1. JOURNAL OF VIROLOGY, Aug. 2000, p. 6893–6910 Vol. 74, No. 15
0022-538X/00/$04.00ϩ0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Use of Inhibitors To Evaluate Coreceptor Usage by Simian and
Simian/Human Immunodeficiency Viruses and Human
Immunodeficiency Virus Type 2 in Primary Cells
YI-JUN ZHANG,1 BERNARD LOU,1 RENU B. LAL,2 AGEGNEHU GETTIE,1
PRESTON A. MARX,1,3 AND JOHN P. MOORE1*
Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York 100161; National Center
for Infectious Diseases, DASTLR, Centers for Disease Control and Prevention, Atlanta, Georgia 303332;
and Tulane University Medical Center, New Orleans, Louisiana 704333
Received 21 January 2000/Accepted 2 May 2000
We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immuno-
deficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus
type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is
specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and
AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates
tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human
PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative
coreceptor that is expressed in the cells of some individuals. SIVmac239 and SIVmac251 (from macaques) were
also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors.
The replication in human PBMC of SIVrcm (from a red-capped mangabey), a virus which uses CCR2 but not
CCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for this
virus in primary cells.
Like human immunodeficiency virus type 1 (HIV-1), simian The use of CCR5 by several other SIV strains, including pri-
immunodeficiency viruses (SIV) and human immunodeficiency mary isolates from the natural host, has since been docu-
virus type 2 (HIV-2) use seven-transmembrane receptors as mented (14, 22, 30, 31, 33, 48, 53, 54, 64, 65, 93). CXCR4 usage
coreceptors during the process of virus-cell fusion (reviewed in by SIV strains is, however, very rare, although an example of
references 6, 8, 9, 25, 74, and 87). Usually, this process requires SIV entry via CXCR4 is known, albeit for an isolate obtained
an initial interaction of the viral envelope glycoproteins with from mandrills (SIVmnd) (98). However, the initial reports of
CD4 (48, 107, 114). However, rare examples of CD4-indepen- SIVmac entry via CCR5 concluded that additional coreceptors
dent HIV-1 isolates have been described (29, 47), and several used by SIV may be substitutes for CXCR4 (12, 18, 38). Since
HIV-2 and SIV strains can interact with coreceptors quite then, several seven-transmembrane receptors have been re-
efficiently in the absence of CD4 (14, 30, 33, 35, 65, 89, 97). The
ported to support SIV entry in vitro, often with an efficiency
first coreceptor described for HIV-1 was CXCR4, which serves
comparable to that of CCR5 (3, 22, 31, 32, 38, 93, 96). Argu-
to mediate the entry of the so-called T-cell line-tropic or syn-
cytium-inducing (SI) viruses (39), now designated X4 isolates ably, the most efficient among these SIV coreceptors are the
(7). The major coreceptor for the macrophage-tropic, non- ones variously designated BOB/GPR15 and Bonzo/STRL33/
syncytium-inducing (NSI) viruses, now designated R5 isolates, TYMSTR (3, 22, 31, 62).
was found to be CCR5 (2, 20, 22, 27, 28). A plethora of other The question arises, however, as to whether these other
coreceptors has also been described to function for HIV-1 coreceptors are as important as CCR5 and (for HIV-1 and
entry, especially for SI viruses, at least in the context of core- HIV-2) CXCR4 for viral replication in vivo. There is mounting
ceptor-transfected cells in vitro (5, 19, 20, 22, 27, 31, 32, 38, 40, evidence that many, if not all, of the other coreceptors have
50, 60, 62, 84, 85, 92, 93, 96, 102, 104, 105, 116). only limited, if any, relevance to viral replication in primary
HIV-2 isolates can also use CCR5 and CXCR4 for entry into cells and hence in vivo, except perhaps in specialized tissues
coreceptor-transfected cells in vitro. In general, coreceptor and cell types (13, 31, 43, 70, 81, 86, 101, 102, 117). Care must
usage by HIV-2 is broader than that for HIV-1, in that many always be taken when evaluating viral entry mediated via trans-
seven-transmembrane receptors have been reported to support fected seven-transmembrane receptors (66). We have contin-
HIV-2 entry when transfected into cell lines (10, 14, 22, 35, 45, ued to address this issue here using inhibitors targeted at
48, 52, 68, 77, 80, 89, 106). CCR5 and CXCR4 (117). Our conclusion is that CCR5 is the
The first coreceptor to be identified as supporting SIV entry most important SIVmac coreceptor in primary CD4ϩ T cells
was CCR5, which was shown to function with SIVmac isolates but that an alternative coreceptor(s) may indeed be relevant, at
soon after it was found to be an HIV-1 coreceptor (12, 18, 38). least in cells from some macaques. SIVrcm, however, uses
CCR2 and not CCR5. These observations may be useful in
* Corresponding author. Present address: Weill Medical College of
studies of SIV-infected nonhuman primates as model systems
Cornell University, 1300 York Ave., New York, NY 10021. Phone: for the development of HIV-1 vaccines (23, 57, 73). For the
(212) 746-4462. Fax: (212) 746-8340. E-mail: jpm2003@med.cornell same reason, we have also studied the coreceptor usage of
.edu. selected simian/human immunodeficiency viruses (SHIV).
6893

2. 6894 ZHANG ET AL. J. VIROL.
MATERIALS AND METHODS ing only CD4 (GHOST-CD4 cells) served as controls; they were cultured in the
same medium, except that puromycin was omitted.
Coreceptor inhibitors. The bicyclam AMD3100, a small-molecule inhibitor of GHOST cells (105/ml; 500 ␮l per well) were maintained in 24-well plates for
HIV-1 entry via CXCR4 (26, 37, 56, 100), and TAK-779, a small-molecule 24 h. The medium was then removed, and 200 ␮l of fresh medium was added,
inhibitor of HIV-1 entry via CCR5 (4), were both gifts from Annette Bauer, along with a viral inoculum of 1,000 TCID50. On the next day, residual virus was
Michael Miller, Susan Vice, Bahige Baroudy, and Stuart McCombie (Schering removed and the cells were washed once with 1 ml of medium. A 750-␮l aliquot
Plough Research Institute, Bloomfield, N.J.). Aminooxypentane-RANTES of fresh complete medium containing the selection antibiotics was then added.
(AOP-RANTES), a derivatized CC-chemokine that interacts with CCR5, was At approximately day 5 postinfection, Gag antigen production in 100 ␮l of
provided by Amanda Proudfoot, Serono Pharmaceutical Research Institute, harvested culture supernatant was measured. For a few slowly replicating SIV
Geneva, Switzerland (34, 63, 103, 113). The human chemokines monocyte che- isolates, it was necessary to replenish the cultures and repeat the antigen assay on
day 7 or 10 postinfection. In all cases, the amount of antigen produced in control
motactic peptide (MCP) 1 (MCP-1), MCP-3, and stromal cell-derived factor 1␣
GHOST-CD4 cells was subtracted from the amount produced in coreceptor-
(SDF-1␣) were purchased from Peprotech Inc. (Norwood, Mass.).
transfected GHOST-CD4 cells. Whether this is a sufficient correction for use by
Viral isolates and preparation of virus stocks. The HIV-1 primary isolates
some isolates of the low level of endogenous CXCR4 in GHOST-CD4 cells is
5160 and 5073, derived from individuals with AIDS, have been described previ-
discussed in Results. Attempts were made to quantify CXCR4 expression on the
ously (115), as have two other primary isolates, M6-v3 and P6-v3, obtained from
various coreceptor-transfected GHOST-CD4 cell lines. All the lines do express
an HIV-1-infected mother-child transmission pair (116, 117). All these viruses
CXCR4, but at very low levels that are difficult to quantify accurately by fluo-
have the SI phenotype, except for P6-v3. Six HIV-2 primary isolates have also
rescence-activated cell sorting (FACS). Thus, we could not accurately quantitate
been described elsewhere (41, 80). Three of these (7924A, 77618, and GB122)
the extent to which CXCR4 expression varied among the various lines. This
were isolated from individuals with AIDS, one (7312A) was isolated from an
situation is consistent with the experience of others (Dan Littman, personal
individual with lymphadenopathy, and two (310340 and 310342) were isolated communication).
from blood donors whose clinical conditions were unrecorded (80). The origins Effect of coreceptor-targeted inhibitors on viral replication. Human PBMC
of HIV-1 SF162, DH123, and NL4-3 have been described elsewhere, as have were used with HIV-1, HIV-2, and SIVrcm, rhesus macaque PBMC were used
their coreceptor usage profiles (116, 117). All HIV-1 and HIV-2 isolates were with other SIV isolates, and both human and macaque PBMC were used with
propagated and titrated in phytohemagglutinin-activated human peripheral SHIV. Stimulated PBMC (75 ␮l) were cultured in 96-well plates at 2 ϫ 105 per
blood mononuclear cells (PBMC) before use. well for human cells and 1 ϫ 105 per well for macaque cells. A range of
The SIV strains SIVmac251, SIVmac239, SIVmac251/1390, SIVmac239/5501, concentrations of inhibitors (75 ␮l) was incubated with the cells, in duplicate or
SIVsm (variant SIVsmpbj), and SIVrcm were all provided by Preston Marx and triplicate wells, for 1 h at 37°C before addition of the viral inoculum (100 TCID50
Zhiwei Chen (14, 15). SIVmac251/1390 and SIVmac239/5501 were isolated from in 75 ␮l). The final inhibitor concentrations used, unless otherwise specified,
macaques which progressed to AIDS after infection with SIVmac251 and were as follows: AMD3100, 400, 40, and 4 nM; AOP-RANTES, 40, 4, and 0.4
SIVmac239, respectively (14, 67). SIVrcm was originally isolated from a red- nM; TAK-779, 3.3 ␮M, 330 nM, and 33 nM; and MCP-1 and MCP-3, 400, 40, and
capped mangabey by cocultivation with human PBMC (15). All SIV strains were 4 nM. For each virus tested, five wells without drugs and five wells containing
propagated and titrated in rhesus macaque PBMC, except for SIVrcm, for which only virus served as positive and negative controls for virus production, respec-
human PBMC were used (15). tively. Culture supernatants (200 ␮l) were harvested for measurement of Gag
SHIV strains 89.6, 89.6P, and 89.6PD were obtained from David Montefiori antigen content (in 100 ␮l) by an enzyme-linked immunosorbent assay on days 4,
(90, 91). SHIV strain SF33A was obtained from Cecilia Cheng-Mayer (46), and 7, and 10. Inhibitors were added back each time. Only when sufficient antigen
SHIV strain KU-2 was obtained from Opendra Narayan (51). All SHIV stocks had been produced was the effect of the inhibitors on virus production calcu-
were prepared in macaque PBMC, except for a second stock of 89.6PD, which lated.
was grown in human PBMC for comparison (89.6PD-hu). To determine the specificity of the inhibitors, GHOST-CD4 cells and a core-
Virus replication in PBMC. Human PBMC were isolated from various healthy ceptor were used. The cells were cultured as described above. Briefly, 24 h after
blood donors by Ficoll-Hypaque separation and stimulated for 3 days with the cells were plated, inhibitors in a total volume of 200 ␮l were added to each
phytohemagglutinin (5 ␮g/ml) and interleukin-2 (IL-2; 100 U/ml) (a gift from well of a 24-well plate. AMD3100 was used at 1.2 ␮M, AOP-RANTES was used
Hofmann-La Roche, Inc., Nutley, N.J.). These donors were all homozygous for at 120 nM, and TAK-779 was used at 10 ␮M. After incubation for 1 h at 37°C,
the CCR5 wild-type allele. PBMC from three individuals known to be homozy- a viral inoculum of 1,000 TCID50 was added for overnight incubation. The cells
gous for the CCR5 ⌬32 allele (⌬32-CCR5) were also used. Activated PBMC (2 ϫ were then washed, and 750 ␮l of fresh medium was added. The production of p24
105/well) were cultured in 96-well plates with 150 ␮l of RPMI 1640 medium antigen and the effect of the inhibitors were determined as for the PBMC
containing 10% fetal calf serum and IL-2. Virus inocula (100 or 1,000 50% tissue cultures, except that the supernatants were harvested on days 3, 6, and 10.
culture infective doses [TCID50] in 75 ␮l) were added to duplicate or triplicate
wells. Three wells lacked cells to provide a control for the viral antigen input.
Rhesus macaque PBMC were prepared by similar procedures, except that they RESULTS
were stimulated for 3 days with staphylococcal enterotoxin B (Sigma Chemical
Co., St. Louis, Mo.) at 5 ␮g/ml in RPMI 1640 growth medium containing IL-2 Coreceptor usage by HIV-1, HIV-2, SHIV, and SIV in trans-
(46).
CEMx174 cells in RPMI 1640 growth medium were used at concentrations of fected cells. We assembled a panel of HIV-1, HIV-2, SHIV,
4 ϫ 104/well. Culture supernatants were harvested on days 7 and 11 postinfec- and SIV isolates to study their replication in primary cells. We
tion, and fresh medium was added to replenish the cultures. first determined which coreceptors these viruses could use, at
Viral antigen detection. Virus production was measured using a Gag antigen least under artificial conditions, by measuring their replication
capture enzyme-linked immunosorbent assay. A commercial diagnostic kit (Cel-
lular Products Inc., Buffalo, N.Y.) was used, with modifications, to quantitate
in human GHOST-CD4 cell lines stably transfected with one
HIV-2 and SIV p27 antigen. Briefly, p27 antigen in a 100-␮l volume was captured of several seven-transmembrane receptors (Table 1).
onto wells of a 96-well plate by the adsorbed anti-p27 monoclonal antibody CCR5 and CXCR4 were clearly the coreceptors most widely
provided with the kit. The captured p27 antigen was then detected using the and efficiently used by HIV-1, HIV-2, and SHIV isolates. None
biotin-labeled anti-SIV Gag polyclonal antibodies provided with the kit. To
increase the sensitivity of antigen detection, we used a modified protocol that
of the SIV used CXCR4, a feature that distinguishes SIV from
involved streptavidin-conjugated alkaline phosphatase (DAKO, Carpinteria, HIV-1 and HIV-2 (3, 10, 22, 31, 32, 34, 38, 45, 48, 68, 77, 80,
Calif.) and a chemiluminescent alkaline phosphatase substrate (ELISA-Light; 89, 93, 96, 106), but all the SIV except for SIVrcm used CCR5
Tropix Inc., Bedford, Mass.). The plates were read with a microtiter plate (Table 1). SIVrcm was originally isolated from a red-capped
luminometer (Dynex Technologies Inc.), and the amount of antigen detected was mangabey, a monkey species with a high frequency of a mu-
calculated using a standard antigen curve prepared in each assay. The use of the
chemiluminescent detection system increased the sensitivity of HIV-2 or SIV p27 tated, inactive CCR5 gene, the ⌬24-CCR5 allele (15). SIVrcm
detection by more than 100-fold. HIV-1 p24 antigen was detected as described has a unique pattern of coreceptor usage in that it uses CCR2
previously (109, 111), except that the chemiluminescent detection system was and not CCR5 as its major coreceptor (15). We confirmed this
used. fact and found that SIVrcm can also use Bonzo/STRL33, V28,
Determination of coreceptor usage by viral isolates using GHOST cells ex-
pressing CD4 and coreceptors. Coreceptor usage was determined essentially as and US28 efficiently (Table 1).
described previously (109, 116, 117). Human osteosarcoma (GHOST) cells ex- Consistent with previous reports, some HIV-2 and SIV iso-
pressing CD4 and one of the following coreceptors were obtained from Dan lates were able to enter cells expressing several other corecep-
Littman and Vineet KewalRamani (Skirball Institute, New York University tors (3, 10, 22, 31, 32, 34, 38, 45, 48, 68, 77, 80, 89, 93, 96, 106).
School of Medicine, New York, N.Y.): CCR1, CCR2, CCR3, CCR4, CCR5,
CCR8, CXCR4, BOB, Bonzo, GPR1, APJ, V28, and US28. These cells were
For instance, some SIV isolates were able to use BOB/GPR15
cultured in complete Dulbecco’s minimal essential medium containing G418 (5 and Bonzo/STRL33 efficiently—notably, SIVmac239/5501 (Ta-
␮g/ml), hygromycin (1 ␮g/ml), and puromycin (1 ␮g/ml). GHOST cells express- ble 1). HIV-1 and SHIV isolates of the SI phenotype, i.e.,

3. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6895
TABLE 1. Coreceptor usage by HIV-1, SHIV, HIV-2, and SIV isolates in GHOST-CD4 cells expressing a transfected seven-transmembrane,
G-protein-coupled receptor
Inoculum Replication in the presence of the following receptora:
Viral isolate
(TCID50) CCR1 CCR2 CCR3 CCR4 CCR5 CCR8 CXCR4 BOB Bonzo GPR1 V28 APJ US28
HIV-1 P6-v3 1,000 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ
HIV-1 P6-v3 1,000 Ϫ Ϫ Ϫ ϩϩϩ ϩ ϩϩϩ Ϫ ϩϩϩ Ϫ ϩ ϩ Ϫ
HIV-1 5073 1,000 ϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ ϩ ϩ ϩ
HIV-1 5160 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ ϩ ϩ ϩ
HIV-1 NL4-3 1,000 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ
SHIV 89.6PD 500 Ϫ Ϫ ϩϩϩ Ϫ ϩϩϩϩϩ ϩϩϩ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩϩ ϩϩ ϩϩϩ
SHIV 89.6PD-hu 500 Ϫ Ϫ ϩϩ Ϫ ϩϩϩϩϩ ϩϩ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ ϩϩ
SHIV KU-2 500 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ ϩϩ
SHIV SF33A 500 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩ Ϫ
HIV-2 310340 1,000 Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ
HIV-2 310342 1,000 Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ
HIV-2 7312A 1,000 Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Ϫ
HIV-2 GB122 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩϩ
HIV-2 77618 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩ
HIV-2 7924A 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩϩϩ ϩϩϩ
SIVrcm 100 Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ ϩϩϩϩ Ϫ ϩϩϩϩϩ
SIVrcm 500 Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ ϩϩϩϩϩ Ϫ ϩϩϩϩϩ
SIVmac239 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ ϩϩ ϩϩϩ ϩ Ϫ ϩϩ Ϫ
SIVmac251 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Ϫ
SIVmac239/5501 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ ϩϩϩϩ ϩϩϩϩ ϩϩϩ Ϫ ϩϩϩ Ϫ
SIVmac251/1390 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Ϫ
SIVsmpbj 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ
a
Ability to replicate in GHOST-CD4 cells expressing the seven-transmembrane, G-protein-coupled receptor indicated. The extent of replication (Gag antigen
production) is recorded as follows: Ϫ, Ͻ0.1 ng/ml; ϩ, 0.1 to 1 ng/ml; ϩϩ, 1 to 5 ng/ml; ϩϩϩ, 5 to 20 ng/ml; ϩϩϩϩ, 20 to 100 ng/ml; and ϩϩϩϩϩ, Ͼ100 ng/ml. For
each CXCR4-utilizing virus, the amount of p24 antigen produced in the parental GHOST-CD4 cells was subtracted from the amount produced in the coreceptor-
expressing GHOST-CD4 cells. Whether this is always a sufficient correction for the use of the CXCR4 that is endogenous to GHOST-CD4 cells is discussed in the text.
viruses that could use CXCR4 efficiently, were usually able to fected with other coreceptors was only rarely comparable to
replicate in GHOST-CD4 cells expressing various coreceptors the replication of the same viruses in CCR5- or CXCR4-ex-
(Table 1). Any differences in coreceptor usage patterns be- pressing cells. Examples of relatively efficient replication in-
tween this and previous reports (80, 115) probably arises from clude that of HIV-1 P6-v3 and M6-v3 in Bonzo-transfected
the use of different GHOST-CD4 cell clones and/or isolates cells, HIV-2 7924A in APJ- or US28-transfected cells, and
with a different passage history. SHIV 89.6PD in V28-transfected cells (Table 1). Whether
The broad tropism of SI viruses in coreceptor-transfected
cell lines is well known (5, 19, 20, 22, 27, 31, 32, 38, 40, 50, 60,
62, 84, 85, 92, 93, 96, 104, 105, 116). However, the growth of
HIV-1, HIV-2, and SHIV isolates in GHOST-CD4 cells trans- TABLE 3. Replication of HIV-2 isolates in PBMC from wild-type
and ⌬32-CCR5 donors and in CEMx174 cells
Virus (ng of p27 antigen/ml) produced in the following
TABLE 2. Replication of HIV-1 and SHIV isolates in PBMC from cells on the indicated day postinfectiona:
wild-type and ⌬32-CCR5 donors and in CEMx174 cells
HIV-2
TCID50 Wild-type ⌬32-CCR5
Virus (ng of p24 or p27 antigen/ml) produced in the isolate CEMx174
PBMC PBMC
following cells on the indicated day postinfectiona:
HIV-1 or 7 11 7 11 7 11
Wild-type ⌬32-CCR5
SHIV isolate CEMx174
PBMC PBMC 310340 100 1,250 1,509 0 0 0 0
1,000 1,329 1,717 0 0 0 0
7 11 7 11 7 11
SF162 15.2 13.4 0 0 0 0 7312A 100 2.3 10.3 0 0.7 0.4 0.8
P6-v3 10.2 14.4 0 0 0 0 1,000 15 69.8 0.2 5.5 1.5 8.9
NL4-3 2.6 10.1 5.7 14.3 8 17.3
DH123 14.1 11.6 13.2 6.2 7.3 15 GB122 100 58 87 85 130 121 114
M6-v3 12.6 13.9 6.5 15.5 4.1 18.2 1,000 82.9 105 108 133 116 135
5073 7 13.2 5.6 13.8 2.9 17.3
89.6PD 85.2 134.2 53.3 154.3 41.5 181.9 77618 100 37.7 58.2 42.6 159 167 186
KU-2 37 102 15.7 70 94 134 1,000 125 182 113 195 168 237
SF33A 73 131 72 107 51.4 155
7924A 100 88 141 78 141 29.9 89
a
The inoculum was 1,000 TCID50, but an identical pattern of data was found 1,000 84 68 91 99 59 161
at 100 TCID50 (data not shown). PBMC were from donors A (wild type) and 1
a
(⌬32-CCR5). PBMC were from donors B (wild type) and 1 (⌬32-CCR5).

4. 6896 ZHANG ET AL. J. VIROL.
TABLE 4. Replication of SIV isolates in PBMC from wild-type and validate the use of the ⌬32-CCR5 cells for subsequent studies
⌬32-CCR5 donors and in CEMx174 cells of HIV-2 and SIV replication. HIV-1 SF162 and P6-v3 also
Virus (ng of p24 or p27 antigen/ml) produced in failed to replicate in CEMx174 cells (Table 2). In contrast, the
the following cells on the indicated day X4 HIV-1 clone NL4-3 and the multitropic HIV-1 isolates
postinfectiona: DH123, M6-v3, and 5073 all replicated in both wild-type and
SIV
isolate
TCID50
Wild-type ⌬32-CCR5 ⌬32-CCR5 PBMC (donor 1) as well as in CEMx174 cells. This
CEMx174 finding was also true of the three SHIV tested, 89.6PD, KU-2,
PBMC PBMC
and SF33A (Table 2). Hence, all seven of these HIV-1 and
7 11 7 11 7 11
SHIV isolates can use a coreceptor other than CCR5 to enter
SIVmac239 100 69.9 414.8 0.4 4.1 146 444 PBMC and CEMx174 cells, consistent with their replication
500 373 289 5.2 1.9 209 555 patterns in the various GHOST-CD4 cell lines (Table 1).
1,000 303 381 8 5.6 755 470 One of the five HIV-2 isolates tested, 310340, failed to
replicate in ⌬32-CCR5 PBMC from donor 1 and in CEMx174
SIVmac239/5501 100 394 366 4.5 7 923 455
cells (Table 3). This virus was also unable to use any coreceptor
SIVmac251 100 121 606 6.8 6.8 47 402 other than CCR5 to enter GHOST-CD4 cells (Table 1). An-
other HIV-2 isolate, 7312A, grew very poorly, but detectably,
SIVmac251/1390 100 22 299 0.9 0.5 20.9 442 in ⌬32-CCR5 PBMC and CEMx174 cells; the extent of 7312A
production in ⌬32-CCR5 PBMC was 5 to 10% that in wild-type
SIVrcm 100 2,536 2,413 266 678 12 4 PBMC (Table 3). Of note is that HIV-2 7312A could use
1,000 7,550 2,975 5,016 2,707 79 46 BOB/GPR15 and Bonzo/STRL33 inefficiently; the amount of
a
PMBC were from donors C (wild type) and 1 (⌬32-CCR5). p24 produced from GHOST-CD4 cells expressing BOB or
Bonzo was approximately 5% that derived from GHOST-CD4
cells expressing CCR5 (Table 1; also data not shown). The
virus entry into the various GHOST-CD4 cell lines actually remaining three HIV-2 isolates, GB122, 77618, and 7924A, all
occurs via the transfected coreceptor is discussed below. replicated to comparable extents in the wild-type and ⌬32-
Replication of HIV-1, HIV-2, SHIV, and SIV isolates in CCR5 PBMC and replicated efficiently in CEMx174 cells (Ta-
PBMC from donors expressing or not expressing CCR5 and in ble 3). These results are consistent with the ability of these
CEMx174 cells. The above experiments showed that many of three isolates to use CXCR4 and other coreceptors (Table 1).
the test isolates can apparently use multiple coreceptors to en- The replication of SIVmac239 and SIVmac251 in human
ter transfected human cell lines. To gain insights into the impor- PBMC from an individual homozygous for the ⌬32-CCR5 al-
tance of CCR5 for viral replication in primary cells, we compared lele has been taken as strong evidence that these viruses can
the abilities of the isolates to replicate in human PBMC from use a coreceptor other than CCR5 to enter primary, CD4ϩ
either donors who had wild-type CCR5 alleles or donors who cells (18). We sought to confirm this. In the first experiment,
were homozygous for the ⌬32-CCR5 mutation and so did not the extent of SIVmac239, SIVmac251, SIVmac239/5501, and
express functional CCR5 proteins (21, 61, 95). We also used SIVmac251/1390 replication in ⌬32-CCR5 PBMC from donor 1
the CEMx174 human B/T-hybrid line because these cells can was never more than 5% and usually was less than 1% the
support high-level SIV replication. CEMx174 cells are CXCR4ϩ replication of the same viruses in wild-type PBMC (Table 4). A
but CCR5Ϫ (18, 58, 110) and strongly express the SIVmac251 second experiment also included ⌬32-CCR5 PBMC from two
and SIVmac239 coreceptor BOB/GPR15 (22, 31, 86). more donors, 2 and 3. There was, again, little or no production
Among the six HIV-1 isolates tested, the R5, NSI viruses of SIVmac251 and SIVmac239 in ⌬32-CCR5 PBMC from donor
SF162 and P6-v3 were unable to replicate in the ⌬32-CCR5 1 (Table 5). However, both isolates replicated well in PBMC
PBMC from donor 1 (Table 2). Similar results were obtained from ⌬32-CCR5 donors 2 and 3, although antigen production
with PBMC from two other ⌬32-CCR5 donors (data not from SIVmac251 in cells from donor 2 was lower than that from
shown; see also Table 5). These observations are consistent typical CCR5 wild-type donors (Table 5). Thus, PBMC from
with the known dependence of these viruses on CCR5, so they some, but not all, human donors must express a coreceptor
TABLE 5. Replication of SIV and HIV-1 isolates in PBMC from wild-type and ⌬32-CCR5 donors and in CEMx174 cells in two
different experiments
Virus (ng of p24 or p27 antigen/ml) produced in the following cells on the indicated day postinfectiona:
SIV or HIV-1 Wild-type ⌬32-CCR5 ⌬32-CCR5 Wild-type ⌬32-CCR5
TCID50
isolate PBMC (D) PBMC (1) PBMC (2) PBMC (E) PBMC (3)
7 11 7 11 7 11 7 11 7 11
SIVmac239 100 10.9 87.7 0.2 0.5 3 44.2 34 383 8.7 201
500 27.4 112 0.5 0.8 7.7 53.4 134 460 112 298
1000 37.6 105 1.6 3.4 7.6 52 ND ND ND ND
SIVmac251 100 4.4 146 0.5 1.4 0.6 9.9 85 481 46 309
SIVrcm 100 154 707 65 710 55 637 ND ND 294 358
SF162 100 18.1 16.8 0 0 0 0 0 0 0 0
a
Two experiments are recorded: one that compared donors D, 1, and 2 and the other that compared donors E and 3 (donor designations in parentheses after cell
types). ND, not done.

5. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6897
FIG. 1. Testing of the specificity of coreceptor-targeted inhibitors. The replication of the test viruses in GHOST-CD4 cells expressing the coreceptor indicated in
the presence and absence of AMD3100 (1.2 ␮M), AOP-RANTES (AOP-R) (120 nM), TAK-779 (10 ␮M), or SDF-1␣ (500 nM) was evaluated. The extent to which
replication was inhibited by each agent was recorded.

6. 6898 ZHANG ET AL. J. VIROL.
FIG. 2. Effects of coreceptor-targeted inhibitors on HIV-1 replication in human PBMC. The replication of the HIV-1 isolates P6-v3 and M6-v3 (a) and 5073 and
5160 (b) in human PBMC in the presence and absence of AOP-RANTES (AOP-R) (40 nM [left bar], 4 nM [middle bar], and 0.4 nM [right bar]), TAK-779 (3.3 ␮M,
330 nM, and 33 nM), or AMD3100 (400 nM, 40 nM, and 4 nM) or with combinations of AMD3100 and either AOP-RANTES or TAK-779 was evaluated. When
combinations were used, the concentration of each agent was the same as when the agents were used alone. The extent to which replication was inhibited by each agent
or combination was recorded. The coreceptors that can be used by each isolate in GHOST-CD4 cells are indicated below the isolate designation in parentheses.

7. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6899
other than CCR5 that can be used with reasonable efficiency by AMD3100. As expected, the efficient replication of SHIV
members of the SIVmac group of viruses. 89.6PD in GHOST-CD4 cells expressing CXCR4 was blocked
SIVrcm replicated efficiently in wild-type and ⌬32-CCR5 by AMD3100 (Fig. 1a). However, AMD3100 also prevented
PBMC (Tables 4 and 5), consistent with its lack of dependence the inefficient replication of SHIV 89.6PD in GHOST-CD4
on CCR5 for entry into PBMC (15). SIVmac239 and SIVmac251 cells expressing either CCR3, V28, APJ, US28, or CCR8 and
also replicated efficiently in CEMx174 cells, as found previ- significantly inhibited the limited replication of HIV-2 7924A
ously (18), but SIVrcm replication was inefficient in these cells in GHOST-CD4 cells expressing V28, APJ, or US28 (Fig. 1b;
(Table 4). Thus, neither the major coreceptor for SIVrcm, also data not shown). However, AMD3100 had no detectable
CCR2, nor the minor ones Bonzo, US28, and V28 are ex- effect on SIVmac239 entry into GHOST-CD4 cells expressing
pressed in CEMx174 cells. BOB, Bonzo, GPR1, or APJ or on SIVrcm entry into GHOST-
Evaluation of the specificity of coreceptor-targeted inhibi- CD4 cells expressing CCR2, Bonzo, V28, or US28 (Fig. 1a).
tors. Coreceptor-targeted inhibitors are useful for evaluating The replication of HIV-1 P6-v3 in GHOST-CD4 cells express-
which coreceptors are relevant for viral entry into PBMC. ing Bonzo was also unaffected by AMD3100 (data not shown).
One suitable inhibitor of entry via CXCR4 is the bicyclam Thus, entry via V28, US28, and APJ in GHOST-CD4 cell lines
AMD3100 (26, 37, 56, 100). Inhibitors of entry via CCR5 are can apparently be either sensitive or insensitive to AMD3100,
the TAK-779 molecule (4) or the CC-chemokine derivative depending upon the test virus.
AOP-RANTES (62, 103, 113). The specificity of these There are two possible explanations for the unusual pattern
agents is an important issue. Previous studies have found of inhibition shown by AMD3100. One is that AMD3100 is
that AMD3100 is specific for CXCR4 (26, 37, 56, 100) and broadly reactive with multiple coreceptors but that certain
that TAK-779 can interact with both CCR5 and CCR2 (4). viruses, particularly SIV, can still interact with some of these
Although RANTES fully activates all of its receptors, AOP- coreceptors even in the presence of AMD3100. The other is
RANTES is able to do this only for CCR5; it has half the that the apparent cross-reactivity of AMD3100 is an artifact of
activity of RANTES for CCR3 and is very inefficient at acti- the presence of low levels of endogenous CXCR4 in corecep-
vating CCR1 (79, 88). AOP-RANTES is therefore a moderate tor-transfected GHOST-CD4 cells (109, 110). To address this
inhibitor of CCR3-mediated HIV-1 infection, compared to its possibility, we tested the sensitivity of SHIV 89.6PD and HIV-
effect on entry mediated by CCR5 (34). 2 7924A replication in several GHOST-CD4 cell lines to SDF-
To confirm these specificities, we determined whether 1␣. In all cases, whenever AMD3100 inhibited the replication
AMD3100, TAK-779, and AOP-RANTES could inhibit viral of the test viruses, so did SDF-1␣ (Fig. 1b; also data not shown).
entry into GHOST-CD4 cells transfected with other corecep- Since SDF-1␣ is specific for CXCR4 (6, 8, 9, 71, 84), these
tors by using viruses that were broadly tropic in these cells. For findings strongly suggest that the entry of SHIV 89.6PD and
each test virus, AMD3100 was used at 1.2 ␮M, AOP-RANTES HIV-2 7924A into several coreceptor-transfected GHOST-
was used at 120 nM, and TAK-779 was used at 10 ␮M (Fig. 1). CD4 cell lines occurs via endogenous CXCR4. This coreceptor
SHIV 89.6PD replication in GHOST-CD4 cells expressing may well be expressed to different levels in different individual
CCR5 was sensitive to both TAK-779 and AOP-RANTES but GHOST-CD4 cell lines, although we were unable to accurately
not to AMD3100, as expected (Fig. 1a). We also found that quantitate this expression by FACS.
AOP-RANTES, but not TAK-779, inhibited SHIV 89.6PD The inhibitory effect of AMD3100 in coreceptor-transfected
entry into GHOST-CD4 cells expressing CCR3, consistent GHOST-CD4 cell lines is, therefore, most probably explained
with an interaction between AOP-RANTES and CCR3, a by its antagonism of viral entry via endogenous CXCR4. The
known RANTES receptor (data not shown). However, neither coreceptor usage information presented in Table 1 should be
TAK-779 nor AOP-RANTES had any significant effect on interpreted with this caveat in mind. Overall, we can find no evi-
SHIV 89.6PD replication in GHOST-CD4 cells expressing dence that AMD3100 is anything other than specific for CXCR4.
CXCR4, CCR8, V28, US28, or APJ (Fig. 1a; also data not Effect of coreceptor-targeted inhibitors on HIV-1, SHIV, and
shown). Both TAK-779 and AOP-RANTES inhibited SIV- HIV-2 replication in PBMC. The replication of each test virus
mac239 entry into GHOST-CD4 cells expressing CCR5, but the in mitogen-stimulated PBMC in the presence and absence of
entry of this virus into GHOST-CD4 cells expressing either AMD3100, TAK-779, or AOP-RANTES was evaluated. Com-
BOB, Bonzo, GPR1, or APJ was unaffected by TAK-779 or binations of AMD3100 with TAK-779 and AMD3100 with
AOP-RANTES (Fig. 1a). The entry of SIVrcm into GHOST- AOP-RANTES were also tested. Each inhibitor, alone and in
CD4 cells expressing CCR2 was completely inhibited by TAK- combination, was used at three different concentrations: 400,
779, whereas AOP-RANTES had only a marginal effect on 40, and 4 nM for AMD3100; 3.3 ␮M, 330 nM, and 33 nM for
entry via CCR2 (Fig. 1a). SIVrcm replication in GHOST-CD4 TAK-779; and 40, 4, and 0.4 nM for AOP-RANTES. Prelim-
cells expressing Bonzo, V28, or US28 was, however, insensitive inary experiments had indicated that the effects of the inhibi-
to TAK-779 or AOP-RANTES (Fig. 1a), as was HIV-2 7924A tors usually titrated out over these ranges. Human PBMC from
replication in cells expressing V28, APJ, or US28 (data not CCR5 wild-type donors were used in experiments with HIV-1
shown). Neither TAK-779 nor AOP-RANTES inhibited the and HIV-2 isolates and SIVrcm; rhesus macaque PBMC were
replication of HIV-1 P6-v3 in GHOST-CD4 cells expressing used with other SIV; and both human and macaque PBMC
Bonzo (data not shown). were used with SHIV.
Taken together, these data suggest that AOP-RANTES can Four HIV-1 primary isolates that could use multiple core-
block viral entry via CCR5 and CCR3 and that TAK-779 in- ceptors, as determined by the GHOST-CD4 cell assays (Table
hibits entry via CCR5 and CCR2. The latter result is consistent 1), were evaluated with human PBMC (Fig. 2). P6-v3, a virus
with the report that TAK-779 binds to both CCR2 and CCR5 able to use CCR5 and Bonzo, was completely inhibited by both
but not to CCR1, CCR3, or CCR4 (4). TAK-779 and AOP- TAK-779 and AOP-RANTES but not by AMD3100 (Fig. 2a).
RANTES have no effect on viral replication in GHOST-CD4 The more broadly tropic virus M6-v3 was partially sensitive to
cells expressing any one of the eight coreceptors that we were each of the three inhibitors, but its replication was fully
able to evaluate: CXCR4, CCR8, V28, US28, APJ, BOB, blocked by combinations of either TAK-779 or AOP-RANTES
Bonzo, or GPR1. with AMD3100 (Fig. 2a). Isolates 5073 and 5060 were able to
A less clear-cut pattern of inhibition was observed with replicate in several different coreceptor-expressing GHOST-

8. 6900 ZHANG ET AL. J. VIROL.
FIG. 3. Effects of coreceptor-targeted inhibitors on SHIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SHIV
isolates evaluated were 89.6PD in macaque and human PBMC (a) and KU-2 and SF33A in human PBMC (b).

9. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6901
CD4 cell lines, including GHOST-CD4 cells expressing CXCR4, 5b). Taken together with the insensitivity of HIV-2 7924A to
but their replication was completely inhibited in PBMC by TAK-779 and AOP-RANTES (Fig. 4b), the limited or nonex-
AMD3100 (Fig. 2b). Thus, none of the tested HIV-1 isolates istent effect of AMD3100 and SDF-1␣ on HIV-2 7924A rep-
appeared to enter PBMC from the donors included in these lication suggests that this virus uses an undefined coreceptor
studies via a coreceptor other than CCR5 or CXCR4. other than CXCR4 to enter PBMC. An alternative explanation
Results similar to those obtained with the broadly tropic is that HIV-2 7924A uses CXCR4 in a highly unusual, inhibi-
HIV-1 isolates were found when SHIV were evaluated (Fig. 3). tor-insensitive manner. If this is so, how this virus uses CXCR4
Thus, SHIV 89.6PD replication in either macaque or human must be cell type dependent, since we determined that the IC50
PBMC was fully inhibited by AMD3100, while TAK-779 and of AMD3100 for this virus in GHOST-CD4 cells was 0.47 ␮M.
AOP-RANTES had no effect (Fig. 3a). The same was true of This value contrasts markedly with the IC50s of 2.1 to 34 ␮M
SHIV KU-2 and SHIV SF33A in human PBMC (Fig. 3b) and for the same virus in PBMC.
also of SHIV 89.6 and SHIV 89.6P (data not shown). The Effect of coreceptor inhibitors on SIV replication in ma-
paramount, and most probably exclusive, coreceptor for all of caque PBMC. To evaluate the inhibitor sensitivities of SIV
these SHIV in PBMC therefore appears to be CXCR4. This isolates, we used macaque PBMC. In cells from the first donor
finding was unexpected for SHIV 89.6, 89.6P, and 89.6PD, con- macaque tested, SIVmac251, SIVmac239, SIVmac251/1390, and
sidering that these viruses efficiently use CCR5 in transfected SIVmac239/5501 were all inhibited by both TAK-779 and AOP-
GHOST-CD4 cells (Table 1 and Fig. 1a; also data not shown). RANTES to an extent that was complete, or virtually so (Ͼ95%),
Among the HIV-2 isolates tested, 310342 and 7312A were whereas AMD3100 had no effect (Fig. 6a and b; also data not
both completely inhibited by TAK-779 and AOP-RANTES but shown). Thus, these SIV isolates all use CCR5, and only CCR5,
were insensitive to AMD3100 (Fig. 4a). Although HIV-2 7312A to enter PBMC from this macaque donor. However, there are
can use BOB and Bonzo, to a limited extent, in GHOST-CD4 issues of donor cell dependency to consider (see below).
cells (Table 1), this property does not allow the virus to evade Because SIVrcm uses CCR2 but neither CCR5 nor CXCR4
CCR5-directed inhibitors in PBMC (Fig. 4a). HIV-2 77618 and for entry (Table 1), we tested chemokine ligands of CCR2 for
GB122 were almost completely (Ͼ95%) blocked by AMD3100, their abilities to inhibit SIVrcm replication in human PBMC. Of
whereas TAK-779 and AOP-RANTES had no effect on these these, MCP-1 almost completely inhibited SIVrcm replication,
viruses (Fig. 4b; also data not shown). All of these HIV-2 whereas MCP-3 had only a limited effect (Fig. 6c). TAK-779
isolates probably use only CCR5 or CXCR4 to enter PBMC. was also an effective inhibitor of SIVrcm replication in human
An exception was, however, noted with HIV-2 7924A. This PBMC (Fig. 6c), just as it was in GHOST-CD4 cells expressing
virus was partially sensitive to AMD3100, but the extent of CCR2 (Fig. 1a). However, AOP-RANTES had no effect on
inhibition did not exceed 30% even at the highest AMD3100 SIVrcm replication in human PBMC (data not shown). This
concentration, 400 nM (Fig. 4b). HIV-2 7924A was completely virus appears to make truly exclusive use of CCR2 as a core-
insensitive to TAK-779 or AOP-RANTES, and combining ceptor in primary human PBMC.
these agents with AMD3100 did not increase the extent of The effect of TAK-779 on SIVmac239 replication in macaque
inhibition caused by AMD3100 alone (Fig. 4b). PBMC is donor dependent. We showed above that there is a
HIV-2 isolate 7924A has an unusual pattern of sensitivity to donor dependency in the ability of SIVmac239 and SIVmac251
coreceptor-targeted inhibitors. The insensitivity of HIV-2 to replicate in human PBMC from ⌬32-CCR5 homozygous
7924A to AMD3100 is unusual, since this virus can use individuals (Table 4). There is also a donor dependency in the
CXCR4, and perhaps only CXCR4, to enter GHOST-CD4 potency with which CCR5-targeted inhibitors inhibit SIV-
cells (Table 1 and Fig. 1b). Usually, 50% inhibitory concentra- mac239 replication in macaque PBMC. Thus, the extent to
tions (IC50s) of AMD3100 against viruses that use CXCR4 in which TAK-779, at 3.3 ␮M, inhibited SIVmac239 replication
PBMC are 4 to 40 nM (Fig. 2b, 3a and b, and 4b; also data not varied from Ͼ99% to Ͻ50% in PBMC from four different
shown). To evaluate whether the insensitivity of HIV-2 7924A macaques (Fig. 7a). The IC50s of TAK-779 ranged from 240
to AMD3100 in PBMC was donor dependent, we tested much nM (macaque 3) to 12.6 ␮M (macaque 1), a 60-fold variation.
higher AMD3100 concentrations in cells from four CCR5 wild- However, at the very high concentration of 33 ␮M, TAK-779
type donors (Fig. 5a). Donor-to-donor variation in the potency completely inhibited SIVmac239 replication in all four donors
of AMD3100 was significant, with IC50s ranging from 2.1 ␮M (Fig. 7a). Similar results were obtained with SIVmac251 in the
(donor 1) to 34 ␮M (donor 2). However, if sufficient AMD3100 two donors tested; the IC50s were 0.18 ␮M (donor 3) and 20
(40 ␮M) was used, inhibition of HIV-2 7924A was complete in ␮M (donor 1) (Fig. 7a).
cells from three of the four donors. Whether at a concentration There was less variation in the potency of TAK-779 against
as high as 40 ␮M AMD3100 remains specific for CXCR4 is not HIV-1 replication in human PBMC. For instance, HIV-1 P6-v3
known, although no overt toxicity was observed. was inhibited by TAK-779 in PBMC from four donors at IC50s
We also tested AMD3100 (4 ␮M) against HIV-2 7924A in ranging from 15 nM to 24 nM (Fig. 7b). This result suggests
PBMC from a ⌬32-CCR5 homozygous donor (donor 1). For that major variations in inhibition potency are not an inherent
the first 4 days of culturing, AMD3100 at 4 ␮M completely feature of TAK-779.
suppressed HIV-2 7924A replication; however, by day 7, the When the inhibitor sensitivities of SIVmac239 and SIVmac251
virus had broken through, and the extent of inhibition was were evaluated with CEMx174 cells, both viruses were insen-
negligible thereafter. In contrast, HIV-1 5160 was completely sitive (Ͻ5% inhibition) to AMD3100 (400 nM), TAK-779 (3.3
inhibited by 4 ␮M AMD3100 throughout the duration of cul- ␮M), or AOP-RANTES (40 nM), alone or in combination
turing (data not shown). (data not shown). In contrast, HIV-1 NL4-3 replication in
To gain more insight into whether HIV-2 7924A could use these cells was completely blocked by AMD3100 but not by
CXCR4 for entry into PBMC, we determined its sensitivity to TAK-779 or AOP-RANTES (data not shown). Thus, what-
SDF-1␣ in cells from the same four CCR5 wild-type donors as ever coreceptor(s) SIVmac239 and SIVmac251 use to enter
those used in the AMD3100 experiment. Even at the highest CEMx174 cells, it is not CCR2, CCR3, CCR5, or CXCR4.
concentration tested (400 nM), SDF-1␣ did not inhibit HIV-2 Whether this is the same coreceptor that these viruses can use
7924A replication in PBMC from any of the four donors, to enter human or macaque PBMC from some donors is not
whereas HIV-1 NL4-3 replication was efficiently blocked (Fig. yet known.

10. FIG. 4. Effects of coreceptor-targeted inhibitors on HIV-2 replication in human PBMC. The experimental design was like that described in the legend to Fig. 2.
The HIV-2 isolates evaluated were 310342 and 7312A (a) and 77618 and 7924A (b).
6902

11. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6903
FIG. 5. Effects of coreceptor-targeted inhibitors on HIV-2 isolate 7924A in PBMC from different donors. The replication of HIV-2 7924A in PBMC from four
different human donors in the presence of AMD3100 at 40 ␮M, 4 ␮M, 400 nM, and 40 nM (a) and SDF-1␣ at 400 nM, 40 nM, 4 nM, and 0.4 nM (b) was evaluated.
HIV-1 NL4-3 was also tested with SDF-1␣. In each case, replication was measured after 7 and 10 days. IC50s of AMD3100 were calculated and are shown in panel
a. The data shown were obtained on day 10, but values from day 7 were similar.

12. 6904 ZHANG ET AL. J. VIROL.
FIG. 6. Effects of coreceptor-targeted inhibitors on SIV replication in PBMC. The experimental design was like that described in the legend to Fig. 2. The SIV
isolates evaluated were SIVmac251 and SIVmac239 in macaque PBMC (a), SIVmac251/1390 and SIVmac239/5501 in macaque PBMC (b), and SIVrcm in human PBMC
(c). MCP-1 and MCP-3 were used at 400, 40, and 4 nM (left to right); TAK-779 was used at 3.3 ␮M, 330 nM, and 33 nM.

13. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6905
FIG. 6—Continued.
DISCUSSION accurately quantitate such variation by FACS. Whatever the
Primate lentiviruses can use about 12 different seven-trans- explanation, ambiguities can arise when the coreceptor usage
membrane receptors as coreceptors in transfected cell lines. of CXCR4-tropic viruses is determined with transfected
However, questions have been raised as to whether corecep- GHOST-CD4 cell lines. Many, if not all, of the “positives” for
tors other than CCR5 and CXCR4 are relevant for viral entry use of coreceptors other than CCR5 and CXCR4 by CXCR4-
into primary cells and, hence, for viral replication in vivo (31, tropic viruses in GHOST-CD4 cell lines (Table 1) may simply
43, 70, 86, 101, 116, 117). This issue affects the development of reflect entry via endogenous CXCR4 and not the transfected
antiviral drugs aimed at coreceptors. Must multiple corecep- coreceptor. This caveat may also apply to other studies that
tors be targeted, or just CCR5 and CXCR4 (117)? Does the have used these cell lines. A similar conclusion was recently
ability of SIV and SHIV to use multiple coreceptors in vitro reached by others (59).
influence the interpretation of vaccine experiments with pri- We previously concluded from an inhibitor-based study that
mates (73)? coreceptors other than CCR5 and CXCR4 made, at most, only
We addressed these issues by using coreceptor-targeted in- a limited contribution to HIV-1 replication in PBMC (117).
hibitors to block viral replication in primary PBMC, focusing Our inhibitor studies are now strengthened by the recent avail-
here on HIV-2 and SIV isolates. As inhibitors, we used ability of TAK-779 (4). This CCR5-targeted inhibitor does not
AMD3100 for CXCR4 and TAK-779 and AOP-RANTES for inhibit viral entry via CCR3, whereas AOP-RANTES can do
CCR5. These agents are not completely specific: TAK-779 and so, albeit inefficiently compared to its effect on CCR5-medi-
AOP-RANTES also inhibit viral entry via CCR2 and via ated entry (34). Since TAK-779, by itself, is able to block the
CCR3, respectively. However, we could find no evidence that replication in PBMC of all of the R5, NSI HIV-1 and HIV-2
AMD3100 is anything other than specific for CXCR4, as found isolates that we tested, CCR3 is not relevant to their entry. Any
previously with other assay systems and test viruses (26, 37, 56, possible use of CCR2 that might be masked by TAK-779 is not
100). supported by the complete inhibition of the same isolates by
The low-level entry of viruses such as SHIV 89.6PD and AOP-RANTES. This chemokine derivative does not block vi-
HIV-2 7924A into GHOST-CD4 cells expressing V28, US28, ral entry via CCR2, at least for SIVrcm, which is the only truly
APJ, and others actually occurs via endogenous CXCR4 and CCR2-tropic virus yet identified (15). Another advantage of
not via the transfected coreceptor, since it is inhibited by both TAK-779 is that it avoids the potential complications of AOP-
SDF-1␣ and AMD3100. Of note is that SHIV 89.6PD and RANTES-induced enhancement of attachment and entry of
HIV-2 7924A use CXCR4 very efficiently, so they may be able X4 HIV-1 isolates (44, 108). However, we did not observe
to enter coreceptor-transfected GHOST-CD4 cells that ex- infectivity enhancement with human or macaque PBMC at the
press very low levels of CXCR4; the levels of expression of this AOP-RANTES concentrations tested in this study. Overall,
coreceptor may also vary slightly among different GHOST- the use of TAK-779 reinforces our previous conclusion about
CD4 clones, making some transfected cell lines particularly the paramount role of CCR5 and CXCR4 in HIV-1 replication
susceptible to viruses that use CXCR4, although we could not in PBMC (116). This is not to say that other coreceptors are

14. 6906 ZHANG ET AL. J. VIROL.
FIG. 7. Donor-dependent variation in the effects of coreceptor-targeted inhibitors in PBMC. (a) SIVmac239 replication in PBMC from four different macaques was
evaluated in the presence of TAK-779 at 33 ␮M, 3.3 ␮M, 330 nM, and 33 nM. SIVmac251 was similarly evaluated with cells from two donors. (b) HIV-1 P6-v3 replication
in PBMC from four different human donors was evaluated in the presence of TAK-779 at 3.3 ␮M, 330 nM, 33 nM, and 3 nM. In each case, replication was measured
after 7 and 10 days, and IC50s of the inhibitor were calculated. The data shown were obtained on day 10, but values from day 7 were similar.

15. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6907
completely irrelevant; Bonzo/STRL33 can be used by rare negligible. However, in cells from a second such individual, the
HIV-1 isolates for entry into a minor subset of PBMC in a virus replicated fairly efficiently, as observed previously (18).
donor-dependent manner (102), and CCR3 and CCR8 are po- Furthermore, there was considerable variation in the potency
tential coreceptors expressed on some T-cell subsets (94, 118). with which TAK-779 inhibited the replication of SIVmac239 in
The SHIV isolates that we evaluated—89.6, 89.6P, 89.6PD, PBMC from different macaques. This result might be ac-
SF33A, and KU-2—all exclusively used CXCR4 in human and counted for by the use of an additional coreceptor that is
macaque PBMC; AMD3100 was sufficient to completely inhibit expressed in PBMC from only a subset of macaques or that is
their replication, while neither TAK-779 nor AOP-RANTES expressed in cells from all macaques but at different levels that
had any effect. Thus, although HIV-1 89.6 can enter trans- are sometimes below a threshold needed for infection. The
fected cells via several coreceptors, including CCR5 (27), the expression of both CCR5 and Bonzo/STRL33 varies from do-
SHIV derived from it use only CXCR4 to enter PBMC. Of nor to donor, in both humans and macaques, to an extent that
note is that SHIV 89.6, SHIV 89.6P, and SHIV 89.6PD very can affect infection efficiency (102, 107). The ability of SIV-
efficiently enter GHOST-CD4 cells expressing CCR5 (Table 1; mac239 to use a coreceptor other than CCR5, perhaps Bonzo/
also data not shown). Thus, these viruses can use CCR5 for STRL33, in an animal-dependent manner might influence the
entry, at least in CCR5-transfected cells, but CXCR4 is pre- highly variable rates at which different infected macaques
ferred in primary cells. Why this should be the case and progress to disease and death (23, 57, 73). However, at least for
whether it matters for transmission and pathogenesis studies SIVmne, CCR5 usage is maintained throughout the course of
with these viruses in macaques are open questions. disease progression in infected macaques (53). This is also true
We also conclude that, for most HIV-2 strains, CCR5 and/or of SIVmac239 and SIVmac251 (14).
CXCR4 are the principal coreceptors relevant to the replica- One coreceptor used efficiently by SIVmac239 in vitro is
tion of these strains in PBMC. Thus, TAK-779, AOP-RAN- BOB/GPR15 (22, 38). Pohlmann et al. have, however, shown
¨
TES, and AMD3100, alone or in combination, completely or that this coreceptor has no relevance to SIVmac239 replication
very substantially inhibited the replication of almost all of our in vivo, at least in some macaques (86). An unknown, alterna-
test viruses. HIV-2 isolate 7924A is an apparent exception. The tive coreceptor(s) also mediates the AMD3100-, TAK-779-,
replication of this broadly tropic virus in PBMC was inhibited and AOP-RANTES-insensitive entry of SIVmac239 into
only by very high concentrations of AMD3100 and was com- CEMx174 cells; this coreceptor cannot, therefore, be CCR2,
pletely insensitive to SDF-1␣, TAK-779, or AOP-RANTES. CCR3, CCR5, or CXCR4. It is not known whether this is the
One possibility is that HIV-2 7924A is able to use an alterna- same coreceptor as the one used by SIVmac239 to enter human
tive coreceptor to enter human PBMC, perhaps the CXCR5 or macaque PBMC from some donors.
receptor reported recently to function with some HIV-2 iso- We could not distinguish SIVmac239 from the closely related
lates but not with HIV-1 or SIV isolates (52). Alternatively, SIVmac251 in terms of their sensitivity to coreceptor inhibitors.
HIV-2 7924A may use CXCR4 in a manner that is relatively Although SIVmac251 but not SIVmac239 replicates efficiently in
insensitive to AMD3100. The latter explanation would be con- macrophages, there is no correlation between the coreceptor
sistent with the observation that very high concentrations of usage profiles of these viruses in transfected cells and their
AMD3100 do completely inhibit the replication of HIV-2 tropism for primary cells (53, 75, 76, 86). There is also no
7924A, although there may be concerns about the specificity of relationship between the in vitro tropisms of SIVmac strains
AMD3100 for CXCR4 at such concentrations. Escape mutants and their abilities to be transmitted to uninfected animals (36,
of HIV-1 NL4-3 that continue to use CXCR4, but in a drug- 71, 72). We have not yet performed coreceptor inhibitor stud-
insensitive manner, are known to emerge in response to selec- ies with these viruses and purified macrophages and CD4ϩ T
tion pressure from AMD3100 and SDF-1␣ (24, 99). It has been cells from macaques, as opposed to unfractionated PBMC.
suggested that CXCR4 can exist in different isoforms on dif- SIVrcm clearly uses CCR2 as its primary coreceptor (15), in
ferent cell types (69); this property might be one explanation a manner that we have shown is sensitive to TAK-779. The
for why AMD3100 is a potent inhibitor of HIV-2 7924A in ability of SIVrcm to enter GHOST-CD4 cells expressing US28
GHOST-CD4 cells expressing CXCR4 (IC50 ϭ 0.47 ␮M) but and V28 in vitro is likely to be of limited relevance to the
can be such a weak one in PBMC (IC50 ϭ 2.1 to 34 ␮M, de- replication of this virus in red-capped mangabeys.
pending upon the donor). Additional studies of HIV-2 7924A Overall, we conclude that there is a greater complexity to
are warranted. coreceptor usage by SIV strains in PBMC than there is for
Our conclusions for SIVmac isolates are more complicated. HIV-1 and HIV-2, for which CCR5 and CXCR4 are usually
The CCR5 proteins from multiple primate species can function the paramount coreceptors. An unknown coreceptor(s) can
as viral coreceptors (55, 78), and our inhibitor studies are con- perhaps be used by SIVmac239 and HIV-2 7924A to enter
sistent with an important role of CCR5 in SIVmac entry into PBMC, at least from some macaque and human donors. In-
primary cells. One aspect of coreceptor usage that distinguishes volvement of the same coreceptor in the entry of both
SIV from HIV-2 isolates is the inability of almost all SIV to use SIVmac239 and HIV-2 7924A might conceivably have rele-
CXCR4. This property contrasts with the efficient use of CXCR4 vance to cross-species viral transmission and the evolution of
by many HIV-2 isolates. In this sense, HIV-2 more closely HIV-2 from SIVsm (16, 17, 41, 42, 44, 49).
resembles HIV-1 than it does SIV, an unexpected finding given
the genetic relationships among these virus families and the
evolution of HIV-2 from SIVsm (16, 17, 41, 42, 44, 49). The ACKNOWLEDGMENTS
minimal use of CXCR4 by SIV strains is mirrored by that of
HIV-1 isolates from genetic subtype C (1, 11, 82, 83, 112), We thank Annette Bauer, Michael Miller, Susan Vice, Bahige Ba-
although SI primary viruses from this subtype are known (111). roudy, and Stuart McCombie for AMD3100 and TAK-779; Amanda
Proudfoot, Robin Offord, and Brigitte Dufour for AOP-RANTES;
Although CCR5 is important and CXCR4 is unimportant Zhiwei Chen for SIV isolates; David Montefiori, Cecilia Cheng-Mayer,
for SIVmac entry, we found indications that SIVmac239 could and Opendra Narayan for SHIV isolates; Dan Littman and Vineet
use a coreceptor other than CCR5 to enter PBMC from some KewalRamani for GHOST cells; and James Hoxie and Nelson Michael
human and macaque donors. Thus, in PBMC from one ⌬32- for preferring hard liquor to blood. We appreciate helpful comments
CCR5 homozygous human donor, SIVmac239 replication was by Amanda Proudfoot and Bob/GPR15 Doms.

16. 6908 ZHANG ET AL. J. VIROL.
This study was supported by NIH grant RO1 AI41420 and by the Donfield, D. Vlahov, R. Kaslow, A. Saah, C. Rinaldo, R. Detels, Hemophilia
Pediatric AIDS Foundation, of which J.P.M. is an Elizabeth Glaser Growth and Development Study, Multicenter AIDS Cohort Study, Multi-
Scientist. center Hemophilia Cohort Study, San Francisco City Cohort, ALIVE
Study, and S. J. O’Brien. 1996. Genetic restriction of HIV-1 infection and
progression to AIDS by a deletion of the CKR5 structural allele. Science
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