coreceptor usage

JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

Aug. 2000, p. 6893–6910 Vol. 74, No. 15

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Use of Inhibitors To Evaluate Coreceptor Usage by Simian andSimian/Human Immunodeficiency Viruses and Human

Immunodeficiency Virus Type 2 in Primary CellsYI-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 Centerfor 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 virustype 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which isspecific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, andAMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolatestested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in humanPBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternativecoreceptor that is expressed in the cells of some individuals. SIVmac239 and SIVmac251 (from macaques) werealso 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 notCCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for thisvirus in primary cells.

Like human immunodeficiency virus type 1 (HIV-1), simianimmunodeficiency viruses (SIV) and human immunodeficiencyvirus type 2 (HIV-2) use seven-transmembrane receptors ascoreceptors during the process of virus-cell fusion (reviewed inreferences 6, 8, 9, 25, 74, and 87). Usually, this process requiresan initial interaction of the viral envelope glycoproteins withCD4 (48, 107, 114). However, rare examples of CD4-indepen-dent HIV-1 isolates have been described (29, 47), and severalHIV-2 and SIV strains can interact with coreceptors quiteefficiently in the absence of CD4 (14, 30, 33, 35, 65, 89, 97). Thefirst coreceptor described for HIV-1 was CXCR4, which servesto mediate the entry of the so-called T-cell line-tropic or syn-cytium-inducing (SI) viruses (39), now designated X4 isolates(7). The major coreceptor for the macrophage-tropic, non-syncytium-inducing (NSI) viruses, now designated R5 isolates,was found to be CCR5 (2, 20, 22, 27, 28). A plethora of othercoreceptors has also been described to function for HIV-1entry, especially for SI viruses, at least in the context of core-ceptor-transfected cells in vitro (5, 19, 20, 22, 27, 31, 32, 38, 40,50, 60, 62, 84, 85, 92, 93, 96, 102, 104, 105, 116).

HIV-2 isolates can also use CCR5 and CXCR4 for entry intocoreceptor-transfected cells in vitro. In general, coreceptorusage by HIV-2 is broader than that for HIV-1, in that manyseven-transmembrane receptors have been reported to supportHIV-2 entry when transfected into cell lines (10, 14, 22, 35, 45,48, 52, 68, 77, 80, 89, 106).

The first coreceptor to be identified as supporting SIV entrywas CCR5, which was shown to function with SIVmac isolatessoon after it was found to be an HIV-1 coreceptor (12, 18, 38).

The use of CCR5 by several other SIV strains, including pri-mary isolates from the natural host, has since been docu-mented (14, 22, 30, 31, 33, 48, 53, 54, 64, 65, 93). CXCR4 usageby SIV strains is, however, very rare, although an example ofSIV entry via CXCR4 is known, albeit for an isolate obtainedfrom mandrills (SIVmnd) (98). However, the initial reports ofSIVmac entry via CCR5 concluded that additional coreceptorsused by SIV may be substitutes for CXCR4 (12, 18, 38). Sincethen, several seven-transmembrane receptors have been re-ported to support SIV entry in vitro, often with an efficiencycomparable to that of CCR5 (3, 22, 31, 32, 38, 93, 96). Argu-ably, the most efficient among these SIV coreceptors are theones variously designated BOB/GPR15 and Bonzo/STRL33/TYMSTR (3, 22, 31, 62).

The question arises, however, as to whether these othercoreceptors are as important as CCR5 and (for HIV-1 andHIV-2) CXCR4 for viral replication in vivo. There is mountingevidence that many, if not all, of the other coreceptors haveonly limited, if any, relevance to viral replication in primarycells and hence in vivo, except perhaps in specialized tissuesand cell types (13, 31, 43, 70, 81, 86, 101, 102, 117). Care mustalways be taken when evaluating viral entry mediated via trans-fected seven-transmembrane receptors (66). We have contin-ued to address this issue here using inhibitors targeted atCCR5 and CXCR4 (117). Our conclusion is that CCR5 is themost important SIVmac coreceptor in primary CD41 T cellsbut that an alternative coreceptor(s) may indeed be relevant, atleast in cells from some macaques. SIVrcm, however, usesCCR2 and not CCR5. These observations may be useful instudies of SIV-infected nonhuman primates as model systemsfor the development of HIV-1 vaccines (23, 57, 73). For thesame reason, we have also studied the coreceptor usage ofselected simian/human immunodeficiency viruses (SHIV).

* Corresponding author. Present address: Weill Medical College ofCornell University, 1300 York Ave., New York, NY 10021. Phone:(212) 746-4462. Fax: (212) 746-8340. E-mail: [email protected].

6893

MATERIALS AND METHODS

Coreceptor inhibitors. The bicyclam AMD3100, a small-molecule inhibitor ofHIV-1 entry via CXCR4 (26, 37, 56, 100), and TAK-779, a small-moleculeinhibitor of HIV-1 entry via CCR5 (4), were both gifts from Annette Bauer,Michael Miller, Susan Vice, Bahige Baroudy, and Stuart McCombie (ScheringPlough Research Institute, Bloomfield, N.J.). Aminooxypentane-RANTES(AOP-RANTES), a derivatized CC-chemokine that interacts with CCR5, wasprovided by Amanda Proudfoot, Serono Pharmaceutical Research Institute,Geneva, Switzerland (34, 63, 103, 113). The human chemokines monocyte che-motactic peptide (MCP) 1 (MCP-1), MCP-3, and stromal cell-derived factor 1a(SDF-1a) were purchased from Peprotech Inc. (Norwood, Mass.).

Viral isolates and preparation of virus stocks. The HIV-1 primary isolates5160 and 5073, derived from individuals with AIDS, have been described previ-ously (115), as have two other primary isolates, M6-v3 and P6-v3, obtained froman HIV-1-infected mother-child transmission pair (116, 117). All these viruseshave the SI phenotype, except for P6-v3. Six HIV-2 primary isolates have alsobeen described elsewhere (41, 80). Three of these (7924A, 77618, and GB122)were isolated from individuals with AIDS, one (7312A) was isolated from anindividual with lymphadenopathy, and two (310340 and 310342) were isolatedfrom blood donors whose clinical conditions were unrecorded (80). The originsof HIV-1 SF162, DH123, and NL4-3 have been described elsewhere, as havetheir coreceptor usage profiles (116, 117). All HIV-1 and HIV-2 isolates werepropagated and titrated in phytohemagglutinin-activated human peripheralblood mononuclear cells (PBMC) before use.

The SIV strains SIVmac251, SIVmac239, SIVmac251/1390, SIVmac239/5501,SIVsm (variant SIVsmpbj), and SIVrcm were all provided by Preston Marx andZhiwei Chen (14, 15). SIVmac251/1390 and SIVmac239/5501 were isolated frommacaques which progressed to AIDS after infection with SIVmac251 andSIVmac239, respectively (14, 67). SIVrcm was originally isolated from a red-capped mangabey by cocultivation with human PBMC (15). All SIV strains werepropagated and titrated in rhesus macaque PBMC, except for SIVrcm, for whichhuman PBMC were used (15).

SHIV strains 89.6, 89.6P, and 89.6PD were obtained from David Montefiori(90, 91). SHIV strain SF33A was obtained from Cecilia Cheng-Mayer (46), andSHIV strain KU-2 was obtained from Opendra Narayan (51). All SHIV stockswere prepared in macaque PBMC, except for a second stock of 89.6PD, whichwas grown in human PBMC for comparison (89.6PD-hu).

Virus replication in PBMC. Human PBMC were isolated from various healthyblood donors by Ficoll-Hypaque separation and stimulated for 3 days withphytohemagglutinin (5 mg/ml) and interleukin-2 (IL-2; 100 U/ml) (a gift fromHofmann-La Roche, Inc., Nutley, N.J.). These donors were all homozygous forthe CCR5 wild-type allele. PBMC from three individuals known to be homozy-gous for the CCR5 D32 allele (D32-CCR5) were also used. Activated PBMC (2 3105/well) were cultured in 96-well plates with 150 ml of RPMI 1640 mediumcontaining 10% fetal calf serum and IL-2. Virus inocula (100 or 1,000 50% tissueculture infective doses [TCID50] in 75 ml) were added to duplicate or triplicatewells. Three wells lacked cells to provide a control for the viral antigen input.

Rhesus macaque PBMC were prepared by similar procedures, except that theywere stimulated for 3 days with staphylococcal enterotoxin B (Sigma ChemicalCo., St. Louis, Mo.) at 5 mg/ml in RPMI 1640 growth medium containing IL-2(46).

CEMx174 cells in RPMI 1640 growth medium were used at concentrations of4 3 104/well. Culture supernatants were harvested on days 7 and 11 postinfec-tion, and fresh medium was added to replenish the cultures.

Viral antigen detection. Virus production was measured using a Gag antigencapture enzyme-linked immunosorbent assay. A commercial diagnostic kit (Cel-lular Products Inc., Buffalo, N.Y.) was used, with modifications, to quantitateHIV-2 and SIV p27 antigen. Briefly, p27 antigen in a 100-ml volume was capturedonto wells of a 96-well plate by the adsorbed anti-p27 monoclonal antibodyprovided with the kit. The captured p27 antigen was then detected using thebiotin-labeled anti-SIV Gag polyclonal antibodies provided with the kit. Toincrease the sensitivity of antigen detection, we used a modified protocol thatinvolved streptavidin-conjugated alkaline phosphatase (DAKO, Carpinteria,Calif.) and a chemiluminescent alkaline phosphatase substrate (ELISA-Light;Tropix Inc., Bedford, Mass.). The plates were read with a microtiter plateluminometer (Dynex Technologies Inc.), and the amount of antigen detected wascalculated using a standard antigen curve prepared in each assay. The use of thechemiluminescent detection system increased the sensitivity of HIV-2 or SIV p27detection by more than 100-fold. HIV-1 p24 antigen was detected as describedpreviously (109, 111), except that the chemiluminescent detection system wasused.

Determination of coreceptor usage by viral isolates using GHOST cells ex-pressing CD4 and coreceptors. Coreceptor usage was determined essentially asdescribed previously (109, 116, 117). Human osteosarcoma (GHOST) cells ex-pressing CD4 and one of the following coreceptors were obtained from DanLittman and Vineet KewalRamani (Skirball Institute, New York UniversitySchool of Medicine, New York, N.Y.): CCR1, CCR2, CCR3, CCR4, CCR5,CCR8, CXCR4, BOB, Bonzo, GPR1, APJ, V28, and US28. These cells werecultured in complete Dulbecco’s minimal essential medium containing G418 (5mg/ml), hygromycin (1 mg/ml), and puromycin (1 mg/ml). GHOST cells express-

ing only CD4 (GHOST-CD4 cells) served as controls; they were cultured in thesame medium, except that puromycin was omitted.

GHOST cells (105/ml; 500 ml per well) were maintained in 24-well plates for24 h. The medium was then removed, and 200 ml of fresh medium was added,along with a viral inoculum of 1,000 TCID50. On the next day, residual virus wasremoved and the cells were washed once with 1 ml of medium. A 750-ml aliquotof fresh complete medium containing the selection antibiotics was then added.At approximately day 5 postinfection, Gag antigen production in 100 ml ofharvested culture supernatant was measured. For a few slowly replicating SIVisolates, it was necessary to replenish the cultures and repeat the antigen assay onday 7 or 10 postinfection. In all cases, the amount of antigen produced in controlGHOST-CD4 cells was subtracted from the amount produced in coreceptor-transfected GHOST-CD4 cells. Whether this is a sufficient correction for use bysome isolates of the low level of endogenous CXCR4 in GHOST-CD4 cells isdiscussed in Results. Attempts were made to quantify CXCR4 expression on thevarious coreceptor-transfected GHOST-CD4 cell lines. All the lines do expressCXCR4, but at very low levels that are difficult to quantify accurately by fluo-rescence-activated cell sorting (FACS). Thus, we could not accurately quantitatethe extent to which CXCR4 expression varied among the various lines. Thissituation is consistent with the experience of others (Dan Littman, personalcommunication).

Effect of coreceptor-targeted inhibitors on viral replication. Human PBMCwere used with HIV-1, HIV-2, and SIVrcm, rhesus macaque PBMC were usedwith other SIV isolates, and both human and macaque PBMC were used withSHIV. Stimulated PBMC (75 ml) were cultured in 96-well plates at 2 3 105 perwell for human cells and 1 3 105 per well for macaque cells. A range ofconcentrations of inhibitors (75 ml) was incubated with the cells, in duplicate ortriplicate wells, for 1 h at 37°C before addition of the viral inoculum (100 TCID50in 75 ml). The final inhibitor concentrations used, unless otherwise specified,were as follows: AMD3100, 400, 40, and 4 nM; AOP-RANTES, 40, 4, and 0.4nM; TAK-779, 3.3 mM, 330 nM, and 33 nM; and MCP-1 and MCP-3, 400, 40, and4 nM. For each virus tested, five wells without drugs and five wells containingonly virus served as positive and negative controls for virus production, respec-tively. Culture supernatants (200 ml) were harvested for measurement of Gagantigen content (in 100 ml) by an enzyme-linked immunosorbent assay on days 4,7, and 10. Inhibitors were added back each time. Only when sufficient antigenhad been produced was the effect of the inhibitors on virus production calcu-lated.

To determine the specificity of the inhibitors, GHOST-CD4 cells and a core-ceptor were used. The cells were cultured as described above. Briefly, 24 h afterthe cells were plated, inhibitors in a total volume of 200 ml were added to eachwell of a 24-well plate. AMD3100 was used at 1.2 mM, AOP-RANTES was usedat 120 nM, and TAK-779 was used at 10 mM. After incubation for 1 h at 37°C,a viral inoculum of 1,000 TCID50 was added for overnight incubation. The cellswere then washed, and 750 ml of fresh medium was added. The production of p24antigen and the effect of the inhibitors were determined as for the PBMCcultures, except that the supernatants were harvested on days 3, 6, and 10.

RESULTS

Coreceptor usage by HIV-1, HIV-2, SHIV, and SIV in trans-fected cells. We assembled a panel of HIV-1, HIV-2, SHIV,and SIV isolates to study their replication in primary cells. Wefirst determined which coreceptors these viruses could use, atleast under artificial conditions, by measuring their replicationin human GHOST-CD4 cell lines stably transfected with oneof several seven-transmembrane receptors (Table 1).

CCR5 and CXCR4 were clearly the coreceptors most widelyand efficiently used by HIV-1, HIV-2, and SHIV isolates. Noneof the SIV used CXCR4, a feature that distinguishes SIV fromHIV-1 and HIV-2 (3, 10, 22, 31, 32, 34, 38, 45, 48, 68, 77, 80,89, 93, 96, 106), but all the SIV except for SIVrcm used CCR5(Table 1). SIVrcm was originally isolated from a red-cappedmangabey, a monkey species with a high frequency of a mu-tated, inactive CCR5 gene, the D24-CCR5 allele (15). SIVrcmhas a unique pattern of coreceptor usage in that it uses CCR2and not CCR5 as its major coreceptor (15). We confirmed thisfact and found that SIVrcm can also use Bonzo/STRL33, V28,and US28 efficiently (Table 1).

Consistent with previous reports, some HIV-2 and SIV iso-lates were able to enter cells expressing several other corecep-tors (3, 10, 22, 31, 32, 34, 38, 45, 48, 68, 77, 80, 89, 93, 96, 106).For instance, some SIV isolates were able to use BOB/GPR15and Bonzo/STRL33 efficiently—notably, SIVmac239/5501 (Ta-ble 1). HIV-1 and SHIV isolates of the SI phenotype, i.e.,

6894 ZHANG ET AL. J. VIROL.

viruses that could use CXCR4 efficiently, were usually able toreplicate in GHOST-CD4 cells expressing various coreceptors(Table 1). Any differences in coreceptor usage patterns be-tween this and previous reports (80, 115) probably arises fromthe use of different GHOST-CD4 cell clones and/or isolateswith a different passage history.

The broad tropism of SI viruses in coreceptor-transfectedcell 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 ofHIV-1, HIV-2, and SHIV isolates in GHOST-CD4 cells trans-

fected with other coreceptors was only rarely comparable tothe replication of the same viruses in CCR5- or CXCR4-ex-pressing cells. Examples of relatively efficient replication in-clude that of HIV-1 P6-v3 and M6-v3 in Bonzo-transfectedcells, HIV-2 7924A in APJ- or US28-transfected cells, andSHIV 89.6PD in V28-transfected cells (Table 1). Whether

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

Viral isolate Inoculum(TCID50)

Replication in the presence of the following receptora:

CCR1 CCR2 CCR3 CCR4 CCR5 CCR8 CXCR4 BOB Bonzo GPR1 V28 APJ US28

HIV-1 P6-v3 1,000 2 2 2 2 111 2 2 2 111 2 2 2 2HIV-1 P6-v3 1,000 2 2 2 111 1 111 2 111 2 1 1 2HIV-1 5073 1,000 1 2 2 2 2 2 11 2 2 2 1 1 1HIV-1 5160 1,000 11 2 2 2 2 2 11 2 2 2 1 1 1HIV-1 NL4-3 1,000 2 2 2 2 2 2 111 2 2 2 2 2 2

SHIV 89.6PD 500 2 2 111 2 11111 111 11111 2 2 2 1111 11 111SHIV 89.6PD-hu 500 2 2 11 2 11111 11 11111 2 2 2 111 11 11SHIV KU-2 500 2 2 2 2 2 2 1111 2 2 2 111 11 11SHIV SF33A 500 2 2 2 2 2 2 111 2 2 2 11 1 2

HIV-2 310340 1,000 2 2 2 11111 2 2 2 2 2 2 2 2HIV-2 310342 1,000 2 2 2 111 2 2 2 2 2 2HIV-2 7312A 1,000 2 2 2 111 2 2 1 1 2 2 2 2HIV-2 GB122 1,000 11 2 2 2 2 2 1111 2 2 2 11 11 11HIV-2 77618 1,000 11 2 2 2 2 2 111 2 2 2 11 11 1HIV-2 7924A 1,000 11 2 2 2 2 2 11111 2 2 2 11 1111 111

SIVrcm 100 2 11111 2 2 2 2 2 2 111 2 1111 2 11111SIVrcm 500 2 11111 2 2 2 2 2 2 11111 2 11111 2 11111SIVmac239 500 2 2 2 2 1111 2 2 11 111 1 2 11 2SIVmac251 500 2 2 2 2 111 2 2 1 1 2 2 2 2SIVmac239/5501 500 2 2 2 2 1111 2 2 1111 1111 111 2 111 2SIVmac251/1390 500 2 2 2 2 111 2 2 1 1 2 2 2 2SIVsmpbj 500 2 2 2 2 1111 2 2 2 2 2 2 2 2

a Ability to replicate in GHOST-CD4 cells expressing the seven-transmembrane, G-protein-coupled receptor indicated. The extent of replication (Gag antigenproduction) is recorded as follows: 2, ,0.1 ng/ml; 1, 0.1 to 1 ng/ml; 11, 1 to 5 ng/ml; 111, 5 to 20 ng/ml; 1111, 20 to 100 ng/ml; and 11111, .100 ng/ml. Foreach 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.

TABLE 2. Replication of HIV-1 and SHIV isolates in PBMC fromwild-type and D32-CCR5 donors and in CEMx174 cells

HIV-1 orSHIV isolate

Virus (ng of p24 or p27 antigen/ml) produced in thefollowing cells on the indicated day postinfectiona:

Wild-typePBMC

D32-CCR5PBMC CEMx174

7 11 7 11 7 11

SF162 15.2 13.4 0 0 0 0P6-v3 10.2 14.4 0 0 0 0NL4-3 2.6 10.1 5.7 14.3 8 17.3DH123 14.1 11.6 13.2 6.2 7.3 15M6-v3 12.6 13.9 6.5 15.5 4.1 18.25073 7 13.2 5.6 13.8 2.9 17.389.6PD 85.2 134.2 53.3 154.3 41.5 181.9KU-2 37 102 15.7 70 94 134SF33A 73 131 72 107 51.4 155

a The inoculum was 1,000 TCID50, but an identical pattern of data was foundat 100 TCID50 (data not shown). PBMC were from donors A (wild type) and 1(D32-CCR5).

TABLE 3. Replication of HIV-2 isolates in PBMC from wild-typeand D32-CCR5 donors and in CEMx174 cells

HIV-2isolate TCID50

Virus (ng of p27 antigen/ml) produced in the followingcells on the indicated day postinfectiona:

Wild-typePBMC


7 11 7 11 7 11

310340 100 1,250 1,509 0 0 0 01,000 1,329 1,717 0 0 0 0

7312A 100 2.3 10.3 0 0.7 0.4 0.81,000 15 69.8 0.2 5.5 1.5 8.9

GB122 100 58 87 85 130 121 1141,000 82.9 105 108 133 116 135

77618 100 37.7 58.2 42.6 159 167 1861,000 125 182 113 195 168 237

7924A 100 88 141 78 141 29.9 891,000 84 68 91 99 59 161

a PBMC were from donors B (wild type) and 1 (D32-CCR5).

VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6895

virus entry into the various GHOST-CD4 cell lines actuallyoccurs via the transfected coreceptor is discussed below.

Replication of HIV-1, HIV-2, SHIV, and SIV isolates inPBMC from donors expressing or not expressing CCR5 and inCEMx174 cells. The above experiments showed that many ofthe test isolates can apparently use multiple coreceptors to en-ter transfected human cell lines. To gain insights into the impor-tance of CCR5 for viral replication in primary cells, we comparedthe abilities of the isolates to replicate in human PBMC fromeither donors who had wild-type CCR5 alleles or donors whowere homozygous for the D32-CCR5 mutation and so did notexpress functional CCR5 proteins (21, 61, 95). We also usedthe CEMx174 human B/T-hybrid line because these cells cansupport high-level SIV replication. CEMx174 cells are CXCR41

but CCR52 (18, 58, 110) and strongly express the SIVmac251and SIVmac239 coreceptor BOB/GPR15 (22, 31, 86).

Among the six HIV-1 isolates tested, the R5, NSI virusesSF162 and P6-v3 were unable to replicate in the D32-CCR5PBMC from donor 1 (Table 2). Similar results were obtainedwith PBMC from two other D32-CCR5 donors (data notshown; see also Table 5). These observations are consistentwith the known dependence of these viruses on CCR5, so they

validate the use of the D32-CCR5 cells for subsequent studiesof HIV-2 and SIV replication. HIV-1 SF162 and P6-v3 alsofailed to replicate in CEMx174 cells (Table 2). In contrast, theX4 HIV-1 clone NL4-3 and the multitropic HIV-1 isolatesDH123, M6-v3, and 5073 all replicated in both wild-type andD32-CCR5 PBMC (donor 1) as well as in CEMx174 cells. Thisfinding was also true of the three SHIV tested, 89.6PD, KU-2,and SF33A (Table 2). Hence, all seven of these HIV-1 andSHIV isolates can use a coreceptor other than CCR5 to enterPBMC and CEMx174 cells, consistent with their replicationpatterns in the various GHOST-CD4 cell lines (Table 1).

One of the five HIV-2 isolates tested, 310340, failed toreplicate in D32-CCR5 PBMC from donor 1 and in CEMx174cells (Table 3). This virus was also unable to use any coreceptorother than CCR5 to enter GHOST-CD4 cells (Table 1). An-other HIV-2 isolate, 7312A, grew very poorly, but detectably,in D32-CCR5 PBMC and CEMx174 cells; the extent of 7312Aproduction in D32-CCR5 PBMC was 5 to 10% that in wild-typePBMC (Table 3). Of note is that HIV-2 7312A could useBOB/GPR15 and Bonzo/STRL33 inefficiently; the amount ofp24 produced from GHOST-CD4 cells expressing BOB orBonzo was approximately 5% that derived from GHOST-CD4cells expressing CCR5 (Table 1; also data not shown). Theremaining three HIV-2 isolates, GB122, 77618, and 7924A, allreplicated to comparable extents in the wild-type and D32-CCR5 PBMC and replicated efficiently in CEMx174 cells (Ta-ble 3). These results are consistent with the ability of thesethree isolates to use CXCR4 and other coreceptors (Table 1).

The replication of SIVmac239 and SIVmac251 in humanPBMC from an individual homozygous for the D32-CCR5 al-lele has been taken as strong evidence that these viruses canuse a coreceptor other than CCR5 to enter primary, CD41

cells (18). We sought to confirm this. In the first experiment,the extent of SIVmac239, SIVmac251, SIVmac239/5501, andSIVmac251/1390 replication in D32-CCR5 PBMC from donor 1was never more than 5% and usually was less than 1% thereplication of the same viruses in wild-type PBMC (Table 4). Asecond experiment also included D32-CCR5 PBMC from twomore donors, 2 and 3. There was, again, little or no productionof SIVmac251 and SIVmac239 in D32-CCR5 PBMC from donor1 (Table 5). However, both isolates replicated well in PBMCfrom D32-CCR5 donors 2 and 3, although antigen productionfrom SIVmac251 in cells from donor 2 was lower than that fromtypical CCR5 wild-type donors (Table 5). Thus, PBMC fromsome, but not all, human donors must express a coreceptor

TABLE 4. Replication of SIV isolates in PBMC from wild-type andD32-CCR5 donors and in CEMx174 cells

SIVisolate TCID50

Virus (ng of p24 or p27 antigen/ml) produced inthe following cells on the indicated day

postinfectiona:

Wild-typePBMC


7 11 7 11 7 11

SIVmac239 100 69.9 414.8 0.4 4.1 146 444500 373 289 5.2 1.9 209 555

1,000 303 381 8 5.6 755 470

SIVmac239/5501 100 394 366 4.5 7 923 455

SIVmac251 100 121 606 6.8 6.8 47 402

SIVmac251/1390 100 22 299 0.9 0.5 20.9 442

SIVrcm 100 2,536 2,413 266 678 12 41,000 7,550 2,975 5,016 2,707 79 46

a PMBC were from donors C (wild type) and 1 (D32-CCR5).

TABLE 5. Replication of SIV and HIV-1 isolates in PBMC from wild-type and D32-CCR5 donors and in CEMx174 cells in twodifferent experiments

SIV or HIV-1isolate TCID50

Virus (ng of p24 or p27 antigen/ml) produced in the following cells on the indicated day postinfectiona:

Wild-typePBMC (D)

D32-CCR5PBMC (1)

D32-CCR5PBMC (2)

Wild-typePBMC (E)

D32-CCR5PBMC (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 201500 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 celltypes). ND, not done.


FIG. 1. Testing of the specificity of coreceptor-targeted inhibitors. The replication of the test viruses in GHOST-CD4 cells expressing the coreceptor indicated inthe presence and absence of AMD3100 (1.2 mM), AOP-RANTES (AOP-R) (120 nM), TAK-779 (10 mM), or SDF-1a (500 nM) was evaluated. The extent to whichreplication was inhibited by each agent was recorded.


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 and5160 (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 mM,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. Whencombinations 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 agentor combination was recorded. The coreceptors that can be used by each isolate in GHOST-CD4 cells are indicated below the isolate designation in parentheses.


other than CCR5 that can be used with reasonable efficiency bymembers of the SIVmac group of viruses.

SIVrcm replicated efficiently in wild-type and D32-CCR5PBMC (Tables 4 and 5), consistent with its lack of dependenceon CCR5 for entry into PBMC (15). SIVmac239 and SIVmac251also replicated efficiently in CEMx174 cells, as found previ-ously (18), but SIVrcm replication was inefficient in these cells(Table 4). Thus, neither the major coreceptor for SIVrcm,CCR2, nor the minor ones Bonzo, US28, and V28 are ex-pressed in CEMx174 cells.

Evaluation of the specificity of coreceptor-targeted inhibi-tors. Coreceptor-targeted inhibitors are useful for evaluatingwhich coreceptors are relevant for viral entry into PBMC.One suitable inhibitor of entry via CXCR4 is the bicyclamAMD3100 (26, 37, 56, 100). Inhibitors of entry via CCR5 arethe TAK-779 molecule (4) or the CC-chemokine derivativeAOP-RANTES (62, 103, 113). The specificity of theseagents is an important issue. Previous studies have foundthat AMD3100 is specific for CXCR4 (26, 37, 56, 100) andthat TAK-779 can interact with both CCR5 and CCR2 (4).Although RANTES fully activates all of its receptors, AOP-RANTES is able to do this only for CCR5; it has half theactivity of RANTES for CCR3 and is very inefficient at acti-vating CCR1 (79, 88). AOP-RANTES is therefore a moderateinhibitor of CCR3-mediated HIV-1 infection, compared to itseffect on entry mediated by CCR5 (34).

To confirm these specificities, we determined whetherAMD3100, TAK-779, and AOP-RANTES could inhibit viralentry into GHOST-CD4 cells transfected with other corecep-tors by using viruses that were broadly tropic in these cells. Foreach test virus, AMD3100 was used at 1.2 mM, AOP-RANTESwas used at 120 nM, and TAK-779 was used at 10 mM (Fig. 1).

SHIV 89.6PD replication in GHOST-CD4 cells expressingCCR5 was sensitive to both TAK-779 and AOP-RANTES butnot to AMD3100, as expected (Fig. 1a). We also found thatAOP-RANTES, but not TAK-779, inhibited SHIV 89.6PDentry into GHOST-CD4 cells expressing CCR3, consistentwith an interaction between AOP-RANTES and CCR3, aknown RANTES receptor (data not shown). However, neitherTAK-779 nor AOP-RANTES had any significant effect onSHIV 89.6PD replication in GHOST-CD4 cells expressingCXCR4, CCR8, V28, US28, or APJ (Fig. 1a; also data notshown). Both TAK-779 and AOP-RANTES inhibited SIV-mac239 entry into GHOST-CD4 cells expressing CCR5, but theentry of this virus into GHOST-CD4 cells expressing eitherBOB, Bonzo, GPR1, or APJ was unaffected by TAK-779 orAOP-RANTES (Fig. 1a). The entry of SIVrcm into GHOST-CD4 cells expressing CCR2 was completely inhibited by TAK-779, whereas AOP-RANTES had only a marginal effect onentry via CCR2 (Fig. 1a). SIVrcm replication in GHOST-CD4cells expressing Bonzo, V28, or US28 was, however, insensitiveto TAK-779 or AOP-RANTES (Fig. 1a), as was HIV-2 7924Areplication in cells expressing V28, APJ, or US28 (data notshown). Neither TAK-779 nor AOP-RANTES inhibited thereplication of HIV-1 P6-v3 in GHOST-CD4 cells expressingBonzo (data not shown).

Taken together, these data suggest that AOP-RANTES canblock viral entry via CCR5 and CCR3 and that TAK-779 in-hibits entry via CCR5 and CCR2. The latter result is consistentwith the report that TAK-779 binds to both CCR2 and CCR5but not to CCR1, CCR3, or CCR4 (4). TAK-779 and AOP-RANTES have no effect on viral replication in GHOST-CD4cells expressing any one of the eight coreceptors that we wereable to evaluate: CXCR4, CCR8, V28, US28, APJ, BOB,Bonzo, or GPR1.

A less clear-cut pattern of inhibition was observed with

AMD3100. As expected, the efficient replication of SHIV89.6PD in GHOST-CD4 cells expressing CXCR4 was blockedby AMD3100 (Fig. 1a). However, AMD3100 also preventedthe inefficient replication of SHIV 89.6PD in GHOST-CD4cells expressing either CCR3, V28, APJ, US28, or CCR8 andsignificantly inhibited the limited replication of HIV-2 7924Ain GHOST-CD4 cells expressing V28, APJ, or US28 (Fig. 1b;also data not shown). However, AMD3100 had no detectableeffect on SIVmac239 entry into GHOST-CD4 cells expressingBOB, Bonzo, GPR1, or APJ or on SIVrcm entry into GHOST-CD4 cells expressing CCR2, Bonzo, V28, or US28 (Fig. 1a).The replication of HIV-1 P6-v3 in GHOST-CD4 cells express-ing Bonzo was also unaffected by AMD3100 (data not shown).Thus, entry via V28, US28, and APJ in GHOST-CD4 cell linescan apparently be either sensitive or insensitive to AMD3100,depending upon the test virus.

There are two possible explanations for the unusual patternof inhibition shown by AMD3100. One is that AMD3100 isbroadly reactive with multiple coreceptors but that certainviruses, particularly SIV, can still interact with some of thesecoreceptors even in the presence of AMD3100. The other isthat the apparent cross-reactivity of AMD3100 is an artifact ofthe presence of low levels of endogenous CXCR4 in corecep-tor-transfected GHOST-CD4 cells (109, 110). To address thispossibility, we tested the sensitivity of SHIV 89.6PD and HIV-2 7924A replication in several GHOST-CD4 cell lines to SDF-1a. In all cases, whenever AMD3100 inhibited the replicationof the test viruses, so did SDF-1a (Fig. 1b; also data not shown).Since SDF-1a is specific for CXCR4 (6, 8, 9, 71, 84), thesefindings strongly suggest that the entry of SHIV 89.6PD andHIV-2 7924A into several coreceptor-transfected GHOST-CD4 cell lines occurs via endogenous CXCR4. This coreceptormay well be expressed to different levels in different individualGHOST-CD4 cell lines, although we were unable to accuratelyquantitate this expression by FACS.

The inhibitory effect of AMD3100 in coreceptor-transfectedGHOST-CD4 cell lines is, therefore, most probably explainedby its antagonism of viral entry via endogenous CXCR4. Thecoreceptor usage information presented in Table 1 should beinterpreted with this caveat in mind. Overall, we can find no evi-dence that AMD3100 is anything other than specific for CXCR4.

Effect of coreceptor-targeted inhibitors on HIV-1, SHIV, andHIV-2 replication in PBMC. The replication of each test virusin mitogen-stimulated PBMC in the presence and absence ofAMD3100, TAK-779, or AOP-RANTES was evaluated. Com-binations of AMD3100 with TAK-779 and AMD3100 withAOP-RANTES were also tested. Each inhibitor, alone and incombination, was used at three different concentrations: 400,40, and 4 nM for AMD3100; 3.3 mM, 330 nM, and 33 nM forTAK-779; and 40, 4, and 0.4 nM for AOP-RANTES. Prelim-inary experiments had indicated that the effects of the inhibi-tors usually titrated out over these ranges. Human PBMC fromCCR5 wild-type donors were used in experiments with HIV-1and HIV-2 isolates and SIVrcm; rhesus macaque PBMC wereused with other SIV; and both human and macaque PBMCwere used with SHIV.

Four HIV-1 primary isolates that could use multiple core-ceptors, as determined by the GHOST-CD4 cell assays (Table1), were evaluated with human PBMC (Fig. 2). P6-v3, a virusable to use CCR5 and Bonzo, was completely inhibited by bothTAK-779 and AOP-RANTES but not by AMD3100 (Fig. 2a).The more broadly tropic virus M6-v3 was partially sensitive toeach of the three inhibitors, but its replication was fullyblocked by combinations of either TAK-779 or AOP-RANTESwith AMD3100 (Fig. 2a). Isolates 5073 and 5060 were able toreplicate in several different coreceptor-expressing GHOST-


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 SHIVisolates evaluated were 89.6PD in macaque and human PBMC (a) and KU-2 and SF33A in human PBMC (b).


CD4 cell lines, including GHOST-CD4 cells expressing CXCR4,but their replication was completely inhibited in PBMC byAMD3100 (Fig. 2b). Thus, none of the tested HIV-1 isolatesappeared to enter PBMC from the donors included in thesestudies via a coreceptor other than CCR5 or CXCR4.

Results similar to those obtained with the broadly tropicHIV-1 isolates were found when SHIV were evaluated (Fig. 3).Thus, SHIV 89.6PD replication in either macaque or humanPBMC was fully inhibited by AMD3100, while TAK-779 andAOP-RANTES had no effect (Fig. 3a). The same was true ofSHIV KU-2 and SHIV SF33A in human PBMC (Fig. 3b) andalso of SHIV 89.6 and SHIV 89.6P (data not shown). Theparamount, and most probably exclusive, coreceptor for all ofthese SHIV in PBMC therefore appears to be CXCR4. Thisfinding was unexpected for SHIV 89.6, 89.6P, and 89.6PD, con-sidering that these viruses efficiently use CCR5 in transfectedGHOST-CD4 cells (Table 1 and Fig. 1a; also data not shown).

Among the HIV-2 isolates tested, 310342 and 7312A wereboth completely inhibited by TAK-779 and AOP-RANTES butwere insensitive to AMD3100 (Fig. 4a). Although HIV-2 7312Acan use BOB and Bonzo, to a limited extent, in GHOST-CD4cells (Table 1), this property does not allow the virus to evadeCCR5-directed inhibitors in PBMC (Fig. 4a). HIV-2 77618 andGB122 were almost completely (.95%) blocked by AMD3100,whereas TAK-779 and AOP-RANTES had no effect on theseviruses (Fig. 4b; also data not shown). All of these HIV-2isolates probably use only CCR5 or CXCR4 to enter PBMC.

An exception was, however, noted with HIV-2 7924A. Thisvirus was partially sensitive to AMD3100, but the extent ofinhibition did not exceed 30% even at the highest AMD3100concentration, 400 nM (Fig. 4b). HIV-2 7924A was completelyinsensitive to TAK-779 or AOP-RANTES, and combiningthese agents with AMD3100 did not increase the extent ofinhibition caused by AMD3100 alone (Fig. 4b).

HIV-2 isolate 7924A has an unusual pattern of sensitivity tocoreceptor-targeted inhibitors. The insensitivity of HIV-27924A to AMD3100 is unusual, since this virus can useCXCR4, and perhaps only CXCR4, to enter GHOST-CD4cells (Table 1 and Fig. 1b). Usually, 50% inhibitory concentra-tions (IC50s) of AMD3100 against viruses that use CXCR4 inPBMC are 4 to 40 nM (Fig. 2b, 3a and b, and 4b; also data notshown). To evaluate whether the insensitivity of HIV-2 7924Ato AMD3100 in PBMC was donor dependent, we tested muchhigher AMD3100 concentrations in cells from four CCR5 wild-type donors (Fig. 5a). Donor-to-donor variation in the potencyof AMD3100 was significant, with IC50s ranging from 2.1 mM(donor 1) to 34 mM (donor 2). However, if sufficient AMD3100(40 mM) was used, inhibition of HIV-2 7924A was complete incells from three of the four donors. Whether at a concentrationas high as 40 mM AMD3100 remains specific for CXCR4 is notknown, although no overt toxicity was observed.

We also tested AMD3100 (4 mM) against HIV-2 7924A inPBMC from a D32-CCR5 homozygous donor (donor 1). Forthe first 4 days of culturing, AMD3100 at 4 mM completelysuppressed HIV-2 7924A replication; however, by day 7, thevirus had broken through, and the extent of inhibition wasnegligible thereafter. In contrast, HIV-1 5160 was completelyinhibited by 4 mM AMD3100 throughout the duration of cul-turing (data not shown).

To gain more insight into whether HIV-2 7924A could useCXCR4 for entry into PBMC, we determined its sensitivity toSDF-1a in cells from the same four CCR5 wild-type donors asthose used in the AMD3100 experiment. Even at the highestconcentration tested (400 nM), SDF-1a did not inhibit HIV-27924A replication in PBMC from any of the four donors,whereas HIV-1 NL4-3 replication was efficiently blocked (Fig.

5b). Taken together with the insensitivity of HIV-2 7924A toTAK-779 and AOP-RANTES (Fig. 4b), the limited or nonex-istent effect of AMD3100 and SDF-1a on HIV-2 7924A rep-lication suggests that this virus uses an undefined coreceptorother than CXCR4 to enter PBMC. An alternative explanationis that HIV-2 7924A uses CXCR4 in a highly unusual, inhibi-tor-insensitive manner. If this is so, how this virus uses CXCR4must be cell type dependent, since we determined that the IC50of AMD3100 for this virus in GHOST-CD4 cells was 0.47 mM.This value contrasts markedly with the IC50s of 2.1 to 34 mMfor the same virus in PBMC.

Effect of coreceptor inhibitors on SIV replication in ma-caque PBMC. To evaluate the inhibitor sensitivities of SIVisolates, we used macaque PBMC. In cells from the first donormacaque tested, SIVmac251, SIVmac239, SIVmac251/1390, andSIVmac239/5501 were all inhibited by both TAK-779 and AOP-RANTES to an extent that was complete, or virtually so (.95%),whereas AMD3100 had no effect (Fig. 6a and b; also data notshown). Thus, these SIV isolates all use CCR5, and only CCR5,to enter PBMC from this macaque donor. However, there areissues of donor cell dependency to consider (see below).

Because SIVrcm uses CCR2 but neither CCR5 nor CXCR4for entry (Table 1), we tested chemokine ligands of CCR2 fortheir abilities to inhibit SIVrcm replication in human PBMC. Ofthese, MCP-1 almost completely inhibited SIVrcm replication,whereas MCP-3 had only a limited effect (Fig. 6c). TAK-779was also an effective inhibitor of SIVrcm replication in humanPBMC (Fig. 6c), just as it was in GHOST-CD4 cells expressingCCR2 (Fig. 1a). However, AOP-RANTES had no effect onSIVrcm replication in human PBMC (data not shown). Thisvirus appears to make truly exclusive use of CCR2 as a core-ceptor in primary human PBMC.

The effect of TAK-779 on SIVmac239 replication in macaquePBMC is donor dependent. We showed above that there is adonor dependency in the ability of SIVmac239 and SIVmac251to replicate in human PBMC from D32-CCR5 homozygousindividuals (Table 4). There is also a donor dependency in thepotency with which CCR5-targeted inhibitors inhibit SIV-mac239 replication in macaque PBMC. Thus, the extent towhich TAK-779, at 3.3 mM, inhibited SIVmac239 replicationvaried from .99% to ,50% in PBMC from four differentmacaques (Fig. 7a). The IC50s of TAK-779 ranged from 240nM (macaque 3) to 12.6 mM (macaque 1), a 60-fold variation.However, at the very high concentration of 33 mM, TAK-779completely inhibited SIVmac239 replication in all four donors(Fig. 7a). Similar results were obtained with SIVmac251 in thetwo donors tested; the IC50s were 0.18 mM (donor 3) and 20mM (donor 1) (Fig. 7a).

There was less variation in the potency of TAK-779 againstHIV-1 replication in human PBMC. For instance, HIV-1 P6-v3was inhibited by TAK-779 in PBMC from four donors at IC50sranging from 15 nM to 24 nM (Fig. 7b). This result suggeststhat major variations in inhibition potency are not an inherentfeature of TAK-779.

When the inhibitor sensitivities of SIVmac239 and SIVmac251were evaluated with CEMx174 cells, both viruses were insen-sitive (,5% inhibition) to AMD3100 (400 nM), TAK-779 (3.3mM), or AOP-RANTES (40 nM), alone or in combination(data not shown). In contrast, HIV-1 NL4-3 replication inthese cells was completely blocked by AMD3100 but not byTAK-779 or AOP-RANTES (data not shown). Thus, what-ever coreceptor(s) SIVmac239 and SIVmac251 use to enterCEMx174 cells, it is not CCR2, CCR3, CCR5, or CXCR4.Whether this is the same coreceptor that these viruses can useto enter human or macaque PBMC from some donors is notyet known.


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

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 fourdifferent human donors in the presence of AMD3100 at 40 mM, 4 mM, 400 nM, and 40 nM (a) and SDF-1a at 400 nM, 40 nM, 4 nM, and 0.4 nM (b) was evaluated.HIV-1 NL4-3 was also tested with SDF-1a. In each case, replication was measured after 7 and 10 days. IC50s of AMD3100 were calculated and are shown in panela. The data shown were obtained on day 10, but values from day 7 were similar.


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 SIVisolates 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 mM, 330 nM, and 33 nM.


DISCUSSION

Primate lentiviruses can use about 12 different seven-trans-membrane receptors as coreceptors in transfected cell lines.However, questions have been raised as to whether corecep-tors other than CCR5 and CXCR4 are relevant for viral entryinto primary cells and, hence, for viral replication in vivo (31,43, 70, 86, 101, 116, 117). This issue affects the development ofantiviral drugs aimed at coreceptors. Must multiple corecep-tors be targeted, or just CCR5 and CXCR4 (117)? Does theability of SIV and SHIV to use multiple coreceptors in vitroinfluence the interpretation of vaccine experiments with pri-mates (73)?

We addressed these issues by using coreceptor-targeted in-hibitors to block viral replication in primary PBMC, focusinghere on HIV-2 and SIV isolates. As inhibitors, we usedAMD3100 for CXCR4 and TAK-779 and AOP-RANTES forCCR5. These agents are not completely specific: TAK-779 andAOP-RANTES also inhibit viral entry via CCR2 and viaCCR3, respectively. However, we could find no evidence thatAMD3100 is anything other than specific for CXCR4, as foundpreviously with other assay systems and test viruses (26, 37, 56,100).

The low-level entry of viruses such as SHIV 89.6PD andHIV-2 7924A into GHOST-CD4 cells expressing V28, US28,APJ, and others actually occurs via endogenous CXCR4 andnot via the transfected coreceptor, since it is inhibited by bothSDF-1a and AMD3100. Of note is that SHIV 89.6PD andHIV-2 7924A use CXCR4 very efficiently, so they may be ableto enter coreceptor-transfected GHOST-CD4 cells that ex-press very low levels of CXCR4; the levels of expression of thiscoreceptor may also vary slightly among different GHOST-CD4 clones, making some transfected cell lines particularlysusceptible to viruses that use CXCR4, although we could not

accurately quantitate such variation by FACS. Whatever theexplanation, ambiguities can arise when the coreceptor usageof CXCR4-tropic viruses is determined with transfectedGHOST-CD4 cell lines. Many, if not all, of the “positives” foruse of coreceptors other than CCR5 and CXCR4 by CXCR4-tropic viruses in GHOST-CD4 cell lines (Table 1) may simplyreflect entry via endogenous CXCR4 and not the transfectedcoreceptor. This caveat may also apply to other studies thathave used these cell lines. A similar conclusion was recentlyreached by others (59).

We previously concluded from an inhibitor-based study thatcoreceptors other than CCR5 and CXCR4 made, at most, onlya limited contribution to HIV-1 replication in PBMC (117).Our inhibitor studies are now strengthened by the recent avail-ability of TAK-779 (4). This CCR5-targeted inhibitor does notinhibit viral entry via CCR3, whereas AOP-RANTES can doso, albeit inefficiently compared to its effect on CCR5-medi-ated entry (34). Since TAK-779, by itself, is able to block thereplication in PBMC of all of the R5, NSI HIV-1 and HIV-2isolates that we tested, CCR3 is not relevant to their entry. Anypossible use of CCR2 that might be masked by TAK-779 is notsupported by the complete inhibition of the same isolates byAOP-RANTES. This chemokine derivative does not block vi-ral entry via CCR2, at least for SIVrcm, which is the only trulyCCR2-tropic virus yet identified (15). Another advantage ofTAK-779 is that it avoids the potential complications of AOP-RANTES-induced enhancement of attachment and entry ofX4 HIV-1 isolates (44, 108). However, we did not observeinfectivity enhancement with human or macaque PBMC at theAOP-RANTES concentrations tested in this study. Overall,the use of TAK-779 reinforces our previous conclusion aboutthe paramount role of CCR5 and CXCR4 in HIV-1 replicationin PBMC (116). This is not to say that other coreceptors are

FIG. 6—Continued.


FIG. 7. Donor-dependent variation in the effects of coreceptor-targeted inhibitors in PBMC. (a) SIVmac239 replication in PBMC from four different macaques wasevaluated in the presence of TAK-779 at 33 mM, 3.3 mM, 330 nM, and 33 nM. SIVmac251 was similarly evaluated with cells from two donors. (b) HIV-1 P6-v3 replicationin PBMC from four different human donors was evaluated in the presence of TAK-779 at 3.3 mM, 330 nM, 33 nM, and 3 nM. In each case, replication was measuredafter 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.


completely irrelevant; Bonzo/STRL33 can be used by rareHIV-1 isolates for entry into a minor subset of PBMC in adonor-dependent manner (102), and CCR3 and CCR8 are po-tential coreceptors expressed on some T-cell subsets (94, 118).

The SHIV isolates that we evaluated—89.6, 89.6P, 89.6PD,SF33A, and KU-2—all exclusively used CXCR4 in human andmacaque PBMC; AMD3100 was sufficient to completely inhibittheir replication, while neither TAK-779 nor AOP-RANTEShad any effect. Thus, although HIV-1 89.6 can enter trans-fected cells via several coreceptors, including CCR5 (27), theSHIV derived from it use only CXCR4 to enter PBMC. Ofnote is that SHIV 89.6, SHIV 89.6P, and SHIV 89.6PD veryefficiently enter GHOST-CD4 cells expressing CCR5 (Table 1;also data not shown). Thus, these viruses can use CCR5 forentry, at least in CCR5-transfected cells, but CXCR4 is pre-ferred in primary cells. Why this should be the case andwhether it matters for transmission and pathogenesis studieswith these viruses in macaques are open questions.

We also conclude that, for most HIV-2 strains, CCR5 and/orCXCR4 are the principal coreceptors relevant to the replica-tion of these strains in PBMC. Thus, TAK-779, AOP-RAN-TES, and AMD3100, alone or in combination, completely orvery substantially inhibited the replication of almost all of ourtest viruses. HIV-2 isolate 7924A is an apparent exception. Thereplication of this broadly tropic virus in PBMC was inhibitedonly by very high concentrations of AMD3100 and was com-pletely insensitive to SDF-1a, TAK-779, or AOP-RANTES.One possibility is that HIV-2 7924A is able to use an alterna-tive coreceptor to enter human PBMC, perhaps the CXCR5receptor reported recently to function with some HIV-2 iso-lates but not with HIV-1 or SIV isolates (52). Alternatively,HIV-2 7924A may use CXCR4 in a manner that is relativelyinsensitive to AMD3100. The latter explanation would be con-sistent with the observation that very high concentrations ofAMD3100 do completely inhibit the replication of HIV-27924A, although there may be concerns about the specificity ofAMD3100 for CXCR4 at such concentrations. Escape mutantsof HIV-1 NL4-3 that continue to use CXCR4, but in a drug-insensitive manner, are known to emerge in response to selec-tion pressure from AMD3100 and SDF-1a (24, 99). It has beensuggested that CXCR4 can exist in different isoforms on dif-ferent cell types (69); this property might be one explanationfor why AMD3100 is a potent inhibitor of HIV-2 7924A inGHOST-CD4 cells expressing CXCR4 (IC50 5 0.47 mM) butcan be such a weak one in PBMC (IC50 5 2.1 to 34 mM, de-pending upon the donor). Additional studies of HIV-2 7924Aare warranted.

Our conclusions for SIVmac isolates are more complicated.The CCR5 proteins from multiple primate species can functionas viral coreceptors (55, 78), and our inhibitor studies are con-sistent with an important role of CCR5 in SIVmac entry intoprimary cells. One aspect of coreceptor usage that distinguishesSIV from HIV-2 isolates is the inability of almost all SIV to useCXCR4. This property contrasts with the efficient use of CXCR4by many HIV-2 isolates. In this sense, HIV-2 more closelyresembles HIV-1 than it does SIV, an unexpected finding giventhe genetic relationships among these virus families and theevolution of HIV-2 from SIVsm (16, 17, 41, 42, 44, 49). Theminimal use of CXCR4 by SIV strains is mirrored by that ofHIV-1 isolates from genetic subtype C (1, 11, 82, 83, 112),although SI primary viruses from this subtype are known (111).

Although CCR5 is important and CXCR4 is unimportantfor SIVmac entry, we found indications that SIVmac239 coulduse a coreceptor other than CCR5 to enter PBMC from somehuman and macaque donors. Thus, in PBMC from one D32-CCR5 homozygous human donor, SIVmac239 replication was

negligible. However, in cells from a second such individual, thevirus replicated fairly efficiently, as observed previously (18).Furthermore, there was considerable variation in the potencywith which TAK-779 inhibited the replication of SIVmac239 inPBMC from different macaques. This result might be ac-counted for by the use of an additional coreceptor that isexpressed in PBMC from only a subset of macaques or that isexpressed in cells from all macaques but at different levels thatare sometimes below a threshold needed for infection. Theexpression of both CCR5 and Bonzo/STRL33 varies from do-nor to donor, in both humans and macaques, to an extent thatcan affect infection efficiency (102, 107). The ability of SIV-mac239 to use a coreceptor other than CCR5, perhaps Bonzo/STRL33, in an animal-dependent manner might influence thehighly variable rates at which different infected macaquesprogress to disease and death (23, 57, 73). However, at least forSIVmne, CCR5 usage is maintained throughout the course ofdisease progression in infected macaques (53). This is also trueof SIVmac239 and SIVmac251 (14).

One coreceptor used efficiently by SIVmac239 in vitro isBOB/GPR15 (22, 38). Pohlmann et al. have, however, shownthat this coreceptor has no relevance to SIVmac239 replicationin vivo, at least in some macaques (86). An unknown, alterna-tive coreceptor(s) also mediates the AMD3100-, TAK-779-,and AOP-RANTES-insensitive entry of SIVmac239 intoCEMx174 cells; this coreceptor cannot, therefore, be CCR2,CCR3, CCR5, or CXCR4. It is not known whether this is thesame coreceptor as the one used by SIVmac239 to enter humanor macaque PBMC from some donors.

We could not distinguish SIVmac239 from the closely relatedSIVmac251 in terms of their sensitivity to coreceptor inhibitors.Although SIVmac251 but not SIVmac239 replicates efficiently inmacrophages, there is no correlation between the coreceptorusage profiles of these viruses in transfected cells and theirtropism for primary cells (53, 75, 76, 86). There is also norelationship between the in vitro tropisms of SIVmac strainsand their abilities to be transmitted to uninfected animals (36,71, 72). We have not yet performed coreceptor inhibitor stud-ies with these viruses and purified macrophages and CD41 Tcells from macaques, as opposed to unfractionated PBMC.

SIVrcm clearly uses CCR2 as its primary coreceptor (15), ina manner that we have shown is sensitive to TAK-779. Theability of SIVrcm to enter GHOST-CD4 cells expressing US28and V28 in vitro is likely to be of limited relevance to thereplication of this virus in red-capped mangabeys.

Overall, we conclude that there is a greater complexity tocoreceptor usage by SIV strains in PBMC than there is forHIV-1 and HIV-2, for which CCR5 and CXCR4 are usuallythe paramount coreceptors. An unknown coreceptor(s) canperhaps be used by SIVmac239 and HIV-2 7924A to enterPBMC, at least from some macaque and human donors. In-volvement of the same coreceptor in the entry of bothSIVmac239 and HIV-2 7924A might conceivably have rele-vance to cross-species viral transmission and the evolution ofHIV-2 from SIVsm (16, 17, 41, 42, 44, 49).

ACKNOWLEDGMENTS

We thank Annette Bauer, Michael Miller, Susan Vice, Bahige Ba-roudy, and Stuart McCombie for AMD3100 and TAK-779; AmandaProudfoot, Robin Offord, and Brigitte Dufour for AOP-RANTES;Zhiwei Chen for SIV isolates; David Montefiori, Cecilia Cheng-Mayer,and Opendra Narayan for SHIV isolates; Dan Littman and VineetKewalRamani for GHOST cells; and James Hoxie and Nelson Michaelfor preferring hard liquor to blood. We appreciate helpful commentsby Amanda Proudfoot and Bob/GPR15 Doms.


This study was supported by NIH grant RO1 AI41420 and by thePediatric AIDS Foundation, of which J.P.M. is an Elizabeth GlaserScientist.

REFERENCES

1. Abebe, A., D. Demissie, J. Goudsmit, M. Brouwer, C. L. Kuiken, G. Pol-lakis, H. Schuitemaker, A. L. Fontanet, and T. F. Rinke deWit. 1999. HIV-1subtype C syncytium- and non-syncytium-inducing phenotypes and core-ceptor usage among Ethiopian patients with AIDS. AIDS 13:1305–1311.

2. 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-1a, MIP-1breceptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955–1958.

3. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. C. Peden. 1997.A new SIV coreceptor, STRL33. Nature 388:238.

4. Baba, M., O. Nishimura, N. Kanzaki, M. Okamoto, H. Sawada, Y. Iizawa,M. Shiraishi, Y. Aramaki, K. Okonogi, Y. Ogawa, and K. Meguro. 1999. Asmall molecule nonpeptide CCR5 antagonist with highly potent and selec-tive anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA 96:5698–5703.

5. Bazan, H. A., G. Alkhatib, C. C. Broder, and E. A. Berger. 1998. Patterns ofCCR5, CXCR4, and CCR3 usage by envelope glycoproteins from humanimmunodeficiency virus type 1 primary isolates. J. Virol. 72:4485–4491.

6. Berger, E. A. 1997. HIV entry and tropism: the chemokine receptor con-nection. AIDS 11(Suppl. A):S3–S16.

7. Berger, E. A., R. W. Doms, E.-M. Fenyo, B. T. M. Korber, D. R. Littman,J. P. Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A.Weiss. 1998. A new classification for HIV-1. Nature 391:240.

8. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptorsas HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev.Immunol. 17:657–700.

9. Bieniasz, P. D., and B. R. Cullen. 1998. Chemokine receptors and humanimmunodeficiency virus infection. Front. Biosci. 3:44–58.

10. Bron, R., P. J. Klasse, D. Wilkinson, P. R. Clapham, A. Pelchen Matthews,C. Power, T. N. C. Wells, J. Kim, S. C. Peiper, J. A. Hoxie, and M. Marsh.1997. Promiscuous use of CC and CXC chemokine receptors in cell-to-cellfusion mediated by a human immunodeficiency virus type 2 envelope pro-tein. J. Virol. 71:8405–8415.

11. Bjorndal, A., A. Sonnerborg, C. Tscherning, J. Albert, and E. M. Fenyo.1999. Phenotypic characteristics of human immunodeficiency virus type 1subtype C isolates of Ethiopian AIDS patients. AIDS Res. Hum. Retrovir.15:647–653.

12. Chackerian, B., E. M. Long, P. A. Luciw, and J. Overbaugh. 1997. Humanimmunodeficiency virus type 1 coreceptors participate in postentry stages inthe virus replication cycle and function in simian immunodeficiency virusinfection. J. Virol. 71:3932–3939.

13. Chan, S. Y., R. F. Speck, C. Power, S. L. Gaffen, B. Chesebro, and M. A.Goldsmith. 1999. V3 recombinants indicate a central role for CCR5 as acoreceptor in tissue infection by human immunodeficiency virus type 1.J. Virol. 73:2350–2358.

14. Chen, Z., A. Gettie, D. D. Ho, and P. A. Marx. 1998. Primary SIVsm isolatesuse the CCR5 co-receptor from sooty mangabeys naturally infected in WestAfrica: a comparison of coreceptor usage of primary SIVsm, HIV-2 andSIVmac. Virology 245:113–124.

15. Chen, Z., D. Kwon, Z. Jin, S. Monard, P. Telfer, M. S. Jones, C. Y. Lu, R. F.Aguilar, D. D. Ho, and P. A. Marx. 1998. Natural infection of a homozygousD24-CCR5 red-capped mangabey with an R2b-tropic simian immunodefi-ciency virus. J. Exp. Med. 188:2057–2065.

16. Chen, Z., A. Luckay, D. L. Sodora, P. Telfer, P. Reed, A. Gettie, J. M. Kanu,R. F. Sadek, J. Yee, D. D. Ho, L. Zhang, and P. A. Marx. 1997. Humanimmunodeficiency virus type 2 (HIV-2) seroprevalence and characteriza-tion of a distinct HIV-2 genetic subtype from the natural range of simianimmunodeficiency virus-infected sooty mangabeys. J. Virol. 71:3953–3960.

17. 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 strainswith human immunodeficiency virus type 2 subtypes and genetically diverseviruses from a single feral sooty mangabey troop. J. Virol. 70:3617–3627.

18. Chen, Z.-W., P. Zhou, D. D. Ho, N. R. Landau, and P. A. Marx. 1997.Genetically divergent strains of simian immunodeficiency virus use CCR5as a coreceptor for entry. J. Virol. 71:2705–2714.

19. Choe, H., M. Farzan, M. Konkel, K. Martin, Y. Sun, L. Marcon, M.Cayabyab, M. Berman, M. E. Dorf, N. Gerard, G. Gerard, and J. Sodroski.1998. The orphan seven-transmembrane receptor Apj supports the entry ofprimary T-cell-line-tropic and dualtropic human immunodeficiency virustype 1. J. Virol. 72:6113–6118.

20. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu,C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, G. Gerard, and J.Sodroski. 1996. The b-chemokine receptors CCR3 and CCR5 facilitateinfection by primary HIV-1 isolates. Cell 85:1135–1148.

21. 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, HemophiliaGrowth and Development Study, Multicenter AIDS Cohort Study, Multi-center Hemophilia Cohort Study, San Francisco City Cohort, ALIVEStudy, and S. J. O’Brien. 1996. Genetic restriction of HIV-1 infection andprogression to AIDS by a deletion of the CKR5 structural allele. Science273:1856–1862.

22. Deng, H., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997.Expression cloning of new receptors used by simian and human immuno-deficiency viruses. Nature 388:296–300.

23. Desrosiers, R. C. 1995. Non-human primate models for AIDS vaccines.AIDS 9(Suppl. A):S137–S141.

24. De Vreese, K., V. Kofler-Mongold, C. Leutgeb, V. Weber, K. Vermiere, S.Schacht, J. Anne, E. de Clercq, R. Datema, and G. Werner. 1996. Themolecular target of bicyclams, potent inhibitors of human immunodefi-ciency virus replication. J. Virol. 70:689–696.

25. Doms, R. W., and S. C. Peiper. 1997. Unwelcome guests with master keys:how HIV uses chemokine receptors for cellular entry. Virology 235:179–190.

26. Donzella, G. A., D. Schols, S. W. Lin, J. A. Este, K. A. Nagashima, P. J.Maddon, G. P. Allaway, T. P. Sakmar, G. Henson, E. De Clercq, and J. P.Moore. 1998. AMD3100, a small molecule inhibitor of HIV-1 entry via theCXCR4 co-receptor. Nat. Med. 4:72–77.

27. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. Peiper, M.Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic, primaryHIV-1 isolate that uses fusin and the b-chemokine receptors CKR-5 andCKR-2b as fusion cofactors. Cell 85:1149–1159.

28. 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 CD41 cells is mediated by the chemokinereceptor CC-CKR-5. Nature 381:667–673.

29. Dumonceaux, J., S. Nisole, C. Chanel, L. Quivet, A. Amara, F. Baleux, P.Briand, and U. Hazan. 1998. Spontaneous mutations in the env gene of thehuman immunodeficiency virus type 1 NDK isolate are associated with aCD4-independent entry phenotype. J. Virol. 72:512–519.

30. Edinger, A. L., C. Blanpain, K. J. Kuntsman, S. M. Wolinsky, M. Parmen-tier, and R. W. Doms. 1999. Functional dissection of CCR5 coreceptorfunction through the use of CD4-independent simian immunodeficiencyvirus strains. J. Virol. 73:4062–4073.

31. Edinger, A. L., T. L. Hoffman, M. Sharron, B. Lee, B. O’Dowd, and R. W.Doms. 1998. Use of GPR1, GPR15, and STRL33 as co-receptors by diversehuman immunodeficiency virus type 1 and simian immunodeficiency virusenvelope proteins. Virology 249:367–378.

32. Edinger, A. L., T. L. Hoffman, M. Sharron, B. Lee, Y. Yi, W. Choe, D. C.Kolson, B. Mitrovic, Y. Zhou, D. Faulds, R. G. Collman, J. Hesselgesser, R.Horuk, and R. W. Doms. 1998. An orphan seven-transmembrane domainreceptor expressed widely in the brain functions as a coreceptor for humanimmunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol.72:7934–7940.

33. Edinger, A. L., J. L. Mankowski, B. J. Doranz, B. J. Margulies, B. Lee, J.Rucker, M. Sharron, T. L. Hoffman, J. F. Berson, M. C. Zink, V. M. Hirsch,J. E. Clements, and R. W. Doms. 1997. CD4-independent, CCR5-depen-dent infection of brain capillary endothelial cells by a neurovirulent simianimmunodeficiency virus strain. Proc. Natl. Acad. Sci. USA 94:14742–14747.

34. Elsner, J., M. Mack, H. Bruhl, Y. Dulkys, D. Kimmig, G. Simmons, P. R.Clapham, D. Schlondorff, A. Kapp, T. N. C. Wells, and A. E. I. Proudfoot.2000. Differential activation of CC chemokine receptors by AOP-RANTES.J. Biol. Chem. 275:7787–7794.

35. 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. C. 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 byfusin/CXCR4. Cell 87:745–756.

36. Enose, Y., K. Ibuki, K. Tamaru, M. Ui, T. Kuwata, T. Shimada, and M.Hayami. 1999. Replication capacity of simian immunodeficiency virus incultured macaque macrophages and dendritic cells is not prerequisite for in-travaginal transmission of the virus in macaques. J. Gen. Virol. 80:847–855.

37. Este, J. A., C. Cabrera, J. Blanco, A. Gutierrez, G. Bridger, G. Henson, B.Clotet, D. Schols, and E. DeClerq. 1999. Shift of clinical human immuno-deficiency virus type 1 isolates from X4 to X5 and prevention of emergenceof the syncytium-inducing phenotype by blockade of CXCR4. J. Virol.73:5577–5585.

38. Farzan, M., H. Choe, K. Martin, L. Marcon, W. Hofmann, G. Karlsson, Y.Sun, P. Barrett, N. Marchand, N. Sullivan, N. Gerard, C. Gerard, and J.Sodroski. 1997. Two orphan seven-transmembrane segment receptorswhich are expressed in CD4-positive cells support simian immunodeficiencyvirus infection. J. Exp. Med. 186:405–411.

39. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entrycofactor: functional cDNA cloning of a seven-transmembrane G proteincoupled receptor. Science 272:872–877.

40. Frade, J. M. R., M. Llorente, M. Mellado, J. Alcami, J. C. Gutierrez Ramos,A. Zaballos, G. del Real, and A. C. Martinez. 1997. The amino terminaldomain of the CCR2 chemokine receptor acts as a coreceptor for HIV-1infection. J. Clin. Investig. 100:497–502.


41. Gao, F., L. Yue, D. L. Robertson, S. C. Hill, H. Hui, R. J. Biggar, A. E.Neequaye, T. M. Whelan, D. D. Ho, G. M. Shaw, P. M. Sharp, and B. Hahn.1994. Genetic diversity of human immunodeficiency virus type 2: evidencefor distinct sequence subtypes with differences in virus biology. J. Virol.68:7433–7447.

42. Gao, F., L. Yue, A. T. White, P. G. Pappas, J. Barchue, A. P. Hanson, B. M.Greene, P. M. Sharp, and B. H. Hahn. 1992. Human infection by geneticallydiverse SIVsm-related HIV-2 in West Africa. Nature 358:495–499.

43. Glushakova, S., Y. Yi, J.-C. Grivel, A. Singh, D. Schols, E. De Clercq, R. G.Collman, and L. Margolis. 1999. Preferential coreceptor utilization andcytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo.J. Clin. Investig. 104:R7–R11.

44. Gordon, C. J., M. A. Muesing, A. E. I. Proudfoot, C. A. Power, J. P. Moore,and A. Trkola. 1999. Enhancement of human immunodeficiency virus type1 infection by the CC-chemokine RANTES is independent of the mecha-nism of virus-cell fusion. J. Virol. 73:684–694.

45. Guillon, G., M. E. van der Ende, P. H. M. Boers, R. A. Gruters, M.Schutten, and A. D. M. E. Osterhaus. 1998. Coreceptor usage of humanimmunodeficiency virus type 2 primary isolates and biological clones isbroad and does not correlate with their syncytium-inducing capacities.J. Virol. 72:6260–6263.

46. Harouse, J. M., R. C. Tan, A. Gettie, P. Dailey, P. A. Marx, P. A. Luciw, andC. Cheng-Mayer. 1998. Mucosal transmission of pathogenic CXCR4-utiliz-ing SHIVSF33A variants in rhesus macaques. Virology 248:95–107.

47. Hoffman, T. L., C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I.Chaiken, J. A. Hoxie, and R. W. Doms. 1999. Stable exposure of thecoreceptor binding site in a CD4-independent HIV-1 envelope protein.Proc. Natl. Acad. Sci. USA 96:6359–6364.

48. Hill, C. M., H. Deng, D. Unutmaz, V. N. Kewalramani, L. Bastiani, M. K.Gorny, S. Zolla-Pazner, and D. R. Littman. 1997. Envelope glycoproteinsfrom human immunodeficiency virus types 1 and 2 and simian immunode-ficiency virus can use human CCR5 as a coreceptor for viral entry and makedirect CD4-dependent interactions with this chemokine receptor. J. Virol.71:6296–6304.

49. Hirsch, V. M., R. A. Olmsted, M. Murphey-Corb, R. H. Purcell, and P. R.Johnson. 1989. An African primate lentivirus (SIVsm) closely related toHIV-2. Nature 339:389–392.

50. Horuk, R., J. Hesselgesser, Y. Zhou, D. Faulds, M. Halks-Miller, S. Harvey,D. Taub, M. Samson, M. Parmentier, J. Rucker, B. J. Doranz, and R. W.Doms. 1998. The CC chemokine I-309 inhibits CCR8 dependent infectionby diverse HIV-1 strains. J. Biol. Chem. 273:386–391.

51. Joag, S. V., I. Adany, Z. Li, L. Foresman, D. M. Pinson, C. Wang, E. B.Stephens, R. Raghavan, and O. Narayan. 1997. Animal model of mucosallytransmitted human immunodeficiency virus type 1 disease: intravaginal andoral deposition of simian/human immunodeficiency virus in macaques re-sults in systemic infection, elimination of CD41 T cells, and AIDS. J. Virol.71:4016–4023.

52. Kanbe, K., N. Shimizu, Y. Soda, K. Takagishi, and H. Hoshino. 1999. ACXC chemokine receptor, CXCR5/BLR1, is a novel and specific corecep-tor for human immunodeficiency virus type 2. Virology 265:264–273.

53. Kimata, J. T., L. Kuller, D. B. Anderson, P. Dailey, and J. Overbaugh. 1999.Emerging cytopathic and antigenic simian immunodeficiency virus variantsinfluence AIDS progression. Nat. Med. 5:535–541.

54. Kuhmann, S. E., E. J. Platt, S. L. Kozak, and D. Kabat. 1997. Polymor-phisms in the CCR5 genes of African green monkeys and mice implicatespecific amino acids in infections by simian and human immunodeficiencyviruses. J. Virol. 71:8642–8656.

55. Kunstman, K., Z. Chen, B. Korber, J. Oprondek, J. Stanton, M. Agy, R.Shibata, A. Yoder, S. Pillai, C. Kuiken, P. Marx, and S. Wolinsky. Nonhu-man primate CCR5 homologues and their usage by simian and humanimmunodeficiency viruses. AIDS Res. Hum. Retrovir., in press.

56. Labrosse, B., A. Brelot, N. Heveker, N. Sol, D. Schols, E. De Clercq, and M.Alizon. 1998. Determinants for sensitivity of human immunodeficiency viruscoreceptor CXCR4 to the bicyclam AMD3100. J. Virol. 72:6381–6388.

57. Lamb-Wharton, R. J., S. V. Joag, E. B. Stephens, and O. Narayan. 1997. Pri-mate models of AIDS vaccine development. AIDS 11(Suppl. A):S121–S126.

58. Lee, B., M. Sharron, L. J. Montaner, D. Weissman, and R. W. Doms. 1999.Quantification of CD4, CCR5 and CXCR4 levels on lymphocyte subsets,dendritic cells, and differently conditioned monocyte derived macrophages.Proc. Natl. Acad. Sci. USA 96:5215–5220.

59. Li, S., J. Juarez, M. Alali, D. Dwyer, R. Collman, A. Cunningham, andH. M. Naif. 1999. Persistent CCR5 utilization and enhanced macrophagetropism by primary blood human immunodeficiency virus type 1 isolatesfrom advanced stages of disease and comparison to tissue-derived isolates.J. Virol. 73:9741–9755.

60. Liao, F., G. Alkhatib, K. W. C. Peden, G. Sharma, E. A. Berger, and J. M.Farber. 1997. STRL33, a novel chemokine receptor-like protein, functionsas a fusion cofactor for both macrophage-tropic and T cell line-tropicHIV-1. J. Exp. Med. 185:2015–2023.

61. 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 86:367–378.62. Loetscher, M., A. Amara, E. Oberlin, N. Brass, D. F. Legler, P. Loetscher,

M. D’Apuzzo, E. Meese, D. Rousset, J.-L. Virelizier, M. Baggiolini, F.Arenzana-Seisdedos, and B. Moser. 1997. TYMSTR, a putative chemokinereceptor selectively expressed in activated T cells, exhibits HIV-1 corecep-tor function. Curr. Biol. 7:652–660.

63. Mack, M., B. Luckow, P. J. Nelson, J. Lihak, G. Simmons, P. R. Clapham,N. Signoret, M. Marsh, M. Stangassinger, F. Borlat, T. N. C. Wells, D.Schlondorff, and A. E. I. Proudfoot. 1998. Aminooxypentane-RANTESinduces CCR5 internalization but inhibits recycling: a novel inhibitorymechanism of HIV infectivity. J. Exp. Med. 187:1215–1224.

64. Marcon, L., H. Choe, K. A. Martin, M. Farzan, P. D. Ponath, L. Wu, W.Newman, N. Gerard, C. Gerard, and J. Sodroski. 1997. Utilization of C-Cchemokine receptor 5 by the envelope glycoproteins of a pathogenic simianimmunodeficiency virus, SIVmac239. J. Virol. 71:2522–2527.

65. Martin, K. A., R. Wyatt, M. Farzan, H. Choe, L. Marcon, E. Desjardins, J.Robinson, J. Sodroski, C. Gerard, and N. P. Gerard. 1997. CD4 indepen-dent binding of SIV gp120 to rhesus CCR5. Science 278:1470–1473.

66. Martin, V., P. Rondes, D. Unett, A. Wong, T. L. Hoffman, A. L. Edinger,R. W. Doms, and C. D. Funk. 1999. Leukotriene binding, signaling andanalysis of HIV coreceptor function in mouse and human leukotriene B4receptor-transfected cells. J. Biol. Chem. 274:8597–8603.

67. Marx, P. A., A. I. Spira, A. Gettie, P. J. Dailey, R. S. Veazey, A. A. Lackner,C. J. Mahoney, C. J. Miller, L. E. Claypool, D. D. Ho, and N. J. Alexander.1996. Progesterone implants enhance SIV vaginal transmission and earlyvirus load. Nat. Med. 2:1084–1089.

68. McKnight, A., M. T. Dittmar, J. Moniz-Periera, K. Ariyoshi, J. D. Reeves,S. Hibbitts, D. Whitby, E. Aarons, A. E. I. Proudfoot, H. Whittle, and P. R.Clapham. 1998. A broad range of chemokine receptors are used by primaryisolates of human immunodeficiency virus type 2 as coreceptors with CD4.J. Virol. 72:4065–4071.

69. 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 immu-nodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4)is both cell type and virus strain dependent. J. Virol. 71:1692–1696.

70. Michael, N. L., J. A. E. Nelson, V. N. KewalRamani, G. Chang, S. J.O’Brien, J. R. Mascola, B. Volsky, M. Louder, G. C. White II, D. R.Littman, R. Swanstrom, and T. R. O’Brien. 1998. Exclusive and persistentuse of the entry coreceptor CXCR4 by human immunodeficiency virus type1 from a subject homozygous for CCR5 D32. J. Virol. 72:6040–6047.

71. Miller, C. J., and J. Hu. 1999. T cell tropic simian immunodeficiencyvirus (SIV) and simian-human immunodeficiency viruses are readilytransmitted by vaginal inoculation of rhesus macaques, and Langerhans’cells of the female genital tract are infected with SIV. J. Infect. Dis. 179:S413–S417.

72. Miller, C. J., M. Marthas, J. Greenier, D. Lu, P. J. Dailey, and Y. Lu. 1998.In vivo replication capacity rather than in vitro macrophage tropism pre-dicts efficiency of vaginal transmission of simian immunodeficiency virus orsimian/human immunodeficiency virus in rhesus macaques. J. Virol. 72:3248–3258.

73. Moore, J. P., and A. Trkola. 1997. HIV-1 co-receptors, neutralization se-rotypes and vaccine development. AIDS Res. Hum. Retrovir. 13:733–736.

74. Moore, J. P., A. Trkola, and T. Dragic. 1997. Co-receptors for HIV-1 entry.Curr. Opin. Immunol. 9:551–562.

75. Mori, K., D. J. Ringler, and R. C. Desrosiers. 1993. Restricted replicationof simian immunodeficiency virus strain 239 in macrophages is determinedby env but is not due to restricted entry. J. Virol. 67:2807–2814.

76. Mori, K., D. J. Ringler, T. Kodama, and R. C. Derosiers. 1992. Complexdeterminants of macrophage tropism in env of simian immunodeficiencyvirus. J. Virol. 66:2067–2075.

77. Morner, A., A. Bjorndal, J. Albert, V. N. KewalRamani, D. R. Littman, R.Inoue, R. Thorstensson, E. M. Fenyo, and E. Bjorling. 1999. Primary humanimmunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, fre-quently use CCR5 but show promiscuity in coreceptor usage. J. Virol.73:2343–2349.

78. Muller-Trutwin, M. C., S. Corbet, J. Hansen, M.-C. Georges-Courbot, O.Diop, J. Rigoulet, F. Barre-Sinoussi, and A. Fomsgaard. 1999. Mutations inCCR5-coding sequences are not associated with SIV carrier status in Af-rican nonhuman primates. AIDS Res. Hum. Retrovir. 15:931–939.

79. Oppermann, M., M. Mack, A. E. I. Proudfoot, and H. Olbrich. 1999.Differential effect of CC chemokines on CC chemokine receptor 5 (CCR5)phosphorylation and identification of phosphorylation sites on the CCR5carboxyl terminus. J. Biol. Chem. 274:8875–8885.

80. Owen, S. M., D. Ellenberger, M. Rayfield, S. Wiktor, P. Michel, M. H.Grieco, F. Gao, B. H. Hahn, and R. B. Lal. 1998. Genetically divergentstrains of human immunodeficiency virus type 2 use multiple coreceptorsfor viral entry. J. Virol. 72:5425–5432.

81. Park, I.-W., J.-F. Wang, and J. E. Groopman. 1999. Expression and utili-zation of co-receptors in HIV and simian immunodeficiency virus infectionof megakaryocytes. AIDS 13:2023–2032.

82. Peeters, M., R. Vincent, J. L. Perret, M. Lasky, D. Patrel, F. Liegeois, V.Courgnaud, R. Seng, T. Matton, S. Molinier, and E. Delaporte. 1999.


Evidence for differences in MT2 cell tropism according to genetic subtypesof HIV-1: syncytium-inducing variants seem rare among subtype C HIV-1viruses. J. Acquir. Immune Defic. Syndr. 20:115–121.

83. Ping, L. H., J. A. Nelson, I. F. Hoffman, J. Schock, S. L. Lamers, M.Goodman, P. Vernazza, P. Kazembe, M. Maida, D. Zimba, M. M. Good-enow, J. J. Eron, Jr., S. A. Fiscus, M. S. Cohen, and R. Swanstrom. 1999.Characterization of V3 sequence heterogeneity in subtype C human immu-nodeficiency virus type 1 isolates from Malawi: underrepresentation of X4variants. J. Virol. 73:6271–6281.

84. Pleskoff, O., C. Treboute, A. Brelot, N. Heveker, M. Seman, and M. Alizon.1997. Identification of a chemokine receptor encoded by human cytomeg-alovirus as a cofactor for HIV-1 entry. Science 276:1874–1878.

85. Pohlmann, S., M. Krumbiegel, and F. Kirchoff. 1999. Coreceptor usage ofBOB/GPR1 and Bonzo/STRL33 by primary isolates of human immunode-ficiency virus type 1. J. Gen. Virol. 80:1241–1251.

86. Pohlmann, S., N. Stolte, J. Munch, P. Ten Haaft, J. L. Heeney, C. Stahl-Hennig, and F. Kirchhoff. 1999. Co-receptor usage of BOB/GPR15 inaddition to CCR5 has no significant effect on replication of simian immu-nodeficiency virus in vivo. J. Infect. Dis. 180:1494–1502.

87. Premack, B. A., and T. J. Schall. 1996. Chemokine receptors: gateways toinflammation and infection. Nat. Med. 2:1174–1178.

88. Proudfoot, A. E. I., B. Ruser, F. Borlat, S. Alouani, D. Soler, R. E. Offord,J.-M. Schroder, C. A. Power, and T. N. C. Wells. 1999. Amino-terminallymodified RANTES analogues demonstrate differential effects on RANTESreceptors. J. Biol. Chem. 274:32478–32485.

89. 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-independentinfection by HIV-2 (ROD/B): use of the 7-transmembrane receptorsCXCR-4, CCR-3, and V28 for entry. Virology 231:130–134.

90. Reimann, K. A., J. T. Li, R. Veazey, M. Halloran, I. W. Park, G. B.Karlsson, J. Sodroski, and N. L. Letvin. 1996. A chimeric simian/humanimmunodeficiency virus expressing a primary patient human immunodefi-ciency virus type 1 isolate env causes an AIDS-like disease after in vivopassage in rhesus monkeys. J. Virol. 70:6922–6928.

91. Reimann, K. A., J. T. Li, G. Voss, C. Lekutis, K. Tenner-Racz, P. Racz, W.Lin, D. C. Montefiori, D. E. Lee-Parritz, Y. Lu, R. G. Collman, J. Sodroski,and N. L. Letvin. 1996. An env gene derived from a primary human immu-nodeficiency virus type 1 isolate confers high in vivo replicative capacity toa chimeric simian/human immunodeficiency virus in rhesus monkeys. J. Vi-rol. 70:3198–3206.

92. Ross, T. M., and B. R. Cullen. 1998. The ability of HIV type 1 to use CCR-3as a coreceptor is controlled by envelope V1/V2 sequences acting in con-junction with a CCR-5 tropic V3 loop. Proc. Natl. Acad. Sci. USA 95:7682–7686.

93. Rucker, J., A. L. Edinger, M. Sharron, M. Samson, B. Lee, J. F. Berson, Y.Yi, B. Margulies, R. G. Collman, B. J. Doranz, M. Parmentier, and R. W.Doms. 1997. Utilization of chemokine receptors, orphan receptors, andherpesvirus-encoded receptors by diverse human and simian immunodefi-ciency viruses. J. Virol. 71:8999–9007.

94. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression ofthe eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005–2007.

95. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C.-M. Farber,S. Saragosti, C. Lapoumeroulie, J. Cogniaux, C. Forceille, G. Muylder-mans, 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 of Caucasian individuals bearing mu-tant alleles of the CCR5 chemokine receptor gene. Nature 382:722–725.

96. Samson, M., A. L. Edinger, P. Stordeur, J. Rucker, V. Verhasselt, M.Sharron, C. Govaerts, C. Mollereau, G. Vassart, R. W. Doms, and M.Parmentier. 1998. ChemR23, a putative chemoattractant receptor, is expressedin monocyte-derived dendritic cells and macrophages and is a coreceptor forSIV and some primary HIV-1 strains. Eur. J. Immunol. 28:1689–1700.

97. Schenten, D., L. Marcon, G. B. Karlsson, C. Parolin, T. Kodama, N. Gerard,and J. Sodroski. 1999. Effects of soluble CD4 on simian immunodeficiencyvirus infection of CD4-positive and CD4-negative cells. J. Virol. 73:5373–5380.

98. Schols, D., and E. De Clercq. The simian immunodeficiency virus Mnd(GB-1) strain uses CXCR4, not CCR5, as coreceptor for entry in humancells. J. Gen. Virol. 79:2203–2205.

99. Schols, D., J. A. Este, C. Cabrera, and E. De Clercq. 1998. T-cell-line-tropichuman immunodeficiency virus type 1 that is made resistant to stromalcell-derived factor 1a contains mutations in the envelope gp120 but doesnot show a switch in coreceptor use. J. Virol. 72:4032–4037.

100. Schols, D., S. Struyf, J. Van Damme, J. A. Este, G. Henson, and E. De

Clercq. 1997. Inhibition of T-tropic HIV strains by selective antagonizationof the chemokine receptor CXCR4. J. Exp. Med. 186:1383–1388.

101. Schramm, B., M. L. Penn, R. F. Speck, S. Y. Chan, E. De Clercq, D. Schols,R. I. Connor, and M. A. Goldsmith. 2000. Viral entry through CXCR4 is apathogenic factor and therapeutic target in human immunodeficiency virustype 1 disease. J. Virol. 74:184–192.

102. Sharron, M., S. Pohlmann, K. Price, M. Tsang, F. Kirchhoff, R. W. Doms,and B. Lee. Expression and coreceptor activity of STRL33 on primaryperipheral blood lymphocytes. Blood, in press.

103. Simmons, G., P. R. Clapham, C. Picard, R. E. Offord, M. M. Rosenkilde,T. W. Schwartz, R. Buser, T. N. C. Wells, and A. E. I. Proudfoot. 1997.Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes bya novel CCR5 antagonist. Science 276:276–279.

104. 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 vi-rus type 1 isolates are dual-tropic and most can use either Lestr or CCR5as coreceptors for virus entry. J. Virol. 70:8355–8360.

105. Singh, A., G. Besson, A. Mosbacher, and R. G. Collman. 1999. Patterns ofchemokine receptor fusion cofactor utilization by human immunodeficiencyvirus type 1 variants from the lungs and blood. J. Virol. 73:6680–6690.

106. Sol, N., F. Ferchel, J. Braun, O. Pleskoff, C. Treboute, I. Ansart, and M.Alizon. 1997. Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 byprimary and cell line-adapted human immunodeficiency virus type 2. J. Vi-rol. 71:8237–8244.

107. Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson, G. P. Allaway,C. Cheng-Mayer, J. Robinson, P. J. Maddon, and J. P. Moore. 1996.CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 384:184–187.

108. Trkola, A., C. Gordon, J. Matthews, E. Maxwell, T. Ketas, L. Czaplewski,A. E. I. Proudfoot, and J. P. Moore. 1999. The CC-chemokine RANTESincreases the attachment of human immunodeficiency virus type 1 to targetcells via glycosaminoglycans and also activates a signal transduction path-way that enhances viral infectivity. J. Virol. 73:6370–6379.

109. Trkola, A., T. Ketas, V. N. KewalRamani, F. Endorf, J. M. Binley, H.Katinger, J. Robinson, D. R. Littman, and J. P. Moore. 1998. Neutralizationsensitivity of human immunodeficiency virus type 1 primary isolates toantibodies and CD4-based reagents is independent of coreceptor usage.J. Virol. 72:1876–1885.

110. Trkola, A., J. Matthews, C. Gordon, T. Ketas, and J. P. Moore. 1999. A cellline-based neutralization assay for primary human immunodeficiency virustype 1 isolates that use either the CCR5 or the CXCR4 coreceptor. J. Virol.73:8966–8974.

111. Trkola, A., W. A. Paxton, S. P. Monard, J. A. Hoxie, M. A. Siani, D. A.Thompson, L. Wu, C. R. Mackay, R. Horuk, and J. P. Moore. 1998. Geneticsubtype-independent inhibition of human immunodeficiency virus type 1replication by CC and CXC chemokines. J. Virol. 72:396–404.

112. Tscherning, C., A. Alaeus, R. Fredriksson, A. Bjorndal, H. Deng, D. R.Littman, E. M. Fenyo, and J. Albert. 1998. Differences in chemokine co-receptor usage between genetic subtypes of HIV-1. Virology 241:181–188.

113. Wells, T. N. C., C. A. Power, M. Lusti-Narasimhan, A. J. Hoogewerf, R. M.Cooke, C.-W. Chung, M. C. Peitsch, and A. E. I. Proudfoot. 1996. Selectivityand antagonism of chemokine receptors. J. Leukoc. Biol. 59:53–60.

114. Wu, L., N. P. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti,A. A. Cardoso, E. Desjardin, W. Newman, C. Gerard, and J. Sodroski. 1996.CD4-induced interaction of primary HIV-1 gp120 glycoproteins with thechemokine receptor CCR-5. Nature 384:179–183.

115. Xiao, L., D. L. Rudolph, S. M. Owen, T. J. Spira, and R. B. Lal. 1998.Adaptation to promiscuous usage of CC and CXC-chemokine corecep-tors in vivo correlates with HIV-1 disease progression. AIDS 12:F137–F143.

116. Zhang, Y.-J., T. Dragic, Y. Cao, L. Kostrikis, D. S. Kwon, D. R. Littman,V. N. KewalRamani, and J. P. Moore. 1998. Use of coreceptors other thanCCR5 by non-syncytium-inducing adult and pediatric isolates of humanimmunodeficiency virus type 1 is rare in vitro. J. Virol. 72:9337–9344.

117. Zhang, Y.-J., and J. P. Moore. 1999. Will multiple coreceptors need to betargeted by inhibitors of human immunodeficiency virus type 1? J. Virol.73:3443–3448.

118. Zingoni, A., H. Soto, J. A. Hedrick, F. Stoppacciaro, C. T. Storlazzi, F.Sinigaglia, D. D’Ambrosio, A. O’Garra, D. Robinson, M. Rocchi, A. San-toni, A. Zlotnick, and M. Napolitano. 1998. The chemokine receptor CCR8is preferentially expressed in Th2 but not Th1 cells. J. Immunol. 161:547–551.


coreceptor usage

Health & Medicine