A Human Immunodeficiency Virus Type 1 Isolate ... - Journal of Virology

JOURNAL OF VIROLOGY, Apr. 2002, p. 3114–3124 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.7.3114–3124.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 7

A Human Immunodeficiency Virus Type 1 Isolate from an Infected Person Homozygous for CCR5⌬32 Exhibits Dual Tropism by Infecting Macrophages and MT2 Cells via CXCR4 Hassan M. Naif,1* Anthony L. Cunningham,1 Mohammed Alali,1 Shan Li,1 Najla Nasr,1 Marc M. Buhler,2 Dominique Schols,3 Erik de Clercq,3 and Graeme Stewart2 Centre for Virus Research1 and Institute of Immunology and Allergy Research,2 The Westmead Millennium Institute, The University of Sydney, Sydney, New South Wales, Australia, and Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium3 Received 20 August 2001/Accepted 17 December 2001

The mechanisms of human immunodeficiency virus (HIV) infection of a man (VH) homozygous for the CCR5⌬32 mutation were investigated, and coreceptors other than CCR5 used by HIV type 1 (HIV-1) isolated from this individual were identified. In contrast to previous reports, this individual’s rate of disease progression was not accelerated. Homozygosity for CCR5⌬32 mutation was demonstrated by PCR and DNA sequencing (R. Biti et al., Nat. Med. 3:252–253, 1997). CCR5 surface expression was absent on T lymphocytes and macrophages. HIV was isolated by coculture with peripheral blood mononuclear cells (PBMCs) from siblings who were homozygous (VM) or wild type (WT) for the CCR5⌬32 mutation. The virus demonstrated dual tropism for infection of MT2 cell line and primary macrophages. Sequencing of the full HIV genome directly from the patient’s PBMCs revealed 21 nucleotide insertions in the V1 region of gp120. The VH envelope sequence segregated apart from both the T-cell-line-adapted tropic strains NL4-3 and SF2 and M-tropic strain JRFL or YU2 by phylogenetic tree analysis. VH was shown to utilize predominantly CXCR4 for entry into T lymphocytes and macrophages by HOS.CD4 cell infection assay, direct envelope protein fusion, and inhibition by anti-CXCR4 monoclonal antibody (12G5), SDF-1, and AMD3100. Microsatellite mapping demonstrated the separate inheritance of CXCR4 by both homozygote brothers (VH and VM). Our study demonstrates the ability of certain strains of HIV to readily use CXCR4 for infection or entry into macrophages, which is highly relevant to the pathogenesis of late-stage disease and presumably also HIV transmission. Dualtropic strains of HIV utilize CCR5 and CXCR4 (R5X4 strains) to enter macrophages and T-cell lines, respectively, but they can also utilize combinations of major and minor coreceptors (42). Furthermore, there is coexpression of chemokine receptors, especially CCR5 and CXCR4, on most cell types, including blood and tissue macrophages, dendritic cells, and T lymphocytes (8, 37, 57). Some primary, but not laboratoryadapted X4, T-tropic isolates can also enter macrophages via CXCR4 (dualtropic X4 strains) (49, 55). Major and minor coreceptors are also coexpressed, and CCR3, in addition to CCR5, can be utilized by M-tropic strains in fetal microglial cells (24, 48), although there is no consistent agreement as to the relative importance of CCR3 and CCR5 in adult microglial cells (1, 23). Naturally occurring mutations of CCR5 influence susceptibility to HIV infection. A 32-nucleotide deletion in CCR5 (CCR5⌬32) was found to be common in Caucasians, with heterozygosity being present in 20% of the population and homozygosity in 1%. CD4 T lymphocytes and macrophages from individuals homozygous for this mutant do not express functional CCR5 on the surface (CCR5⌬32/⌬32) and cannot usually be infected with non-syncytium-inducing R5 strains of HIV-1 (12, 32, 46). Heterozygotes progress more slowly to AIDS and death than individuals with the wild-type CCR5wt/wt genotype (26, 35) and are overrepresented among long-term nonprogressors (52). Only a few homozygotes infected with HIV-1 (4, 39, 53) have been reported, including the first reported by us (7). The characterization of an HIV strain infect-

The majority of the primary isolates of human immunodeficiency virus type 1 (HIV-1) display a non-syncytium-inducing phenotype regardless of the route of infection and have the ability to enter and infect both macrophages and T lymphocytes (M-tropic) (21, 54) through binding to both CD4 and the chemokine receptor, CCR5 (R5 strains) (3, 10, 15, 17, 30). CCR5 has been shown to be the main coreceptor involved in both vertical and sexual transmission. R5 strains dominate during the early stage of seroconversion and in the asymptomatic phase (11, 47, 59). In ca. 40% of patients at advanced stages of disease, HIV isolates show syncytium-inducing capacity in T lymphoblastoid cells (T-cell-line-adapted-tropic [TCLA] strains) and mainly utilize the chemokine receptor, CXCR4 (X4 strains) (5, 20, 30). Other chemokine receptors, principally CCR3 and CCR2b, function as minor HIV coreceptors (10, 16). Coreceptors have also been shown to mediate the entry of simian immunodeficiency virus and some M-tropic HIV-1 and HIV-2 strains, including Bonzo/STRL33 (2, 14), Bob/GPR15 (14, 19), US28 (41), CCR8 (25), CX3CR1/V28 (43, 44), and CCR9 (APJ) (9, 18). Another coreceptor, GPR1, mediates the entry of simian immunodeficiency virus but not HIV-1 (19).

* Corresponding author. Mailing address: Molecular Pathogenesis Laboratory, Centre for Virus Research, Westmead Millennium Institute, The University of Sydney, Westmead, NSW 2145, Australia. Phone: (61-2) 9845-9118. Fax: (61-2) 9845-9100. E-mail: hassan_naif @wmi.usyd.edu.au. 3114

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ing one of these patients has been demonstrated (33). The viral quasispecies in this patient were homogeneous, T-tropic, syncytium inducing, utilized only CXCR4, and replicated well in T cells but not in macrophages. In the present study, the HIV strain isolated from our patient was characterized for viral phenotype and genotype, macrophage tropism, and replication kinetics in MT2 cell lines and in primary macrophages and T lymphocytes from siblings who were CCR5⌬32/⌬32 or CCR5wt/wt. Coreceptor utilization of the HIV strains isolated from these cells was also determined. In contrast to the isolate described by Michael et al. (33), this strain was tropic for macrophages as well as T-cell lines. MATERIALS AND METHODS Brief case history. The clinical course for the patient evaluated here to late 1996 has been described previously (7). In brief, he presented in 1992 with a seroconversion-like illness of 1 month’s duration, at which time he had an HIV antibody profile to all major gag, env, and pol proteins on Western blot. His previous HIV antibody test, performed 3 years earlier, had been negative. The mode of infection was via homosexual intercourse with no history of intravenous drug use or exposure to blood products. The patient’s CD4⫹-T-cell count was 960 cells per ␮L (41%) at presentation in March 1992 and declined to 320 (20%) by February 1997 when he commenced antiretroviral therapy with zidovudine 250 mg b.i.d. and 3TC 150 mg b.i.d. He has remained on this therapy to the current time. This resulted in an increase of CD4⫹-T-cell count to a peak of 546 cells/␮l (21%), with the most recent values, in April 2001, being 440 cells/␮l (20%). His first HIV-1 RNA concentration (viral load) in plasma was measured in June 1996 at 19,000 copies per ml (prior to that he had been repeatedly negative by HIV p24 antigen assay). His highest viral load, 26,000, was recorded at the start of antiretroviral therapy. This became undetectable (⬍200 copies/ml) until May and August 1999, when values of 700 and 900/ml were recorded due to incomplete adherence to therapy. With full compliance to his dual therapy, his viral load decreased to ⬍50 copies/ml in April 2001. He has remained symptom-free throughout. Primary cell isolation and culture conditions. Blood-derived monocytes were isolated from 150 ml of whole blood from healthy HIV-seronegative donors as previously described (27). Briefly, peripheral blood mononuclear cells (PBMCs) were obtained by differential centrifugation on Ficoll-Hypaque (Pharmacia-Amersham, Sydney, Australia). Monocytes were isolated from PBMCs by countercurrent elutriation (Beckmann centrifuge J-6 M/E fitted with a JE 5.0 elutriation rotor) with OKT3-complement lysis as an extra step to purify monocytes from contamination with T cells. The isolated monocyte populations were ⬎96% positive for nonspecific esterase. Cells were cultured in the absence of growth factors in 1.0 ml of RF10/10 medium, containing RPMI 1640 supplemented with antibiotics, 10% heat-inactivated fetal bovine serum and 10% heat-inactivated pooled AB⫹ human serum at a density of 106 per well in a 24-well tissue culture plate (Nunc). Cultures were replenished with fresh RF10/10 medium every 3 or 4 days. Monocytes were allowed to adhere for either 16 h or 7 days to monocytederived macrophages (MDM), as judged by cell morphology and maturation cell surface markers (CD14 and CD68). T lymphocytes were isolated from the PBMCs and purified from monocytes by plastic adherence. Cells were phytohemagglutinin-stimulated for 48 h and then replenished with fresh RF10 supplemented with 10% human interleukin-2 (Roche/Boehringer Mannheim). Viral isolates, preparation of virus stocks, and detection of replication. The HIV isolate from this patient (VH) was isolated and expanded by coculturing the patient’s PBMCs either with mononuclear cells from his brother (VM), who was also homozygous for CCR5⌬32/⌬32, and from PBMCs from a control sibling (or an unrelated donor) with CCR5wt/wt (WT ⫽ GO sister) genotypes. These two isolates (VM and WT) were passaged once more in the same cells to prepare virus stocks. Titers of the resulting stocks were determined by reverse transcription assay and p24 antigen enzyme-linked immunosorbent assay (ELISA) and for infectious particles on normal donor PBMCs; the 50% tissue culture infective dose (TCID50) was determined by the method of Spearman-Karber. Monocytes, MDM, MT2 cell lines, or activated primary T lymphocytes were infected with the VM or WT virus stock at a multiplicity of infection (MOI) of 0.02 TCID50/cell. The replication kinetics were examined by sampling supernatants from T lymphocytes, monocytes, MDM, and the MT-2 cell line and measuring the extracellular HIV p24 antigen by using a commercial ELISA kit (Coulter Electronics,

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Sydney, Australia) and HIV cDNA (at the stage of reverse transcription) by quantitative PCR. Syncytium induction by MT-2 assay. High-titer virus (VM) was added to 2 ⫻ 105 cells/ml at an MOI of 0.1 TCID50/ml in a 48-well plate. After 4 h of incubation, the inocula were completely washed off, and cultures were replenished with fresh RF10. Cells were passaged twice weekly for 14 days and scored for syncytia as follows: ⫺, no syncytia; ⫹, fewer than 5 syncytial nuclei; ⫹⫹, 5 to 19 syncytial nuclei; ⫹⫹⫹, 20 to 40 syncytial nuclei; and ⫹⫹⫹⫹, ⬎40 syncytial nuclei. After 14 days, the extracellular p24 antigen in the culture supernatants was measured to assess the production of HIV. HIV cDNA detection by quantitative PCR. Cell lysates of different cell types were prepared as previously described (38). Briefly, cells were lysed with DNA lysis buffer containing proteinase K at 60°C for 2 h and then incubated at 95°C for 15 min to inactivate proteinase K and stored at ⫺20°C until used for PCR. HIV-1 DNA was amplified by PCR with 2.5 U of Taq polymerase, 0.2 mM concentrations of each of the four deoxyribonucleotides, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 0.01% gelatin. Primers M667 (58) and gag1 (38) were used to amplify a 320-bp region extending from the R region within the 5⬘ long terminal repeat to the beginning of gag region, representing almost full-length synthesis of HIV cDNA. Samples were subjected to 30 cycles of amplification in a Perkin-Elmer Cetus thermal cycler as follows: 1 min at 95°C, 2 min at 60°C, and 3 min at 72°C, with a final extension at 72°C for 7 min. Concurrent reactions were also performed with primers PCO3 and PCO4 to amplify a 110-bp DNA fragments of human ␤-globin gene (45) to ensure that equivalent amounts of DNA were used in each sample reaction. PCR products were electrophoresed on a 2% agarose gel, visualized by ethidium bromide staining and UV transilluminator, and then photographed. DNA extracted from 8E5 cells containing one integrated copy of HIV-1 DNA per cell (22) was used to construct a standard curve for the quantification of HIV DNA from different cell types. Tenfold dilutions (0, 10, 102, 103, and 104 cells/ reaction) of 8E5 cells were prepared as described above. The differences in cell number between these dilutions were compensated for by the addition of uninfected PBMCs (to a total of 105) as the background. The limit of detection was between 10 and 50 copies, as previously described (36–38). Determination of ␤-chemokine release by ELISA. Production of beta chemokines (macrophage inhibitory protein 1␣ [MIP-1␣], MIP-1␤, and RANTES) were examined in the serum and PBMCs derived from the patient’s blood. The concentrations of these chemokines were measured by an ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, Minn.). Measurement of chemokine receptor surface expression on mononuclear cells by flow cytometry. The cell surface expression of CCR2b, CCR3, CCR5, CXCR4, and CD4 on the patient’s PBMCs after 2 days of stimulation with interleukin-2 was examined by flow cytometry as previously described (37). Briefly, after the cells were collected, they were washed twice with cold fluorescence-activated cell sorting buffer containing 1% fetal bovine serum and 0.01% sodium azide in phosphate-buffered saline and then resuspended in 50 ␮l of human serum and labeled with specific antibody. Cells were examined by using a Becton Dickinson FACScan flow cytometer. Monoclonal antibodies for CCR5 (2D7), CCR3 (7B11), and CCR2B (5A11) were purchased from Becton Dickinson Pharmingen (San Diego, Calif.), and monoclonal antibody 12G5 to CXCR4 was purchased from R&D Systems. Monoclonal antibody to CD4 (OKT4), anti-Leu-M3 (CD14) phycoerythrin conjugate and anti-Leu3a fluorescein isothiocyanate conjugate were purchased from Becton Dickinson (Franklin Lakes, N.J.). Determination of coreceptor usage by viral isolates. Human osteosarcoma cells expressing human CD4 (HOS.CD4 and U87.CD4) were kindly provided by D. Littman (Skirball Institute, New York University School of Medicine, New York). These cells express human CD4 and the chemokine receptors CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, Bob and Bonzo, or pBABE carrying the vector only as parental control. The cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum, 500 ␮g of G418 per ml, 100 ␮g of hygromycin per ml, and 1 ␮g of puromycin per ml; 104 cells were seeded into 24-well cell culture dishes overnight. Cells were exposed to virus-containing media at an MOI of 0.1 TCID50/cell. Cells infected with NL4-3, BaL, and VH strains were lysed for DNA extraction at day 7 after infection and examined for HIV cDNA by PCR. HIV-1 p24 antigen in the culture supernatants at days 7 and 14 was measured by using the p24 antigen ELISA kit (Coulter Electronics). U87.CD4 cells transfected with the same panel of chemokine receptors (CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, and the parental control) were also used to confirm coreceptor usage of the HIV-VH strain. Blocking experiments were used to confirm coreceptor utilization by this strain after in vitro infection of macrophages from individuals who were either wildtype or homozygous for CCR5⌬32/⌬32. Monoclonal antibodies 12G5 (5 ␮g/ml) to CXCR4, and the specific ligand SDF-1 (500 ng/ml) or CXCR4 antagonist,

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AMD3100 (100 nM), were added to cultures for 1 h prior to HIV infection in an attempt to block HIV entry and/or infection in these cells. PCR amplification, cloning, and sequencing of envelope gene. Envelope genes were cloned from cellular DNA of the patient’s PBMCs. DNA lysates were subjected to PCR amplification with nested env primers amplifying the gp120 gene. The first round of amplification was carried out for 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min with the following external primer pair: NENV1 (5⬘-TTAGGCATCTCCTATGGCAGGAAGAAGCGG at positions 5956 to 5985 of HIV-HXB2) as a sense primer and NENV2 (5⬘-TCTTGCCTG GAGCTGTTTGATGCCCCAGAC-3⬘ at positions 7951 to 7921) as an antisense primer. Aliquots (1 to 5 ␮l) of the amplified products were included in the second round of amplification with the internal primer pair NENVA (5⬘-GAAGACAG TGGCAATGAGAGTG-3⬘, at positions 6208 to 6229) as a sense primer and NENVB (5⬘-TCTTGCCTGGAGCTGTTTGATGCCCCAGAC-3⬘, at positions 7951 to 7921) as an antisense primer for 25 cycles and under the same conditions as for the first round of amplification. Both rounds of PCR were preceded by a denaturation step at 94°C for 5 min and ended by extension at 72°C for 7 min. Samples (10 ␮l) of the second round of PCR were electrophoresed on a 1.5% agarose gel, and DNA fragments of 2.0 kb for the gp120 gene were detected. DNA fragments were cloned into the TA cloning vector (Invitrogen), and DNA plasmid was extracted, purified, and tested for the env gene by PCR amplification and agarose gel electrophoresis. The presence of specific env DNA fragments was further confirmed by DNA sequencing. The purified DNA from PCR products was sequenced by using the dyedeoxy terminator method and an ABI automated DNA sequencer (373A DNA Sequencer). Sequence alignment was done with CLUSTAL W, and phylogenetic trees were generated by PHYLIP by using the Australian National Genome Information Service at the University of Sydney. DNA sequence variation outside the gp120 of the envelope region was determined by amplification and sequencing of the full genome of HIV-VH with a panel of 13 primer pairs according to the established method as previously reported (40). These primers have M13 sequence linkers to assist sequencing with M13 universal primer and by using LiCor sequencing technology. The full genome sequence of this strain has been assigned accession number AF146728. Nucleotide sequence accession numbers. The V1 and V2 sequences of four clones (VHO1 to VHO4) of the original HIV-VH after cloning and direct PCR (i.e., VHPCR) from the patient’s PBMCs were assigned accession numbers AF335002 to AF335005 for VHO1 to VHO4, respectively, and AF146728 for VHPCR. env-mediated fusion and viral infectivity assay. env clones were tested for functional activity by measuring their ability to utilize a panel of chemokine receptors in a luciferase reporter virus infection assay, as previously described (16). Briefly, env and NL-luc plasmids were transfected into 293T cells by using calcium phosphate method. Viral supernatants were harvested 2 days posttransfection, cleared of cell debris by low-speed centrifugation, and used to infect feline CCC cells transfected with CD4 and various HIV coreceptors (CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4). Infection of target cells by pseudotyped virus led to the expression of luciferase, which was quantified in cell lysates 2 to 3 days after addition of the virus. Heteroduplex tracking assay. The V3 to V5 region of JRCSF was PCR amplified with the primers V3A and V5B (30) and cloned into pCR2.1 TA cloning vector according to the instructions of the manufacturer (Invitrogen). Then, 1 ␮g of the cloned product was excised from the vector and 3⬘ end labeled with 2.0 ␮Ci of [␣-32P]dATP by using Klenow fragment to produce a standardized probe for the heteroduplex tracking assay. The V3 to V5 regions of HIV-VH, HIV-VM, and HIV-WT isolates derived from the patient’s PBMCs or cultured on PBMCs from VM and WT, respectively, were also PCR amplified as described above from a template containing 50 copies of proviral DNA. Heteroduplexes were formed between PCR products and 32P-labeled probes after they were processed as described by Delwart et al. (13). Annealed products were separated on a 5% nondenaturing polyacrylamide gel, and heteroduplex complex formation was visualized by autoradiography. Microsatellite marker study. The primer sequences for microsatellite markers d2s2215, d2s132, and d2s134 on chromosome 2 and chromosome 3 markers d3s1578, d3s3522, and d3s3559 were obtained from the Genome Database website (http:www.gdb.org). The primer sequences of markers afmb362wb9 and gaat12d11 were as previously described (50), and primer sequences of d3s4579 (IRI3.1) and d3s4580 (IRI3.2) were as described earlier (31). Each forward primer was labeled with 5⬘-Hex. PCR products were run by a urea-denatured polyacrylamide gel electrophoresis for size determination by using the GS-2000 Corbett system. Internal size standards were used alongside GeneScan-350 (Tamra) size standards (Applied Biosystems, Inc.).

J. VIROL.

RESULTS Family genotyping for CCR5 and CXCR4. The patient’s mother (VA) and three of his siblings (VM, VF, and GO) were available for study. As shown in Fig. 1, one brother (VM) was homozygous for CCR5⌬32/⌬32, one was heterozygous (VF, CCR5⌬32/wt), and a sister (GO) was of CCR5wt/wt (WT). Since peripheral blood cells from the CCR5⌬32/⌬32 brother were used for coculture with the patient’s virus, it was considered important to confirm that the CCR5⌬32/⌬32 brother had inherited the full region of chemokine receptor genes on both chromosomes (without recombination) as had his HIV-infected sibling. This was established by microsatellite analysis of the CCR5 region. Conversely, microsatellite analysis of the CXCR4 genetic region revealed that the proband and his CCR5⌬32/⌬32 brother inherited different chromosomal sequences (segments) from each parent. VH strain induced syncytia in MT2 cells and replicated efficiently in macrophages from a homozygote person. The patient’s virus (VH) was cocultured with PBMCs from both the CCR5⌬32/⌬32 (VM) and the CCR5wt/wt (WT) genotypes at the same MOI. Both viral stocks (VM and WT) were passaged in T lymphocytes and MT2 cells and shown to replicate well and with high levels (Fig. 2A). Replication started to increase at day 7 after infection and was maximal at day 12. The level of replication markedly declined at day 12 in T lymphocytes, probably because of a marked cytopathic effect (CPE) and loss of cells from the cultures. This virus caused syncytia in the MT2 cell line, which appeared to be marked at day 7 after infection. For an unknown reason, syncytia were significantly more numerous with HIV-VM than with the WT stock. More importantly, both stocks replicated very well in 7-day-old MDM from VM (Fig. 2A) but to lower levels in 16-h-old monocytes as measured by EC p24 antigen (data not shown). Both TCLA HIV–NL4-3 and HIV-IIIB, but not HIV-BaL, were able to productively infect macrophages from VM at markedly higher levels than the minimal concentration detected in macrophages from normal (CCR5wt/wt) individuals. In general, the rate of replication in the CCR5⌬32/⌬32 cells was consistently slower during the early days compared to the rate of replication in CCR5wt/wt cells (Fig. 2B). The kinetics and levels of p24 antigen were reflected in the level of HIV DNA synthesis (lower panels of Fig. 2), where higher levels of HIV DNA were observed in both MT2 cells and T lymphocytes than in macrophages; they also declined at later stages in T lymphocytes. The levels of replication of HIV-VM in MDM varied markedly between cells obtained from individuals wild type or homozygous for CCR5⌬32, where lower levels of replication were observed in MDM from the former (Fig. 2). The HIV-VH isolate induced a marked CPE of multinucleated giant cells in lymphocytes from both genotypes but the CPE was much greater (and extracellular p24 concentrations were also higher) in the CCR5⌬32/⌬32 cells from VM than those from the CCR5wt/wt. Cross-infection with HIV strains was produced by MT-2 and macrophages. Culture supernatants from HIV-infected MT-2 cell cultures at day 7 were able to productively infect MDM from VM. Similarly, supernatants from HIV-infected VM macrophages at day 14 were also able to productively infect the

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FIG. 1. Genotyping of CCR5 and CXCR4 and nearby microsatellite markers used to assign haplotypes within the patient’s (VH) family. (A) Markers and genes located in 3p21. The allele sizes (in base pairs) are shown for the microsatellite PCR products under the appropriate parental haplotype. Marker and gene locations are not drawn to scale, and cR/cM data was taken from GeneMAp99 (www.ncbi.nim.nih.gov /genemap). (B) Haplotypes for the 3p21 region assigned to family members. The genotype of CCR5⌬32 was assigned for each individual of the family as a clear box for the wild-type CCR5, the partially shaded box indicates CCR5wt/⌬32 heterozygosity, and the fully shaded box indicates CCR ⌬32/⌬32 homozygosity. VA, mother heterozygous for CCR5⌬32; GO, sister wild type for CCR5⌬32; VH, patient homozygous for CCR5⌬32; VM, brother homozygous for CCR5⌬32; and VF, brother heterozygous for CCR5⌬32. (C) Determination of CXCR4 haplotypes with chromosome 2 microsatellites for D2S2215 and D2S314.

MT-2 cell line, but to a lower level than in MDM, as measured by EC p24 antigen concentrations (data not shown). ␤-Chemokine production. The production of chemokines (RANTES, MIP-1␣, and MIP-1␤) in both sera and cultured PBMCs taken directly from VH was within the normal range for healthy individuals. The concentrations of these proteins in sera were 1,805, 45, and 95 pg/ml for RANTES, MIP-1␣, and MIP-1␤, respectively. Similarly, the concentrations in culture supernatants were 2,340, 165, and 392 pg/ml for the same chemokines. These values are consistent with our previous findings (28).

Expression of chemokine receptors on patient’s PBMCs. Flow cytometry analysis of expression of CCR3, CCR5, and CXCR4 by cultured patient’s PBMCs demonstrated a complete absence of CCR5 expression (data not shown). These cells expressed CXCR4 at relatively moderate proportions of 35%, similarly as for healthy individuals (see reference 35). Interestingly, purified 1-day-old monocytes expressed higher levels of CXCR4 at 75% (higher than the average levels in normal individuals). The expression of CCR3 and CCR2b (data not shown) was barely detectable and was shown for both to be ⬍10, but 43% of the patient’s PBMCs expressed CD4,

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J. VIROL.

FIG. 2. Replication characteristics of HIV-1 isolated from VH in his brother’s mononuclear cells (VM) who is also homozygous for CCR5⌬32 and reinoculated into VM’s cells (A) and into cells from an individual wild type for CCR5 (WT ⫽ GO) (B), as well as in MT2 cell lines. Therefore, the graphs represent replication kinetics of HIV-VM in primary PBMCs, T lymphocytes (L), and 7-day-old macrophages (MDM) obtained from a healthy individual homozygous for CCR5⌬32/⌬32 and compared to the same cell types from an individual wild type for CCR5wt/wt and in the MT2 cell line. In parallel, the R5 M-tropic HIV-BaL (BaL) and the X4 T-tropic HIV-IIIB (IIIB) and HIV–NL4-3 (NL43) laboratory-adapted strains were also used to infect the same cells from both genotypes. Cells were infected with all of these HIV-1 strains by using 0.025 infectious viral particles per cell. The levels of replication were measured by extracellular HIV p24 antigen ELISA (top panels) and DNA PCR (lower panels). Symbols: ⫹, HIV DNA from 8E5 cells from 100 cells (i.e., 100 copies of HIV DNA); ⫺, no cellular DNA. M, standard molecular weight marker.

each value being in the normal range. PBMCs from VM expressed levels of CXCR4 and CD4 similar to those of his brother’s cells, and no CCR5 was detected (data not shown). Coreceptor usage in HOS.CD4 and U87.CD4 cells. In HOS.CD4 cells, both viral stocks (HIV-VM and HIV-WT) were examined for coreceptor usage in HOS.CD4 cells. Both stocks used CXCR4 (four of four experiments) alone, as monitored by both extracellular p24 antigen and HIV cDNA (Table 1). Other known coreceptors for HIV entry, such as CCR2b, CCR3, and CCR5, were not used by these viruses, nor were CCR1, CCR4, Bonzo, and Bob. When U87.CD4 cells were used for confirmation, both viral isolates used only CXCR4. The HIV controls, the M-tropic BaL strain, exclusively used CCR5, whereas the TCLA NL4-3 strain exclusively used CXCR4. In addition, the primary syncytium-inducing HIVNBL70 strain used primarily CXCR4 but also CCR3 and Bonzo (30). Coreceptor utilization by HIV strain in T lymphocytes and MDM. To establish which coreceptor was utilized by these isolates to enter PBMCs and MDM, blocking experiments

were performed with neutralizing monoclonal antibodies for CXCR4 (12G5) and CCR5 (2D7) and specific ligands for CXCR4 (SDF-1), CCR3 (eotaxin), and CCR2b (MCP1). Binding and fusion assay revealed that this virus (HIV-VM) bound specifically and entered via CXCR4 but not CCR1, CCR2b, CCR3, or CCR5. There was only partial inhibition of HIV-VM infection of autologous VM PBMCs (or MDM) with 5 ␮g of 12G5/ml and 2.5 ␮g of SDF-1/ml added once to the culture before infection, although the inhibition by 12G5 was greater than that caused by SDF-1. SDF-1 inhibited replication up to day 9 after infection, but this suppression attenuated by day 12 after infection. This inhibition persisted, as decreased p24 antigen levels, for 10 days; the level of replication then returned to the same level as for the untreated cultures at 12 days postinfection (Fig. 3). As expected, no inhibition was observed with MIP-1␤, eotaxin, MCP-1, or the combinations eotaxin– MIP-1␤, eotaxin–MCP-1, and MIP-1␤–MCP-1 (data not shown). Thus, this strain did not appear to use CCR2b or CCR3 to enter PBMCs. Similar levels of inhibition were also observed when cells from the wild-type MDM cultures were preincu-

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FIG. 2—Continued.

bated with 12G5 or SDF-1 (separately or in combination) for 1 h and then infected with the VM stock (data not shown). However, inhibition of this strain was complete when cells from both genotypes were preincubated with AMD3100 at 100 nM (Fig. 3). This indicates that this virus uses CXCR4 as the major coreceptor to infect different cell types, including those derived from patients with wild-type CCR5 or CCR5 ⌬32 homozygous genotype. Direct Envelope binding and infection. After the VH-gp120 genes were subcloned into IIIB and JRFL genomes, they were tested for the ability to mediate cell-cell fusion with cells expressing CD4 and one of the seven different coreceptors. This assay showed that VH gp120 had a strong binding to CXCR4 and negligible binding to CCR3 (Fig. 4). These env constructs

did not bind or infect cells via other coreceptors such as CCR5, CCR1, CCR2, and CCR4. Cloning and sequencing of gp120. The coding region for gp120 of the VH strain originated from the patient’s PBMCs was amplified by using nested PCR, cloned into plasmid DNA, and sequenced. A major insertion of 15 nucleotides and another two insertions of 3 nucleotides were observed in the V1 region of gp120 of the HIV DNA extracted from the patient’s uncultured PBMC (Fig. 5A). The 15 nucleotides (GTAGAG GACTGTGAC), which are translated to TNSSPY amino acids, were inserted in the same site of the original sequence. This insertion persisted when VM stock was passaged in lymphocytes and macrophages of the same genotype (VM cells) or even cells from WT genotype (data not shown). This sequence

TABLE 1. Coreceptor usage of HIV-VH strain cultured in PBMCs from a healthy individual (VM) homozygous for CCR5⌬32a Isolate

VH-VM VH-WT HIV-BaL HIV-NL43 HIV-NBL70

Presence (⫹) or absence (⫺) of coreceptor usage CCR1

CCR2b

CCR3

CCR4

CCR5

CXCR4

Bob

Bonzo

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a Coreceptor usage was determined by using the HOS.CD4 and U87.CD4 cell lines that transfected with chemokine receptors. HIV infection was measured by ELISA and DNA PCR detection of the extracellular HIV p24 antigen and intracellular HIV DNA synthesis, respectively. The laboratory-adapted HIV-BaL strain, the HIV-NL43 strain, and the primary HIV-NBL70 strain were used as controls.

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FIG. 3. Coreceptor utilization by HIV-VM during infection of macrophages from an individual homozygous for CCR5⌬32/⌬32 by inhibition studies with monoclonal antibody to CXCR4 (12G5), with SDF-1 as the ligand specific for CXCR4 and the CXCR4 antagonist AMD3100. The level of inhibition of HIV replication was determined by measuring p24 antigen in the culture supernatants and by DNA PCR.

insertion in this position is unique to this strain, since no other HIV-1 isolate in the database or in the local Australian isolates (Fig. 5A) (30) shows the insertion. It only had homology to a region of the human X chromosome without known functional activity. Insertions of one amino acid on both sides of the major insertion were also observed in the V1 region of gp120 of HIV-VH derived from an uncultured patient’s PBMCs. The V3 sequence of this isolate was highly conserved with no major changes observed and had a net charge of 5. Phylogenetic analysis revealed that the sequences of gp120 of VH derived from patient’s PBMCs) segregated separately and at a considerable difference from the gp120 sequences of both M- and T-tropic LA strains (JRFL and YU2 strains and SF2 and HXB2 strains, respectively) (Fig. 5B). The segregation distance was closer to the TCLA strains rather than to the M-tropic strains. A similar segregation distance was observed whether the sequences were obtained through direct population or cloning sequencing. Heteroduplex tracking assay. The evolution of envelope quasispecies after coculture in VM and WT PBMCs was compared by hybridizing the envelope genes of the original and each passaged quasispecies with that of JRCSF and NL4-3 by using the heteroduplex tracking assay. Heteroduplexes were formed between amplified products of JRCSF and other sim-

ilarly amplified products of the patient’s PBMCs or after passage through sibling VM and WT cells (data not shown). There were greater differences between JRCSF and VH than with NL4-3 and VH, suggesting a CXCR4-using envelope as the dominant species and consistent with the phylogenetic analysis. Furthermore, there were differences between the original VH and the VM and WT envelopes, which are more likely to be attributed to nucleotide substitution and/or frameshift differences after the passage of VH into VM and WT cells. DISCUSSION Our finding in this study that a primary HIV isolate from an HIV-infected CCR5-deficient person can infect both macrophages and T-cell lines via the coreceptor CXCR4 extends the understanding of HIV pathogenesis obtained from the study of these unusual individuals. This study was supported by the availability of siblings of the homozygous CCR5⌬32/⌬32 and wild-type CCR5wt/wt genotypes. We examined the tropism, replication kinetics, and coreceptor utilization of HIV strains isolated from this patient after initial coculture with mononuclear cells from both the CCR5⌬32/⌬32 brother and the CCR5wt/wt sister. The isolates showed dual tropism as syncytium formation in the MT2 cell line and moderately productive

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FIG. 4. HIV-VM env-mediated fusion and functional activity for binding and entry into cells expressing the chemokine receptors. env regions from HIV-VH, after extraction of DNA from PBMCs, were cloned into HIV-IIIB (1c/IIIB, 3c/IIIB, and 5a/IIIB) and HIV-JRFL (3i/JR), and they were tested for functional activity by measuring their ability to utilize a panel of chemokine receptors (CCR5, CCR3, CCR2b, CXCR4, ChemR1, GPR15, US28, and the parental CD4 expressing cells) in a luciferase reporter virus infection assay, as previously described (16). Further details can be found in Materials and Methods. Infection of target cells by pseudotyped virus leads to expression of luciferase, which was quantified in cell lysates 2 to 3 days after the addition of virus.

infection in primary macrophages from the CCR5⌬32/⌬32 and CCR5wt/wt siblings, a finding similar to that in dualtropic X4 clinical isolates. The question is whether this reflects strains with a different tropism for T-cell lines and macrophages within the quasispecies or a predominant dualtropic strain able to utilize CXCR4 on both MT2 cells and macrophages. Crossinfection of macrophages and MT2 cells with the output virus of each suggests true dual tropism of a predominant strain. However, this needs to be confirmed in future studies by biological cloning. The predominant use of CXCR4 by this dualtropic X4 HIV-1 strain was first established by the HOS.CD4/U87.CD4 cell studies. The importance of CXCR4 was further shown in the binding-fusion studies with the VH envelope in HIV-IIIB and HIV-JRFL backgrounds. Furthermore, the 70% neutralization of infection of macrophages (up to days 7 and 10) by the anti-CXCR4 monoclonal antibody (and to a lower extent with SDF-1) but up to 95 to 100% by the CXCR4 antagonist AMD 3100 (up to day 12 after infection) showed this was clearly the dominant receptor for entry into both macrophages and T lymphocytes. AMD3100 was the most potent CXCR4 antagonist. The minor fusion with CCR3 noted in these studies is of uncertain significance. Sequencing of gp120 of HIV directly from the patient’s PBMCs showed that there were insertions of 21 nucleotides in the V1 region of HIV-VH genome. This insertion was also present in HIV produced by coculture of the patient’s PBMCs with the CCR5⌬32/⌬32 or CCR5wt/wt cells and also MT2 cells. The role of these insertions have yet to be defined but may be important in the interaction between viral gp120 and CXCR4, especially since the V1 and V2 regions, in addition to the V3 loop, appear to be important in binding to CXCR4 (6). The ability of the HIV-VM and HIV-WT isolates to infect

MDM through CXCR4 is in marked contrast to the only other report of the characterization of an isolate able to infect CCR5⌬32/⌬32 patients (33); in that report the infection of T cells but not macrophages was seen. A parallel comparative study should be conducted in the future to confirm these differences. The ability to use CXCR4 to enter macrophages may be due to viral or host cell changes, either in CXCR4 or interacting molecules. The insertions in the V1 region, which persisted after passage in both the sibling CCR5⌬32/⌬32 and CCR5wt/wt MDM could be partly responsible for such viral genotypic changes. The isolate reported by Michael et al. (33) lacks such a sequence insertion, as do primary Australian HIV-1 isolates (30), but it does have an extension at the V1 region of different sequences, as well as an insertion of 12 nucleotides in the V2 region, which does not exist in our HIV strain. It is important to determine whether the introduction of any of these sequence insertions into R5 HIV molecular clones would enable them to infect CCR5⌬32/⌬32 lymphocytes and macrophages via CXCR4 (i.e., to switch from R5 to X4). The ability of the HIV-VH and HIV-VM strains to infect PBMCs and MDM from the CCR5wt/wt sibling and from four other CCR5wt/wt donors indicates that entry via CXCR4 on these cells was not impaired by the coexpression of CCR5. However, the replication level of this virus in the wild-type MDM was consistently lower than that obtained with the homozygote MDM. Interestingly, there were changes in the predominant envelope sequences amplified directly from the patient’s cells compared to those cocultured with CCR5⌬32/⌬32 and CCR5wt/wt cells, as detected by the heteroduplex tracking assay and sequencing. This is probably due to nucleotide substitutions and/or frameshift within the amplified regions of the env gene due to passaging into different host cells. Differences were

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FIG. 5. Amino acid sequence alignment (A) and phylogenetic tree analyses (B) of V1V2 region of the envelope gene of HIV-VH from the patient’s PBMCs generated by direct sequencing of PCR-amplified products (VHPCR) or sequencing of multiple clones (VHO1 to VHO4). These sequences were compared to sequences of the same regions of HIV-HXB2, HIV-SF2, HIV-YU2, HIV-ADA, and HIVJRCSF obtained from Los Alamos HIV sequencing database (http: //hiv-web.lanl.gov), as well as two local Australian X4 HIV isolates (NB3 and NB5 [30]).

greater in cocultures with the CCR5wt/wt cells. This supports a major role for viral genotypic changes, but it does not exclude host factors, to explain macrophage infection. In regard to possible host cell changes, the cells of the patient and those of his CCR5⌬32/⌬32 brother (and cells from an unrelated CCR5-deficient donor [data not shown]) supported the growth of HIV-1 in a similar fashion. Studies are under way to recruit a larger number of CCR5-deficient donors to examine host cell genetics effects on infection and replication of this unique viral strain. Since the microsatellite analysis indicates that they inherited en bloc each of the chemokine receptors encoded on chromosome 3p, the possibility cannot be excluded of an uncommon minor coreceptor inherited in each individual. However, the CCR5wt/wt sister’s cells equally supported growth of the patient’s isolate, and microsatellite studies confirmed that the full chemokine receptor region had been inherited without recombination. Furthermore, the data presented here make it highly likely that the dominant receptor, both on T cells and macrophages, was CXCR4. In that regard, the possibility of an uncommon CXCR4 gene with higher affinity for gp120 in this family is made less likely by the fact that each brother inherited different chromosomes from each parent, as shown by microsatellite analysis. We are unable to determine whether this CXCR4 utilizing strain was selected at the time of infection of the CCR5⌬32/ ⌬32 homozygote since this isolate was obtained 4 years after presentation. This strain, however, was probably able to infect macrophages (and presumably dendritic cells), as well as T cells (via CXCR4), present in the rectum. Initial infection with a predominantly CXCR4-utilizing strain has been demonstrated to have occurred in a laboratory worker infected with the laboratory-adapted strain IIIB (56). There was subsequent alteration in tropism but not in the coreceptor usage (34). Furthermore, unlike the HIV strain characterized previously by Michael et al. (33), the HIV strain characterized here

should be capable of infecting the usual range of tissues infected by HIV (i.e., macrophages in brain, bone marrow, etc.), as well as T lymphocytes in lymphoid tissue. Evidence from other published studies has led to the suggestion (33) that HIV-1 infection in CCR5-deficient people may result in a unique clinical course characterized by a rapid decline in CD4⫹-T-cell count (29, 33, 54), associated with an unexpectedly low viral load (33, 54). This was also accompanied by the absence of a significant rise in CD4⫹-T-cell count following highly active antiretroviral therapy despite achieving sustained undetectable viral load (29). The discordance between CD4⫹-T-cell and viral load has led to the speculation that viral strains using CXCR4 as a main coreceptor may predominantly or exclusively result in infection largely confined to the T-cell compartment with limited involvement of macrophages as a reservoir of viral replication (33). This discordance was not as evident in our patient, perhaps reflecting the ability of this virus to infect both T cells and macrophages. Furthermore, this patient has not shown a rapidly progressive clinical course and is somewhat unusual in maintaining a stable CD4⫹-T-cell count and very low viral load despite only dual

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antiretroviral therapy (zidovudine plus 3TC [␤-L-2⬘,3⬘-dideoxy3⬘-thiacytidine]) for 4.5 years. Studies of more of these patients are needed to clarify this issue since it is relevant to our understanding of the pathogenesis of HIV infection in general. The absence of CCR5 is strongly protective against HIV-1 transmission (51), a finding that has raised hopes that blocking this receptor could play a role in prevention strategies for this infection, particularly since CCR5 expression is not essential for normal health. The description of a few individuals infected via CXCR4 suggests a mechanism for resistance to antiretroviral therapy with CCR5 antagonists, although this may only occur in a minority. The results presented here, along with those of Michael et al. (33), support a role for the other major coreceptor, CXCR4, with little evidence for the use of any of the other described minor coreceptors. Since antiviral agents are being developed to block both CCR5 and CXCR4, blocking both receptors may prove to be an adequate therapeutic strategy and could also be useful in preventing HIV transmission. Furthermore, our finding of a virus which appears to have adapted to using CXCR4 to infect macrophages in the naturally occurring absence of CCR5 may in the future help to explain why only a minority of primary X4 isolates infect macrophages in the majority of HIV-infected CCR5⌬32/⌬32 patients. ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia (NHMRC 980417). The provision of clinical data by Tong Liang, Nepean Hospital, is gratefully acknowledged. We also thank the laboratory of R. W. Doms for conducting the env-directed fusion assay, Dan Littman and Vineet KewalRamani for HOS and U87 cells, and Ghalib Alkhatib for helpful comments and constructive criticism. REFERENCES 1. Albright, A. V., J. T. Shieh, T. Itoh, B. Lee, D. Pleasure, M. J. O’Connor, R. W. Doms, and F. Gonzalez-Scarano. 1999. Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates. J. Virol. 73:205– 213. 2. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. Peden. 1997. A new SIV co-receptor, STRL33. Nature 388:238. 3. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272: 1955–1958. 4. Balotta, C., P. Bagnarelli, M. Violin, A. L. Ridolfo, D. Zhou, A. Berlusconi, S. Corvasce, M. Corbellino, M. Clementi, M. Clerici, M. Moroni, and M. Galli. 1997. Homozygous ⌬32 deletion of the CCR-5 chemokine receptor gene in an HIV-1 infected patient. AIDS 11:F67–F71. 5. Berson, J. F., D. Long, B. J. Doranz, J. Rucker, F. R. Jirik, and R. W. Doms. 1996. A seven transmembrane domain receptor involved in fusion and entry of T-cell tropic HIV-1 strains. J. Virol. 70:6288–6295. 6. Berson, J. F., and R. W. Doms. 1998. Structure-function studies of the HIV-1 coreceptors. Semin. Immunol. 10:237–248. 7. Biti, R., R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang. 1997. HIV-1 infection in an individual homozygous for the CCR5 deletion allele. Nat. Med. 3:252–253. 8. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, and T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829–833. 9. Choe, H., M. Farzan, M. Konkel, K. Martin, Y. Sun, L. Marcon, M. Cayabyab, M. Berman, M. E. Dorf, N. Gerard, C. Gerard, and J. Sodroski. 1998. orphan seven-transmembrane receptor apj supports the entry of primary T-cell-line-tropic and dualtropic human immunodeficiency virus type 1. J. Virol. 72:6113–6118. 10. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rowllins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Neuman, N. Gerard, G. Gerard, and J. Sodroski. 1996. The beta chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:621–628.

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