151 bp of DNA carrying the 32-bp CCR5 deletion. Genomic DNA was derived from PBMCs by using a Nucleon Biosciences (Coatbridge, United Kingdom) kit. The primer pair used was 7591 CTT CAT TAC ACC TGC AGC TCT and 9412ACC AGC CCT GTG CCT CTT CTT. The samples were initially denatured at 95°C for 5 min, followed by 30 cycles of a denaturing step at 95°C for 30 s, an annealing step at 45°C for 30 s, and an extension step at 72°C for 30 s. A final step at 72°C for 5 min was done. PCR products were visualized by electrophoresis on a 3% agarose gel.
RESULTS
Coreceptor use by primary isolates of HIV-2.
We
deter-mined the coreceptor use of six primary isolates of HIV-2
grown in PBMCs. ST came from an asymptomatic Senegalese
individual (53). The remaining isolates were from symptomatic
patients. MIR and A-ND were isolated from two symptomatic
Portuguese patients. Three isolates from Gambian individuals
were also included. Two of these Gambian isolates, V9 and
prCBL-20, were from patients with AIDS, while prCBL-23 was
from an individual with AIDS-related complex. To determine
which coreceptors these viruses used, we challenged U87/CD4
cells stably expressing each of the chemokine receptors, CCR1,
CCR2b, CCR3, CCR5, and CXCR4. Virus isolates MIR, ST,
V9, prCBL-20, and prCBL-23 showed efficient use of all five
chemokine receptor molecules in cell-free infection (Fig. 1).
No infection of the parental U87/CD4 cells was detected. MIR,
prCBL-20, and prCBL-23 plated most efficiently on cells
ex-pressing CXCR4, while ST preferentially infects cells
express-ing CCR5. Interestexpress-ingly, A-ND showed no detectable activity
on CCR5-expressing cells.
To verify the infectivity results gained from the set of U87/
CD4 cells expressing coreceptors, we tested three isolates,
MIR, V9, and prCBL-20, on cat CCC/CD4 cells transfected
with and transiently expressing each of the chemokine
recep-tors tested on U87/CD4 cells. Although this procedure yielded
variable results, we were able to confirm that these viruses
were capable of using a broad range of coreceptors. Table 1
shows a representative set of results from one experiment
using MIR. We also tested whether these isolates used CCR4
(not included in the U87/CD4 experiments) in transiently
transfected CCC/CD4 cells. Although MIR used CCR4
con-sistently, low levels of infectivity were observed with the two
other strains, V9 and prCBL-20.
U87 and CCC cells express undefined coreceptors that can
be used by some HIV-2 strains (12, 39). It was possible that the
chemokine receptor-positive subclones of U87/CD4 may
ex-press such undefined coreceptors at higher levels than parental
U87/CD4 cells and therefore confer infection independently of
the expressed recombinant chemokine receptor. To rule out
this possibility, we tested whether specific coreceptor ligands
could inhibit A-ND infection of U87/CD4 cells expressing
CCR1, CCR3, or CXCR4. vMIP-II is a chemokine encoded
by Kaposi’s sarcoma-associated herpesvirus that
preferential-ly blocks HIV-1 infection via CCR3 (35). vMIP-II (1
m
g/ml)
reduced A-ND infectivity for CCR3
1U87/CD4 cells by over
90% but had no effect on infection via CCR1 or CXCR4.
Similarly, a 100-ng/ml concentration of AMD3100, a
bicy-clam derivative that binds CXCR4, blocked A-ND infection
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of CXCR4
1U87/CD4 cells by over 90% but did not inhibit
CCR1- or CCR3-dependent infection (data not shown).
Coreceptor use by T-cell-line-adapted HIV-2 isolates.
One
isolate, prCBL-20, was adapted for replication in a T-cell line
(H9). The virus strain derived, CBL-20 (52), was then tested
for coreceptor use on the U87/CD4 cells expressing different
chemokine receptors. Figure 2 shows that selection of primary
prCBL-20 to grow in T cells resulted in a virus variant which
could infect only CXCR4
1U87/CD4 cells. Unlike the parental
prCBL-20 virus, CBL-20 had lost the capacity to infect U87/
CD4 cells via CCR1, CCR2b, CCR3, or CCR5. Thus, selection
into T-cell lines resulted in restricted coreceptor use for this
virus.
Phenotypes of HIV-2 strains used.
All of the isolates studied
here could use CXCR4 efficiently as a coreceptor molecule.
For HIV-1, use of CXCR4 generally correlates with an SI
phe-notype (54, 61), whereas all NSI isolates use at least CCR5 (3,
10, 18, 21, 22). We tested whether our isolates could be
cate-gorized as NSI or SI according to the conventional criteria
used for HIV (1, 58). Each isolate was plated on MT-2 cells,
PBMCs, and a number of T-cell lines. Syncytium induction on
these cell types indicates the presence of an SI virus. The
re-sults are shown in Table 2. At least two of these isolates, V9
and prCBL-20, did not induce syncytia in any of the cell types
tested. MIR, A-ND, ST, prCBL-23, and CBL-20, however,
in-duced syncytia in at least one of the T-cell lines tested.
Infection of PBMCs from an individual who is homozygous
for the
D
32 CCR5 gene.
We assessed whether the primary
HIV-2 strains could infect PHA- and IL-2-stimulated PBMCs
derived from an individual who was homozygous for the
D
32
CCR5 gene. Figure 3 shows a smaller PCR-amplified DNA
fragment from PBMCs derived from an individual homozygous
for deletions of CCR5. These cells were resistant to the CCR5
with HIV-1 strain SF-162 (55a). All but one of the primary
HIV-2 strains used CCR5 (see above), while all could use
several other coreceptors, including CXCR4. Table 3 shows
that all strains tested replicated as efficiently in
D
32/
D
32 CCR5
PBMCs as in wild-type CCR5 PBMCs and therefore do not
depend on CCR5 for infection of PBMCs.
[image:3.612.53.547.69.358.2]Inhibition of infection of primary HIV-2 isolates by the
ligands to specific chemokine coreceptors.
We next
investi-gated the coreceptor molecules used by A-ND, prCBL-20, and
CBL-20 to infect PBMCs in vitro. PBMCs were derived either
from individuals homozygous for
D
32 CCR5 or from wild-type
donors. We tested for infectivity of cells treated with SDF-1
(400 and 1,600 ng/ml), AOP-RANTES (200 and 800 ng/ml),
and MCP-1 (800 ng/ml). AOP-RANTES is an analog of
RAN-TES and a potent inhibitor of HIV-1 infection (55) and is also
a ligand for CCR5, CCR3, and CCR1 (47). Figure 4 shows that
whatever the source of PBMCs, SDF-1, the natural ligand for
FIG. 1. Titration of primary HIV-2 isolates on chemokine receptor-expressing U87/CD4 cells. After incubation for 3 to 5 days, cells were fixed and immunostained and infection (FFU per milliliter) was estimated. Error bars indicate standard deviations.TABLE 1. HIV-2 MIR infection of CCC/CD4 cells expressing
different chemokine receptors after transfection
Chemokine
receptor Infectivity of HIV-2 MIR(FFU, mean6SD)
Control ...
0
CCR1... 150
6
70
CCR2b ... 440
6
84
CCR3... 530
6
42
CCR4... 440
6
56
CCR5... 990
6
268
CXCR4... 1,050
6
212
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[image:3.612.308.548.634.726.2]CXCR4, inhibited the infection of all three viruses by more
than 90% (at 1,600 ng/ml). In contrast, MCP-1, a natural
li-gand for CCR2b and CCR4, had no effect on the infectivity of
PBMCs by prCBL-20. In some experiments AOP-RANTES
showed some inhibition early on when RT activity was first
detected in control infections after infection of wild-type
PB-MCs (data not shown). This inhibition was not sustained.
These results therefore indicate that CXCR4 is the
predomi-nant coreceptor used in vitro by the three HIV-2 strains tested
for PBMC infection.
Tropism of primary HIV-2 isolates for CD4-expressing cell
lines.
We determined the abilities of the HIV-2 isolates to
in-fect various human cell lines transin-fected with and stably
ex-pressing human CD4. Each of these cell types (HeLa/CD4,
RD/CD4, WI-38T/CD4, SCL/CD4, and U87/CD4/CXCR4)
was tested for expression of CCR5 or CXCR4 by using specific
monoclonal antibodies followed by immunostaining and flow
cytometric analysis (data not shown). As reported previously,
HeLa/CD4 and RD/CD4 cells both express CXCR4 to high
levels (40) but do not express CCR5 (data not shown). The
SCL/CD4 or WI-38/CD4 cells did not express either CXCR4
or CCR5 at the cell surface. All seven virus isolates were
titrat-ed on these cell types as well as on U87/CD4/CXCR4 cells
(Ta-ble 4). MIR, A-ND, V9, prCBL-20, and CBL-20 can efficiently
infect HeLa/CD4 or RD/CD4 cells, presumably through CXCR4,
which is expressed on both of these cell types. Interestingly, ST
and prCBL-23, both of which efficiently infect CXCR4
1U87/
CD4 cells, did not infect either CXCR4
1HeLa/CD4 or RD/
CD4 cells even when the appropriate coreceptor was
ex-pressed. Thus, there is a further restriction to infection of some
isolates on these cell lines. Furthermore, the ST isolate, unlike
the other isolates tested, infected SCL/CD4 and WI-38/CD4
cells. Since ST, like other strains tested here, can use CCR1 to
-5 and CXCR4, these results suggest that another cell surface
receptor may be expressed on these two cell types.
DISCUSSION
[image:4.612.135.467.67.203.2]This study determined the abilities of six primary isolates
and one laboratory-adapted isolate of HIV-2 to use six
che-mokine receptors as coreceptor molecules for fusion and
in-fection of human cells expressing CD4. The results presented
here show that, unlike HIV-1 and SIV, the primary isolates of
HIV-2 tested used a wide range of chemokine receptors. Five
of the isolates, MIR, ST, V9, prCBL-20, and prCBL-23, could
use CCR1, CCR2b, CCR3, CCR5, and CXCR4 expressed on
U87/CD4 cells. This broad coreceptor use was confirmed for
MIR, V9, and prCBL-20 on CCC/CD4 cells transiently
trans-fected with these chemokine receptors. One isolate, A-ND,
FIG. 2. Selection of a primary isolate to grow in T-cell lines restricts coreceptor use. prCBL-20 was selected to grow in C8166 cells, and stocks were made in H9 cells (52). The resulting virus was titrated on either the parental U87/CD4 cells or those stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Infection (FFU per milliliter) was calculated after immunostaining of 5-day-old infected cultures. Error bars indicate standard deviations. [image:4.612.369.486.504.665.2]FIG. 3. Detection of homozygous deleted CCR5. CCR5 DNA was amplified from genomic DNA derived from the PBMCs of an individual homozygous for the CCR5 deletion and an individual homozygous for the wild-type CCR5 gene. PCR products were visualized by electrophoresis on a 3% agarose gel. Lanes: M, 1-kb marker; 1, negative control; 2 and 3, 200 and 400 ng of homozygousD32 CCR5 PBMC DNA, respectively; 4, negative control; 5 and 6, 200 and 400 ng of wild-type CCR5 PBMC DNA, respectively.
TABLE 2. Phenotype determination of primary HIV-2 isolates
aTarget cell
Syncytium formationbby the following virus:
MIR A-ND ST V9 pCBL20 CBL-20 pCBL23
Molt 4
11
11
1
2
2
111
2
Sup T1
1
2
2
2
2
1
2
C8166
1111 111 11
2
2
111
2
MT-2
1
2
2
2
2
2
1111
PBMC
1
2
2
2
2
2
2
aThe phenotypes (SI or NSI) of the primary isolates and laboratory-adapted
CBL-20 were determined. Viruses were analyzed for their ability to induce syncytia in the cell lines indicated. Cells were infected with virus, passaged twice weekly for 2 weeks, and then scored for syncytia.
b2, no syncytia seen;1, at least one syncytium in cultured cells but fewer than
5% of nuclei in syncytia,11,.5% but,19% of nuclei in syncytia;111, 20 to 40% of nuclei in syncytia;1111,.40% of nuclei in syncytia. Supernatants from PBMCs were tested for RT activity (see Materials and Methods) after 2 weeks to verify the presence of virus; for each virus the RT activity was.500 pg/ml.
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[image:4.612.50.290.564.645.2]is peculiar in that we could not detect any infectivity on
CCR5-expressing cells yet it used CCR1, CCR2b, CCR3, and
CXCR4. MIR, V9, and prCBL-20 were also tested for
infec-tion of CCC/CD4 cells transfected with and expressing CCR4
(not tested in the U87/CD4 panel). We found that all of these
virus isolates could also use CCR4 at least to some extent. This
is the first report of any HIV or SIV isolates using CCR4.
Recently, Heredia et al. (30) also showed that primary
HIV-2 isolates used several coreceptors. However in contrast to the
viruses we tested, several isolates that used only CCR5 were
identified, as well as some that additionally used CXCR4,
CCR1, and CCR3 expressed by HOS cells. It is therefore
pos-sible that HIV-2 coreceptor use broadens as disease
pro-gresses, as suggested for HIV-1 by Connor et al. (16). Apart
from ST, all of the HIV-2 strains tested here were derived from
symptomatic patients. Our results therefore support the
hy-pothesis of Connor et al.
For transmission of HIV-1, CCR5 is clearly the most
impor-tant coreceptor. The majority of isolates thus far reported use
CCR5 either exclusively (for NSI isolates) or in addition to
CXCR4 (for SI isolates). Early after HIV-1 infection and in
the asymptomatic phase, NSI CCR5-using viruses are usually
isolated (16), suggesting that there is a strong selective
pres-sure for the spread of CCR5-using viruses. As disease
devel-ops, CXCR4-using viruses can be more frequently isolated,
from about 50% of patients with AIDS. How the broad
core-ceptor use by HIV-2 strains shown here influences their
trans-mission remains to be determined.
[image:5.612.49.291.81.186.2]We tested the coreceptor use of prCBL-20 after its
adap-tation to culture in H9 cells. The isolate derived (CBL-20)
showed a marked loss in its ability to use the broad range of
coreceptors used by the parental virus and could use only
CXCR4 efficiently. Furthermore, T-cell-line-adapted CBL-20
could use CXCR4 expressed on HeLa/CD4 cells, whereas
prCBL-20 could not. In contrast, prCBL-20 but not CBL-20
infected primary macrophages (not shown) even though they
express CXCR4 (40). Recently Bro¨n et al. (6) showed that the
T-cell-line-adapted ROD strain of HIV-2 infected
CXCR4-and CCR3-expressing CD4
1cells. Interestingly, while cell-free
[image:5.612.102.486.389.688.2]FIG. 4. Inhibition of infection by prCBL-20, CBL-20, and A-ND with chemokines on wild-type CCR5 andD32 CCR5 PBMCs. Infectivity assays with prCBL-20 and A-ND were performed in the absence and presence of the chemokines SDF-1, AOP-RANTES, and MCP-1 either alone or together at the concentrations indicated. Cell targets were either homozygous wild-type orD32 CCR5 PBMCs. RT activity in the supernatant on day 5 was determined, and the results are shown as percent inhibition with respect to the control (con.) value. Error bars indicate standard deviations.
TABLE 3. HIV-2 infection of wild-type and
D
32/
D
32 PBMCs
aVirus
RT activity (pg/ml) in supernatants after culture in PBMCs
Day 2 Day 5 Day 9 D32 wt D32 wt D32 wt
MIR
117
292
446
415
.
500
.
500
A-ND
264
283
441
328
.
500
.
500
ST
86
193
577
407
.
500
.
500
prCBL-20
72
121
309
477
.
500
.
500
CBL-20
112
108
484
.
500
.
500
.
500
prCBL-23
11
,
4
158
61
.
500
.
500
aVirus isolates were tested for their ability to infect PBMCs from an individualwho is homozygous for theD32 deletion in the CCR5 gene and from an indi-vidual homozygous for wild-type (wt) CCR5 gene. PBMCs were infected, and supernatants were analyzed for RT activity on days 2, 5, and 9.
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infection was restricted to particular coreceptors, cell-to-cell
fusion was induced by a broad range of chemokine receptors,
including CCR1, CCR2b, and CXCR2. The results of Bro¨n et
al. suggest that cell culture adaptation for CXCR4 use
prefer-entially restricts coreceptor use for virion-to-cell fusion
com-pared with cell-to-cell fusion and support the notion that the
physical requirements for these processes are subtly different
(6, 39).
So far, all primary and T-cell-line-adapted SI isolates of
HIV-1 use CXCR4 (2, 25, 54, 61). We tested the phenotypes of
the HIV-2 isolates described here by criteria used first for
HIV-1 (58) and later for HIV-2 (1). Of the primary isolates,
only MIR induced syncytia in PBMCs, while both MIR and
prCBL-23 induced syncytia in MT-2 cells. Of the four other
primary strains, ST and A-ND induced syncytia in at least one
of the T-cell lines tested, whereas V9 and prCBL-20 showed no
evidence of syncytium formation in any of the cell types tested.
Thus, we did not observe a clear correlation between CXCR4
use and syncytium induction in CXCR4
1T cells by the HIV-2
strains used here.
The determination of coreceptor use by HIV-1 and HIV-2
strains in vitro, using CD4
1cell lines, can be misleading (20).
Primary cell types that are targets for HIV infection in vivo
may express several coreceptors that can be used by HIV
strains in such in vitro assays. It is therefore hard to assess the
predominant coreceptor used on these cell types. It has been
shown previously that stimulated primary lymphocytes express
CCR1 (57), CCR2b (45), CCR3 (49), CCR4 (38), CCR5 (14),
CXCR4 (40), and BONZO (4, 36) as determined by mRNA
expression. In addition we have shown that CCR5
(unpub-lished data) and CXCR4 (40) are expressed at the cell surface
as detected by specific monoclonal antibodies. We assessed the
coreceptors used by HIV-2 strains A-ND, prCBL-20, and
CBL-20 on primary PHA- and IL-2-stimulated PBMCs by
test-ing whether the natural ligands for different coreceptors could
block infection. SDF-1 inhibited infection of PBMCs by all
three virus strains, while AOP-RANTES or MCP-1 had little
effect. Since SDF-1 has so far been shown to bind only to
CXCR4, these results suggest that this coreceptor is
predom-inantly used for PBMC infection.
We have shown that six primary strains of HIV-2 can use a
broad range of different coreceptors. Although the more
re-cently described coreceptors BOB, BONZO, and GPR1 were
not included in this study, it seems likely that they too will be
used by primary HIV-2 strains, at least for some
virus-core-ceptor combinations. How such a broad corevirus-core-ceptor use
influ-ences the cell types infected by HIV-2 strains in vivo remains
to be elucidated. Although HIV-2 is often described as less
pathogenic than HIV-1, many HIV-2-infected individuals do
progress to full-blown AIDS and suffer from encephalitis. It
will be of interest to establish whether the use of a wide
spec-trum of coreceptors available correlates with any disease state.
ACKNOWLEDGMENTS
We thank Robin Weiss and Graham Simmons for critical discussion
and reading of the manuscript. Dan Littman generously provided U87/
CD4 cells expressing different chemokine receptors. Erik de Clercq
and Dominic Schols kindly provided AMD3100. We also thank the
European Community Concerted Action Group on HIV Variability
for providing an intellectual forum for collaboration and discussion.
This work is funded by the Medical Research Council, United
King-dom, and supported partly by an EC Biomed II grant.
REFERENCES
1. Albert, J., P. Stalhandske, S. Marquina, J. Karis, R. A. Fouchier, E. Norrby,
and F. Chiodi.1996. Biological phenotype of HIV type 2 isolates correlates with V3 genotype. AIDS Res. Hum. Retroviruses. 12:821–828.
2. Alkhatib, G., C. C. Broder, and E. A. Berger. 1996. Cell type-specific fusion cofactors determine human immunodeficiency virus type 1 tropism for T-cell lines versus primary macrophages. J. Virol. 70:5487–5494.
3. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M.
Murphy, and E. A. Berger.1996. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Sci-ence 272:1955–1958.
4. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. Peden. 1997. A new SIV co-receptor, STRL33. Nature 388:238.
5. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark Lewis, J. Sodroski, and
T. A. Springer.1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829–833.
6. Bro¨n, R., P. J. Klasse, D. Wilkinson, P. R. Clapham, A. Pelchen-Matthews,
C. Power, T. N. C. Wells, J. Kim, S. Peiper, J. A. Hoxie, and M. Marsh.1997. Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 ROD envelope protein. J. Virol. 71:8405–8415.
7. Chen, Z., P. Telfier, A. Gettie, P. Reed, L. Zhang, D. D. Ho, and P. A. Marx. 1996. Genetic characterization of new West African simian immunodefi-ciency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop. J. Virol. 70:3617–3627. 8. Chen, Z., P. Zhou, D. D. Ho, N. R. Landau, and P. A. Marx. 1997. Genetically
divergent strains of simian immunodeficiency virus use CCR5 as a corecep-tor for entry. J. Virol. 71:2705–2714.
9. Chesebro, B., and K. Wehrly. 1988. Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity. J. Virol. 62:3779– 3788.
10. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu,
C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski.1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135–1148.
11. Clapham, P. R., R. A. Weiss, A. G. Dalgleish, M. Exley, D. Whitby, and N.
Hogg. 1987. Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: receptor modulation and differentiation induced by phorbol ester. Virology 158:44–51.
12. Clapham, P. R., D. Blanc, and R. A. Weiss. 1991. Specific cell surface requirements for the infection of CD4-positive cells by human immunode-ficiency virus types 1 and 2 and by simian immunodeimmunode-ficiency virus. Virology
181:703–715.
13. Clapham, P. R., A´. McKnight, and R. A. Weiss. 1992. Human immunodefi-ciency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4. J. Virol. 66:3531–3537. 14. Clapham, P. R. 1997. HIV and chemokines: ligands sharing cell surface
receptors. Trends Cell Biol. 7:264–268.
15. Cocchi, F., A. L. DeVico, A. Garzino Demo, S. K. Arya, R. C. Gallo, and P.
Lusso.1995. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD81T cells. Science 270: 1811–1815.
16. 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.
17. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R.
Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, S. Donfield, D. Vlahov, R. Kaslow, A. Saah, C. Rinaldo, R. Detels, and S. J. O’Brien.1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter He-mophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science
[image:6.612.50.290.90.189.2]273:1856–1862.
TABLE 4. Tropism of primary HIV-2 isolates for cell lines
expressing human CD4
aVirus
Infectivity (FFU/ml) with the following cell line:
HeLa/CD4 RD/CD4 SCL/CD4 WI-38T/CD4 U87/CD4/CXCR4
MIR 1.03102 3.03102 ,10 ,10 2.03104 A-ND 4.03103 3.03102 ,10 ,10 5.03104 ST ,10 ,10 1.03102 2.03102 2.03103 V9 1.53102 1.33102 ,10 ,10 1.03103 prCBL-20 NTb 1.83103 ,10 20 4.53104 prCBL-23 ,10 ,10 ,10 NT 5.03103
aCells were seeded and infected as described in Materials and Methods. After
4 days of incubation, cells were fixed and immunostained for HIV-2 antigen. Clusters of stained cells were counted as infection foci.
bNT, not tested.
on November 9, 2019 by guest
http://jvi.asm.org/
18. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di
Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, and D. R. Littman.1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661–666.
19. Deng, H., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997. Expression cloning of new receptors used by simian and human immunode-ficiency viruses. Nature 388:296–300.
20. Dittmar, M., A´. McKnight, G. Simmons, P. R. Clapham, R. A. Weiss, and
P. A. Simmonds.1997. HIV-1 tropism and co-receptor use. Nature 385: 495–496.
21. 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 primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149–1158.
22. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A.
Na-gashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton.1996. HIV-1 entry into CD41cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667–673.
23. Endres, M. J., P. R. Clapham, M. Marsh, M. Ahuja, J. D. Turner, A´.
McKnight, J. F. Thomas, B. Stoebenau Haggarty, S. Choe, P. J. Vance, T. N. Wells, C. A. Power, S. S. Sutterwala, R. W. Doms, N. R. Landau, and J. A. Hoxie.1996. CD4-independent infection by HIV-2 is mediated by fusin/ CXCR4. Cell 87:745–756.
24. Farzan, M., H. Choe, K. Martin, L. Marcon, W. Hofmann, G. Karlsson, Y.
Sun, P. Barrett, N. Marchand, S. Nancy, C. Gerard, and J. Sodroski.1997. Two orphan seven-transmembrane segment receptors that are expressed in CD4-positive cells support immunodeficiency virus infection. J. Exp. Med.
186:405–411.
25. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872–877.
26. Gao, F., L. Yue, A. T. White, P. G. Pappas, J. Barchue, A. P. Hanson, B. M.
Greene, P. M. Sharp, G. M. Shaw, and B. H. Hahn.1992. Human infection by genetically diverse SIVSM-related HIV-2 in west Africa. Nature 358: 495–499.
27. Hahn, B. H., L. I. Kong, S. Lee, P. Kumar, M. E. Taylor, S. K. Arya, and
G. M. Shaw.1987. Relation of HTLV-4 to simian and human immunodefi-ciency associated viruses. Nature 300:184–186.
28. Hayflick, L. 1965. Limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614–636.
29. Heiber, M., A. Marchese, T. Nguyen, H. H. Heng, S. R. O. George, and B. F.
Dowd.1996. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3. Genomics 32:462–465.
30. Heredia, A., A. Vallejo, V. Soriano, J. S. Epstein, and I. K. Hewlett. 1997. Chemokine receptors and HIV-2. AIDS 11:1198–1199.
31. Hill, C. M., and D. R. Littman. 1996. Natural resistance to HIV? Nature 382: 668–669.
32. Hoxie, J. A., B. S. Haggarty, S. E. Bonser, J. L. Rackowski, H. Shan, and P. J.
Kanki.1988. Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection. J. Virol. 62:2557–2568.
33. Huang, Y., W. A. Paxton, S. M. Wolinsky, A. U. Neumann, L. Zhang, T. He,
S. Kang, D. Ceradini, Z. Jin, K. Yazdanbakhsh, K. Kunstman, D. Erickson, E. Dragon, N. R. Landau, J. Phair, D. D. Ho, and R. A. Koup.1996. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat. Med. 2:1240–1243.
34. Jansson, M., M. Popovic, A. Karlsson, F. Cocchi, P. Rossi, J. Albert, and H.
Wigzell.1996. Sensitivity to inhibition by beta-chemokines correlates with biological phenotypes of primary HIV-1 isolates. Proc. Natl. Acad. Sci. USA
93:15382–15387.
35. Kledal, T. N., M. M. Rosenkilde, F. Coulin, G. Simmons, A. H. Johnson, S.
Alouani, C. A. Power, H. R. Lu¨ttichau, J. Gerstoft, P. R. Clapham, I. Clark-Lewis, T. N. C. Wells, and T. W. Schwartz.1997. A broad spectrum chemo-kine antagonist encoded by Kaposi’s sarcoma-associated herpesvirus. Sci-ence 277:1656–1659.
36. Liao, F., G. Alkhatib, K. W. Peden, G. Sharma, E. A. Berger, and J. M.
Farber.1997. STRL33, a novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1. J. Exp. Med. 185:2015–2023.
37. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E.
MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau.1996. Homozy-gous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367–377.
38. Mackay, C. R. 1996. Chemokine receptors and T cell chemotaxis. J. Exp. Med. 184:799–802.
39. McKnight, A´., P. R. Clapham, and R. A. Weiss. 1994. HIV-2 and SIV infec-tion of nonprimate cell lines expressing human CD4: restricinfec-tions to replica-tion at distinct stages. Virology 201:8–18.
40. McKnight, A´., D. Wilkinson, G. Simmons, S. Talbot, L. Picard, M. Ahuja,
M. Marsh, J. A. Hoxie, and P. R. Clapham.1997. Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent. J. Virol. 71:1692– 1696.
41. Miyoshi, I., I. Kubonishi, S. Yoshimoto, and Y. Shiraishi. 1981. A T-cell line derived from normal human cord leukocytes by co-culturing with human leukemic T-cells. Gann 72:978–981.
42. Moore, J. P., A. Trkokla, and T. Dragic. 1997. Co-receptors for HIV-1 entry. Curr. Opin. Immunol. 9:551–562.
43. Myers, G., K. MacInnes, and B. Korber. 1992. The emergence of simian/ human immunodeficiency viruses. AIDS Res. Hum. Retroviruses. 8:373–386. 44. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, F. Arenzana
Seisdedos, O. Schwartz, J. M. Heard, I. Clark Lewis, D. F. Legler, M. Loetscher, M. Baggiolini, and B. Moser.1996. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 382:833–835.
45. Oin, S., G. LaRosa, J. J. Campbell, H. Smith Heath, N. Kassam, X. Shi, L.
Zeng, E. C. Buthcher, and C. R. Mackay.1996. Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential. Eur. J. Immunol. 26: 640–647.
46. Paxton, W. A., T. Dragic, R. A. Koup, and J. P. Moore. 1996. The beta-chemokines, HIV type 1 second receptors, and exposed uninfected persons. AIDS Res. Hum. Retroviruses 12:1203–1207.
47. Premack, B., and T. J. Schall. 1996. Chemokine receptors: gateways to inflammation and infection. Nat. Med. 2:1174–1178.
48. Reeves, J. D., A. McKnight, S. Potempa, G. Simmons, P. W. Gray, C. A.
Power, T. Wells, R. A. Weiss, and S. J. Talbot.1997. CD4-independent infection by HIV-2 (ROD/B): use of the 7-transmembrane receptors CXCR-4, CCR-3, and V28 for entry. Virology 231:130–134.
49. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277: 2005–2007.
50. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber,
S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C. Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth, R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier.1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant al-leles of the CCR-5 chemokine receptor gene. Nature 382:722–725. 51. Schols, D., J. A. Este, G. Henson, and E. De Clercq. 1997. Bicyclams, a class
of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/ CXCR-4. Antiviral Res. 35:147–156.
52. Schulz, T. F., D. Whitby, J. G. Hoad, T. Corrah, H. Whittle, and R. A. Weiss. 1990. Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia. J. Virol. 64:5177–5182.
53. Sharp, P. M., D. L. Robertson, and B. H. Hahn. 1995. Cross-species trans-mission and recombination of ‘AIDS’ viruses. Philos. Trans. R. Soc. London B 349:41–47.
54. 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.
55. Simmons, G., P. R. Clapham, L. Picard, R. E. Offord, M. M. Rosenkilde,
T. W. Schwartz, R. Buser, T. N. C. Wells, and A. E. Proudfoot.1997. Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276:276–279.
55a.Simmons, G. Personal communication.
56. Sol, N., F. Ferchal, J. Braun, O. Pleskoff, C. Tre´boute, and M. Alizon. 1997. Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2. J. Virol. 71:8237–8244. 57. Su, S. B., N. Mukaida, J. Wang, H. Nomura, and K. Matsushima. 1996. Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leuko-cytes. J. Leukocyte Biol. 60:658–666.
58. Tersmette, M., R. E. de Goede, B. J. Al, I. N. Winkel, R. A. Gruters, H. T.
Cuypers, H. G. Huisman, and F. Miedema.1988. Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent de-tection of syncytium-inducing isolates in patients with acquired immunode-ficiency syndrome (AIDS) and AIDS-related complex. J. Virol. 62:2026– 2032.
59. Weiss, R. A., and P. R. Clapham. 1996. Hot fusion of HIV. Nature 381: 647–648.
60. Weiss, R. A. 1996. HIV receptors and the pathogenesis of AIDS. Science
272:1885–1886.
61. Zhang, L., Y. Huang, T. He, Y. Cao, and D. D. Ho. 1996. HIV-1 subtype and second receptor use. Nature 383:768.