Coreceptor usage


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Coreceptor usage

  1. 1. JOURNAL OF VIROLOGY, Aug. 2000, p. 6893–6910 Vol. 74, No. 150022-538X/00/$04.00ϩ0Copyright © 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 usagecoreceptors during the process of virus-cell fusion (reviewed in by SIV strains is, however, very rare, although an example ofreferences 6, 8, 9, 25, 74, and 87). Usually, this process requires SIV entry via CXCR4 is known, albeit for an isolate obtainedan initial interaction of the viral envelope glycoproteins with from mandrills (SIVmnd) (98). However, the initial reports ofCD4 (48, 107, 114). However, rare examples of CD4-indepen- SIVmac entry via CCR5 concluded that additional coreceptorsdent HIV-1 isolates have been described (29, 47), and several used by SIV may be substitutes for CXCR4 (12, 18, 38). SinceHIV-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 efficiencyfirst 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 othercoreceptors has also been described to function for HIV-1 coreceptors are as important as CCR5 and (for HIV-1 andentry, especially for SI viruses, at least in the context of core- HIV-2) CXCR4 for viral replication in vivo. There is mountingceptor-transfected cells in vitro (5, 19, 20, 22, 27, 31, 32, 38, 40, evidence that many, if not all, of the other coreceptors have50, 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 tissuescoreceptor-transfected cells in vitro. In general, coreceptor and cell types (13, 31, 43, 70, 81, 86, 101, 102, 117). Care mustusage 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 at48, 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 cellswas CCR5, which was shown to function with SIVmac isolates but that an alternative coreceptor(s) may indeed be relevant, atsoon 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 systemsCornell 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 selected simian/human immunodeficiency viruses (SHIV). 6893
  2. 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 forHIV-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 wasMichael Miller, Susan Vice, Bahige Baroudy, and Stuart McCombie (Schering removed and the cells were washed once with 1 ml of medium. A 750-␮l aliquotPlough 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 ofprovided by Amanda Proudfoot, Serono Pharmaceutical Research Institute, harvested culture supernatant was measured. For a few slowly replicating SIVGeneva, 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 controlmotactic 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 is5160 and 5073, derived from individuals with AIDS, have been described previ- discussed in Results. Attempts were made to quantify CXCR4 expression on theously (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 expressan 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 quantitatebeen described elsewhere (41, 80). Three of these (7924A, 77618, and GB122) the extent to which CXCR4 expression varied among the various lines. Thiswere isolated from individuals with AIDS, one (7312A) was isolated from an situation is consistent with the experience of others (Dan Littman, personalindividual 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 PBMCof 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 usedtheir 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 withpropagated and titrated in phytohemagglutinin-activated human peripheral SHIV. Stimulated PBMC (75 ␮l) were cultured in 96-well plates at 2 ϫ 105 perblood 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 orSIVsm (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 TCID50Zhiwei 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 0.4SIVmac239, 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, andcapped mangabey by cocultivation with human PBMC (15). All SIV strains were 4 nM. For each virus tested, five wells without drugs and five wells containingpropagated 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 antigenSHIV 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 afterblood 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 eachphytohemagglutinin (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 usedHofmann-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 cellsgous 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 p24105/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 PBMCcontaining 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 triplicatewells. Three wells lacked cells to provide a control for the viral antigen input. Rhesus macaque PBMC were prepared by similar procedures, except that they RESULTSwere stimulated for 3 days with staphylococcal enterotoxin B (Sigma ChemicalCo., 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. Wetion, 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 replicationcapture 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 oneHIV-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 widelyprovided with the kit. The captured p27 antigen was then detected using the and efficiently used by HIV-1, HIV-2, and SHIV isolates. Nonebiotin-labeled anti-SIV Gag polyclonal antibodies provided with the kit. Toincrease the sensitivity of antigen detection, we used a modified protocol that of the SIV used CXCR4, a feature that distinguishes SIV frominvolved 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 CCR5Tropix Inc., Bedford, Mass.). The plates were read with a microtiter plate (Table 1). SIVrcm was originally isolated from a red-cappedluminometer (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 thechemiluminescent detection system increased the sensitivity of HIV-2 or SIV p27 tated, inactive CCR5 gene, the ⌬24-CCR5 allele (15). SIVrcmdetection by more than 100-fold. HIV-1 p24 antigen was detected as described has a unique pattern of coreceptor usage in that it uses CCR2previously (109, 111), except that the chemiluminescent detection system was and not CCR5 as its major coreceptor (15). We confirmed thisused. 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/GPR15cultured 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. 3. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6895TABLE 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 US28HIV-1 P6-v3 1,000 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϪHIV-1 P6-v3 1,000 Ϫ Ϫ Ϫ ϩϩϩ ϩ ϩϩϩ Ϫ ϩϩϩ Ϫ ϩ ϩ ϪHIV-1 5073 1,000 ϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ ϩ ϩ ϩHIV-1 5160 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ ϩ ϩ ϩHIV-1 NL4-3 1,000 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϪSHIV 89.6PD 500 Ϫ Ϫ ϩϩϩ Ϫ ϩϩϩϩϩ ϩϩϩ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩϩ ϩϩ ϩϩϩSHIV 89.6PD-hu 500 Ϫ Ϫ ϩϩ Ϫ ϩϩϩϩϩ ϩϩ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ ϩϩSHIV KU-2 500 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ ϩϩSHIV SF33A 500 Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩ ϪHIV-2 310340 1,000 Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϪHIV-2 310342 1,000 Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϪHIV-2 7312A 1,000 Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ ϪHIV-2 GB122 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩϩHIV-2 77618 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩ ϩHIV-2 7924A 1,000 ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ ϩϩ ϩϩϩϩ ϩϩϩSIVrcm 100 Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ ϩϩϩϩ Ϫ ϩϩϩϩϩSIVrcm 500 Ϫ ϩϩϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩϩϩ Ϫ ϩϩϩϩϩ Ϫ ϩϩϩϩϩSIVmac239 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ ϩϩ ϩϩϩ ϩ Ϫ ϩϩ ϪSIVmac251 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ ϪSIVmac239/5501 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩϩ Ϫ Ϫ ϩϩϩϩ ϩϩϩϩ ϩϩϩ Ϫ ϩϩϩ ϪSIVmac251/1390 500 Ϫ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ ϪSIVsmpbj 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 antigenproduction) 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. 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.viruses that could use CXCR4 efficiently, were usually able to fected with other coreceptors was only rarely comparable toreplicate 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-transfectedthe use of different GHOST-CD4 cell clones and/or isolates cells, HIV-2 7924A in APJ- or US28-transfected cells, andwith a different passage history. SHIV 89.6PD in V28-transfected cells (Table 1). Whether 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- 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 followingTABLE 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-CCR5SHIV 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 161at 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. 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 enterSIVmac239 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 CEMx174SIVmac239/5501 100 394 366 4.5 7 923 455 cells (Table 3). This virus was also unable to use any coreceptorSIVmac251 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-typeSIVrcm 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). Thevirus entry into the various GHOST-CD4 cell lines actually remaining three HIV-2 isolates, GB122, 77618, and 7924A, alloccurs 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 theseCEMx174 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 humanter 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 canthe abilities of the isolates to replicate in human PBMC from use a coreceptor other than CCR5 to enter primary, CD4ϩeither 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, andexpress functional CCR5 proteins (21, 61, 95). We also used SIVmac251/1390 replication in ⌬32-CCR5 PBMC from donor 1the CEMx174 human B/T-hybrid line because these cells can was never more than 5% and usually was less than 1% thesupport high-level SIV replication. CEMx174 cells are CXCR4ϩ replication of the same viruses in wild-type PBMC (Table 4). Abut CCR5Ϫ (18, 58, 110) and strongly express the SIVmac251 second experiment also included ⌬32-CCR5 PBMC from twoand 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 donorSF162 and P6-v3 were unable to replicate in the ⌬32-CCR5 1 (Table 5). However, both isolates replicated well in PBMCPBMC from donor 1 (Table 2). Similar results were obtained from ⌬32-CCR5 donors 2 and 3, although antigen productionwith PBMC from two other ⌬32-CCR5 donors (data not from SIVmac251 in cells from donor 2 was lower than that fromshown; see also Table 5). These observations are consistent typical CCR5 wild-type donors (Table 5). Thus, PBMC fromwith 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 celltypes). ND, not done.
  5. 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 inthe 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 whichreplication was inhibited by each agent was recorded.
  6. 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 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 ␮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. 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.
  7. 7. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6899other than CCR5 that can be used with reasonable efficiency by AMD3100. As expected, the efficient replication of SHIVmembers 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 preventedPBMC (Tables 4 and 5), consistent with its lack of dependence the inefficient replication of SHIV 89.6PD in GHOST-CD4on CCR5 for entry into PBMC (15). SIVmac239 and SIVmac251 cells expressing either CCR3, V28, APJ, US28, or CCR8 andalso replicated efficiently in CEMx174 cells, as found previ- significantly inhibited the limited replication of HIV-2 7924Aously (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 detectableCCR2, nor the minor ones Bonzo, US28, and V28 are ex- effect on SIVmac239 entry into GHOST-CD4 cells expressingpressed 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 linesAMD3100 (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 patternagents is an important issue. Previous studies have found of inhibition shown by AMD3100. One is that AMD3100 isthat AMD3100 is specific for CXCR4 (26, 37, 56, 100) and broadly reactive with multiple coreceptors but that certainthat TAK-779 can interact with both CCR5 and CCR2 (4). viruses, particularly SIV, can still interact with some of theseAlthough RANTES fully activates all of its receptors, AOP- coreceptors even in the presence of AMD3100. The other isRANTES is able to do this only for CCR5; it has half the that the apparent cross-reactivity of AMD3100 is an artifact ofactivity 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 thisinhibitor 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 replicationAMD3100, 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), thesetors by using viruses that were broadly tropic in these cells. For findings strongly suggest that the entry of SHIV 89.6PD andeach 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 individualCCR5 was sensitive to both TAK-779 and AOP-RANTES but GHOST-CD4 cell lines, although we were unable to accuratelynot 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-transfectedentry into GHOST-CD4 cells expressing CCR3, consistent GHOST-CD4 cell lines is, therefore, most probably explainedwith an interaction between AOP-RANTES and CCR3, a by its antagonism of viral entry via endogenous CXCR4. Theknown RANTES receptor (data not shown). However, neither coreceptor usage information presented in Table 1 should beTAK-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, andshown). Both TAK-779 and AOP-RANTES inhibited SIV- HIV-2 replication in PBMC. The replication of each test virusmac239 entry into GHOST-CD4 cells expressing CCR5, but the in mitogen-stimulated PBMC in the presence and absence ofentry 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 withAOP-RANTES (Fig. 1a). The entry of SIVrcm into GHOST- AOP-RANTES were also tested. Each inhibitor, alone and inCD4 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 forentry 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 fromreplication in cells expressing V28, APJ, or US28 (data not CCR5 wild-type donors were used in experiments with HIV-1shown). Neither TAK-779 nor AOP-RANTES inhibited the and HIV-2 isolates and SIVrcm; rhesus macaque PBMC werereplication of HIV-1 P6-v3 in GHOST-CD4 cells expressing used with other SIV; and both human and macaque PBMCBonzo (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 (Tablehibits entry via CCR5 and CCR2. The latter result is consistent 1), were evaluated with human PBMC (Fig. 2). P6-v3, a viruswith the report that TAK-779 binds to both CCR2 and CCR5 able to use CCR5 and Bonzo, was completely inhibited by bothbut 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 tocells expressing any one of the eight coreceptors that we were each of the three inhibitors, but its replication was fullyable to evaluate: CXCR4, CCR8, V28, US28, APJ, BOB, blocked by combinations of either TAK-779 or AOP-RANTESBonzo, 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. 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 SHIVisolates evaluated were 89.6PD in macaque and human PBMC (a) and KU-2 and SF33A in human PBMC (b).
  9. 9. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6901CD4 cell lines, including GHOST-CD4 cells expressing CXCR4, 5b). Taken together with the insensitivity of HIV-2 7924A tobut 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 coreceptorstudies 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 CXCR4Thus, SHIV 89.6PD replication in either macaque or human must be cell type dependent, since we determined that the IC50PBMC 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 ␮MSHIV 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 SIVthese SHIV in PBMC therefore appears to be CXCR4. This isolates, we used macaque PBMC. In cells from the first donorfinding was unexpected for SHIV 89.6, 89.6P, and 89.6PD, con- macaque tested, SIVmac251, SIVmac239, SIVmac251/1390, andsidering 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 notboth 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 arecan 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 CXCR4CCR5-directed inhibitors in PBMC (Fig. 4a). HIV-2 77618 and for entry (Table 1), we tested chemokine ligands of CCR2 forGB122 were almost completely (Ͼ95%) blocked by AMD3100, their abilities to inhibit SIVrcm replication in human PBMC. Ofwhereas 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-779isolates 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 expressingvirus was partially sensitive to AMD3100, but the extent of CCR2 (Fig. 1a). However, AOP-RANTES had no effect oninhibition did not exceed 30% even at the highest AMD3100 SIVrcm replication in human PBMC (data not shown). Thisconcentration, 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 macaqueinhibition 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 SIVmac251coreceptor-targeted inhibitors. The insensitivity of HIV-2 to replicate in human PBMC from ⌬32-CCR5 homozygous7924A to AMD3100 is unusual, since this virus can use individuals (Table 4). There is also a donor dependency in theCXCR4, 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 totions (IC50s) of AMD3100 against viruses that use CXCR4 in which TAK-779, at 3.3 ␮M, inhibited SIVmac239 replicationPBMC are 4 to 40 nM (Fig. 2b, 3a and b, and 4b; also data not varied from Ͼ99% to Ͻ50% in PBMC from four differentshown). To evaluate whether the insensitivity of HIV-2 7924A macaques (Fig. 7a). The IC50s of TAK-779 ranged from 240to 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-779type donors (Fig. 5a). Donor-to-donor variation in the potency completely inhibited SIVmac239 replication in all four donorsof 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 againstas high as 40 ␮M AMD3100 remains specific for CXCR4 is not HIV-1 replication in human PBMC. For instance, HIV-1 P6-v3known, 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 suggestsPBMC from a ⌬32-CCR5 homozygous donor (donor 1). For that major variations in inhibition potency are not an inherentthe 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 SIVmac251virus 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 (3.3inhibited by 4 ␮M AMD3100 throughout the duration of cul- ␮M), or AOP-RANTES (40 nM), alone or in combinationturing (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 byCXCR4 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 enterthose 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 use7924A replication in PBMC from any of the four donors, to enter human or macaque PBMC from some donors is notwhereas HIV-1 NL4-3 replication was efficiently blocked (Fig. yet known.
  10. 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. 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 fourdifferent 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 panela. The data shown were obtained on day 10, but values from day 7 were similar.
  12. 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 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 ␮M, 330 nM, and 33 nM.
  13. 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 usagemembrane receptors as coreceptors in transfected cell lines. of CXCR4-tropic viruses is determined with transfectedHowever, questions have been raised as to whether corecep- GHOST-CD4 cell lines. Many, if not all, of the “positives” fortors 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 simply43, 70, 86, 101, 116, 117). This issue affects the development of reflect entry via endogenous CXCR4 and not the transfectedantiviral drugs aimed at coreceptors. Must multiple corecep- coreceptor. This caveat may also apply to other studies thattors be targeted, or just CCR5 and CXCR4 (117)? Does the have used these cell lines. A similar conclusion was recentlyability 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 thatmates (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 notAMD3100 for CXCR4 and TAK-779 and AOP-RANTES for inhibit viral entry via CCR3, whereas AOP-RANTES can doCCR5. 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 theCCR3, respectively. However, we could find no evidence that replication in PBMC of all of the R5, NSI HIV-1 and HIV-2AMD3100 is anything other than specific for CXCR4, as found isolates that we tested, CCR3 is not relevant to their entry. Anypreviously with other assay systems and test viruses (26, 37, 56, possible use of CCR2 that might be masked by TAK-779 is not100). 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 trulyAPJ, and others actually occurs via endogenous CXCR4 and CCR2-tropic virus yet identified (15). Another advantage ofnot 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 ofHIV-2 7924A use CXCR4 very efficiently, so they may be able X4 HIV-1 isolates (44, 108). However, we did not observeto enter coreceptor-transfected GHOST-CD4 cells that ex- infectivity enhancement with human or macaque PBMC at thepress 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 aboutCD4 clones, making some transfected cell lines particularly the paramount role of CCR5 and CXCR4 in HIV-1 replicationsusceptible to viruses that use CXCR4, although we could not in PBMC (116). This is not to say that other coreceptors are
  14. 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 wasevaluated 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 replicationin 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 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.
  15. 15. VOL. 74, 2000 CORECEPTOR INHIBITORS AND HIV-2 AND SIV REPLICATION 6907completely irrelevant; Bonzo/STRL33 can be used by rare negligible. However, in cells from a second such individual, theHIV-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 potencytential 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 ismacaque PBMC; AMD3100 was sufficient to completely inhibit expressed in PBMC from only a subset of macaques or that istheir replication, while neither TAK-779 nor AOP-RANTES expressed in cells from all macaques but at different levels thathad any effect. Thus, although HIV-1 89.6 can enter trans- are sometimes below a threshold needed for infection. Thefected 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 thatnote 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 theentry, at least in CCR5-transfected cells, but CXCR4 is pre- highly variable rates at which different infected macaquesferred in primary cells. Why this should be the case and progress to disease and death (23, 57, 73). However, at least forwhether it matters for transmission and pathogenesis studies SIVmne, CCR5 usage is maintained throughout the course ofwith 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 istion 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 replicationvery 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 intoonly 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 theOne possibility is that HIV-2 7924A is able to use an alterna- same coreceptor as the one used by SIVmac239 to enter humantive 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 relatedlates 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 ininsensitive to AMD3100. The latter explanation would be con- macrophages, there is no correlation between the coreceptorsistent with the observation that very high concentrations of usage profiles of these viruses in transfected cells and theirAMD3100 do completely inhibit the replication of HIV-2 tropism for primary cells (53, 75, 76, 86). There is also no7924A, although there may be concerns about the specificity of relationship between the in vitro tropisms of SIVmac strainsAMD3100 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 Ttion 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), inferent cell types (69); this property might be one explanation a manner that we have shown is sensitive to TAK-779. Thefor why AMD3100 is a potent inhibitor of HIV-2 7924A in ability of SIVrcm to enter GHOST-CD4 cells expressing US28GHOST-CD4 cells expressing CXCR4 (IC50 ϭ 0.47 ␮M) but and V28 in vitro is likely to be of limited relevance to thecan 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 toare 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 usuallyThe CCR5 proteins from multiple primate species can function the paramount coreceptors. An unknown coreceptor(s) canas viral coreceptors (55, 78), and our inhibitor studies are con- perhaps be used by SIVmac239 and HIV-2 7924A to entersistent 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 bothSIV 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 ofby 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 giventhe genetic relationships among these virus families and theevolution of HIV-2 from SIVsm (16, 17, 41, 42, 44, 49). The ACKNOWLEDGMENTSminimal use of CXCR4 by SIV strains is mirrored by that ofHIV-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 Vineetuse a coreceptor other than CCR5 to enter PBMC from some KewalRamani for GHOST cells; and James Hoxie and Nelson Michaelhuman and macaque donors. Thus, in PBMC from one ⌬32- for preferring hard liquor to blood. We appreciate helpful commentsCCR5 homozygous human donor, SIVmac239 replication was by Amanda Proudfoot and Bob/GPR15 Doms.
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