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1 Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA
2 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
3 Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia
Correspondence
Lokesh Agrawal
lokesh.agrawal{at}jefferson.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Feng et al., 1996). T-cell tropic (T-tropic) viruses use CXCR4 as a co-receptor (Feng et al., 1996), whereas macrophage-tropic (M-tropic) HIV-1 uses CCR5 (Choe et al., 1998). Some strains of HIV-1 may bind CCR3 and other chemokine receptors and use them to enter cells that express them following stable transfection (Doranz et al., 1996), but the role of CCR3 in primary cells in HIV-1 infection is unclear.
CCR5 is the major co-receptor for M-tropic HIV-1. It is utilized for HIV-1 entry into macrophages in the peripheral immune system and into brain microglia cells (Albright et al., 1999; Gorry et al., 2001; Shieh et al., 1998; Smit et al., 2001). It is mainly expressed on eosinophils, microglial cells, basophils and some T lymphocytes (Berger et al., 1999). The extent to which it functions as a co-receptor for HIV-1 entry into central nervous system (CNS) cells is conjectural, but considerable data suggest that some forms of HIV-1 envelope may bind to CCR3, so it could act as a minor co-receptor for HIV-1 entry (Alkhatib et al., 1997; Choe et al., 1996; Ghorpade et al., 1998; He et al., 1997; Ho et al., 2004; Martin-Garcia et al., 2002; Peters et al., 2004; Shieh et al., 1998). The cloned envelopes of many primary HIV-1 isolates show comparable tropism for CCR3 and CCR5 (Aasa-Chapman et al., 2006).
We approached the study of the roles of CCR3 and CCR5 in CNS HIV-1 infection from the perspective of the potential therapeutic applicability of targeting CCR3 and CCR5 to protect CNS and monocyte-derived macrophages (MDM) from HIV-1. We used viral vector-delivered transgenes that specifically target CCR3 and CCR5 to decrease their concentrations at the plasma membrane: interfering RNA (RNAi) and single-chain Fv antibodies (SFv).
Post-transcriptional gene silencing by small double-stranded RNAs (siRNA) is highly sequence specific and leads to the degradation of the targeted mRNA (Yu et al., 2002). On the other hand, SFv are the smallest antibody fragments that retain antigen-binding configurations of parent antibodies.
We used primary cells to study co-receptor usage by both CNS-derived and peripheral strains of HIV-1 in infection of microglia and MDM. We compared the effects of downregulating CCR3 and CCR5 using rSV40-delivered RNAi and SFv against these co-receptors, as well as inhibition by ligands (eotaxin for CCR3) or ligand antagonists (TAK-779 for CCR5). SV40-derived vectors were used because they transduce brain microglia and macrophages very efficiently.
We found that some strains of HIV-1 utilize CCR5 exclusively, while for other strains inhibiting either CCR3 or CCR5 alone decreased HIV-1 replication. These data suggest that for some strains of HIV-1, including primary isolates from both the CNS and the periphery, CCR3 may be an important partner co-receptor that is used together with CCR5 in HIV-1 entry into macrophages and brain microglia. Therapeutic strategies targeting both CCR3 and CCR5 may thus be helpful in inhibiting HIV-1 infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Deng et al., 1996) were obtained from NIH-AIDS Research and Reference Reagent Program (AIDS-RRRP). U87-CD4-CCR3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 15 % new born calf serum (NCS) (Hyclone), 1 µg puromycin ml–1, 300 µg G418 ml–1, 2 mM glutamine, 200 U penicillin ml–1 and 100 µg streptomycin ml–1. HOS-CD4-CCR3 cells were cultured in DMEM containing 10 % NCS and 1 µg puromycin ml–1. COS-7 cells (ATCC) were maintained in DMEM, 10 % NCS, 2 mM L-glutamine, 1.5 g NaHCO3 l–1, 4.5 g glucose l–1, 1 mM sodium pyruvate, 200 U penicillin ml–1 and 100 µg streptomycin ml–1. U87-CD4-CCR3 or microglial cells were transfected with either plasmids (5 µg) or soluble siRNAs (0–10 nm) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Cell surface expression of CCR3 and AU1 expression were analysed after 48 h.
Antibodies and proteins.
Antibody to CCR3 (clone 5E8-G9-B4) was developed in the laboratory of Dr Charles Mackay (Heath et al., 1997). Monoclonal anti-CCR5 (clone 2D7), anti-CD68 and anti-CD11B were purchased from BD Biosciences. Polyclonal anti-CCR5 was purchased from Abcam and anti-CD4 (clone RPAT4) from BD Biosciences. Anti-AU1-fluorescein isothiocyanate (FITC) was purchased from Covance. Eotaxin was purchased from Peprotech Inc. TAK-779 was obtained from AIDS-RRRP.
Primary microglial cells.
Microglial cells were isolated from human fetal brain as described previously (Cordelier & Strayer, 2006). Briefly, fetal brain (Human Fetal Tissue Bank, Albert Einstein College of Medicine, Bronx, NY, USA) was homogenized in Ca2+- and Mg2+-free Hanks' balanced salt solution containing 0.05 % trypsin and 100 U DNase, passed through 170 µm nylon mesh, then plated at 50x106 cells per T-150 flask. Non-adherent cells were removed by washing with DMEM/F-12 (Invitrogen). Microglia were harvested as clarified supernatants after 2 weeks and collected on alternate 2 days for 4 weeks. Cell purity was >95 %, as verified by flow cytometry analysis using anti-CD68 or anti-CD11B antibodies.
Preparation of macrophages.
Macrophages were prepared from fresh buffy coat (TJU Blood Center) as described previously (Simmons et al., 1995). Briefly, cells were plated at a density of 108 cells in DMEM with 10 % human serum for 2 h at 37 °C. Adherent cells were incubated in DMEM containing macrophage colony stimulating factor and granulocyte macrophage colony stimulating factor (50 U ml–1; Sigma Chemicals). The nature and purity of these cells were verified by staining with anti-CD68.
SFv against CCR3.
An SFv against CCR3 was constructed using the mouse SFv module according to the manufacturer's instructions (Amersham Biosciences). Briefly, total RNA from CCR3 hybridoma was used with 10 different sets of primers (in the module) for amplification of variable heavy (VH) and variable light (VL) domains using RT-PCR. VH- and VL-coding fragments were cloned into pGEM-T (Promega). VH and VL domains were amplified by PCR to generate an SFv construct containing a (Gly4 Ser)3 spacer, plus a C-terminal AU1 epitope (DTYRYI).
FPH, forward primer (heavy chain): 5'-GCCACCATGGTGCAGCTGCAGCAGTCA-3'; RPH, reverse primer (heavy chain): 5'-AGATCCGCCACCACCGGATCCGCCTCCGCCTGAGGAGACGGTGAC-3'; FPL, forward primer (light chain): 5'-GGTGGTGGCGGATCTGGAGGTGGCGGAAGCGACATCCAGATGACA-3'; RPL, reverse primer (light chain): 5'-CTATCAGATGTATCGGTACGTGTCTTTGATTTCCAGCTTGGTG-3'.
SFv-CCR3-AU1-coding sequences were subcloned into pcDNA3.1(+) mammalian expression vector (Clontech) and also into SV40 vector, pT7A5. SFv-CCR3-AU1 structure has been reported previously to GenBank (accession no. EU019106 [GenBank] ).
siRNA against CCR3.
Synthetic siRNA duplexes were chemically synthesized by Integrated DNA technologies. They were approximately 21 nt in length, with 2 nt deoxythymidine 3'-overhangs.
A 63 nt R3-526-siRNA was synthesized containing both sense and antisense strands for R3(526)-siRNA and cloned into pT7VA. This plasmid contains the adenovirus VA1 promoter (pol III) (Cordelier et al., 2003). All constructs were sequenced to confirm structure.
The sequence below represents the synthetic (R3-526)-siRNA construct that was chemically synthesized for cloning into SV40 vector pT7VA. Restriction site BamHI (ggatcc) with three ‘a’ overhangs was synthesized with the sense sequence of (R3-526)-siRNA followed by the introduction of an EcoRI site (gaattc). This was followed by the antisense sequence of (R3-526)-siRNA and a SacII site (ccgcgg) and two ‘a’ overhangs. BamHI and SacII were used for directional cloning in pT7VA vector and EcoRI was introduced for orientation screening purposes.
AaaggatccGCCTTGTACAGCGAGATCT-gaattc-AGATCTCGCTGTACAAGGCTccgcgggaa
Plasmids and viral expression constructs.
SFv-CCR3-AU1 cDNA was subcloned into pT7A5, with expression driven by cytomegalovirus (CMV) immediate-early promoter (CMV-IEP) (Cordelier et al., 2004; Cordelier et al., 2003). pT7A5 and pT7VA lack the large T-antigen gene (Tag), but carry SV40 capsid genes, ori, packaging and polyadenylation signals. SV(RNAi-R5) is an rSV40 that encodes an siRNA targeting CCR5 mRNA. SV(2C7) contains an SFv versus human CCR5. The specificity of this SFv and its parent hybridoma have been reported previously (Lee et al., 1999; Strayer et al., 2001). Control vectors were SV(HBS), which encodes hepatitis B surface antigen (HBsAg) (Kondo et al., 1998), and SV(Lamin-A/C)-siRNA, which encodes siRNA targeting lamin-A/C (Cordelier et al., 2003, 2004).
Procedures for generating rSV40s have been reported in detail previously (Strayer et al., 2001). Briefly, recombinant viral genomes were transfected into COS-7 cells, which supply all SV40 genes needed for virus packaging. Recombinant viral stocks were prepared as cell lysates, band purified by sucrose cushion (Strayer et al., 2001) and titrated by quantitative PCR (Q-PCR; Stratagene) (Strayer et al., 2006).
Transduction with SV40 vectors.
U87-CD4-CCR3 or primary microglial cells and macrophages were transduced with rSV40s on days 0, 3 and 5 at virus : cell ratios (m.o.i.) of 10, 3 and 3, respectively. Cells were tested for transgene expression by immunostaining for SFv-CCR3-AU1, using anti-AU1, which showed >90 % transduction efficiency (data not shown), and by assessing downmodulation of CCR3 by both the constructs. Cells were maintained for 5 days in DMEM with 2 % fetal bovine serum. All transduced cell groups, whether receiving test or control vectors [SV(HBS) and SV(Lamin-A/C-siRNA)], showed >95 % viability throughout.
Immunostaining and FACS analysis.
For immunostaining and FACS, cells were grown on four-chamber slides and in six-well plates coated with poly-D-lysine, respectively. At the indicated times, cells were fixed with 1 % paraformaldehyde for 30 min on ice and permeabilized with 0.1 % Triton X-100 in sodium citrate buffer. Non-specific binding was blocked using normal serum from the animal species in which the secondary antibody was raised. Cells were immunostained with anti-CCR3 (1 : 50), anti-CCR5 (1 : 100), anti-CD4 (1 : 100) or anti-AU1-FITC (1 : 100) for 1 h on ice. After extensive washing with PBS containing 1 % BSA, secondary antibodies conjugated with rhodamine (Sigma Chemicals) for CCR3, were added. Cells were washed and analysed on a fluorescence imaging microscope (Olympus IX70) or analysed by FACS (FACSort; BD Biosciences) using CellQuest software.
HIV-1 stocks.
HIV-1 ADA (R5) was described previously (Gendelman et al., 1992). Briefly 30x106 monocytes were infected with the original virus preparation. Supernatants were collected every 2–3 days, fresh phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) were added and p24 concentration was measured. For HIV-1 JR-FL(R5), 107 PHA-stimulated PBMCs were infected with viral inoculum containing 10 µg polybrene ml–1. Supernatants were harvested daily for 3 days (O'Brien et al., 1990). Preparation of HIV-1 SF-2(R5/X4) has been described previously (Levy et al., 1984).
Generation of primary HIV-1 isolates.
Primary isolates, 92US657(R5/X4), 92US723(R5/X4), 96USHIPS7(R5) and ASJM108(R5) were propagated in vitro as described in protocols through UNAIDS network http://www.aidsreagent.org/support_docs/virus.pdf. HIV-1 96USSN20(R5/X4) and ASM108(R5) were prepared by co-culture of patient PBMCs with PHA-stimulated PBMCs in RPMI 1640 containing 10 % fetal bovine serum with 100 U interleukin-2 ml–1 (Sullivan et al., 2000). Viruses were used at 10 ng p24 equivalents in infecting both microglia and macrophages.
Full-length envelope genes of primary brain isolates NA176 (B93)(R3/R5) and NA353 (B27)(R3/R5) have been previously amplified from frontal lobes of patients by using PCR (Peters et al., 2004) and full-length replication competent clones were constructed by subcloning brain-derived envelopes into pNL4.3. p24 protein concentrations were measured using an ELISA kit (Zeptometrix).
HIV-1 challenge studies.
After gene transfer, primary microglia or macrophages were cultured at 1x106 cells per well in DMEM containing 10 % NCS. The next day the cells were infected with 10 ng p24 equivalents of cell-free ADA(R5), JR-FL(R5) or SF-2(R5/X4). Primary peripheral isolates 92US657(R5/X4), 92US723(R5/X4), 96USHIPS7(R5), ASJM108(R5) and primary brain isolates NA176 (B93)(R3/R5) and NA353 (B27)(R3/R5) were used at 1 ng p24 equivalents. After 2 h at 37 °C, cells were extensively washed and maintained in growth medium containing 10 % (v/v) NCS. Supernatants were collected every 3 days and p24 antigen levels were determined by ELISA (Zeptrometrix).
Microglia and macrophages were also pre-treated with anti-CCR3 (1 µg ml–1), anti-CCR5 (1 µg ml–1), eotaxin (500 ng ml–1) or TAK-779 (25 µM), and challenged with input doses of virus similar to those described above.
Confocal microscopy.
For co-localization, U87-CD4-CCR3 or microglia cells were grown in poly-D-lysine-coated confocal glass bottom dishes (MatTeK) at 0.5x106 cells per dish. At the indicated times post-transduction, cells were fixed with 1 % paraformaldehyde for 30 min on ice, permeabilized with 0.1 % Triton X-100 in sodium citrate buffer, then immunostained with anti-CCR3 (1 : 50), anti-CCR5 (1 : 100), anti-CD4 (1 : 200) or purified anti-AU1 (1 : 100) for 1 h on ice. After washing in PBS containing 1 % BSA, secondary antibodies conjugated with Alexa-Fluor (Molecular Probes) for CCR3 and CD4 were added. Secondary antibodies conjugated with rhodamine (Sigma) were used to stain AU1. The cells were washed and confocal analysis performed using an imaging system (Radience 2100; Bio-Rad) with Kr/Ar-ion laser source (488 and 568 nm excitation) on an inverted microscope (x40, UApo 340, NA 1.35).
Statistical analysis.
One way ANOVA was used to compute differences between groups (Instat). Significant effects were further analysed using Bonferroni post hoc comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We then characterized and tested the ability of rSV40 vectors carrying these anti-CCR3 transgenes to lower CCR3 in U87-CD4-CCR3 cells, primary brain microglia and macrophages (Figs 2 and 3). Transduction with SV(R3-526)-siRNA and SV(SFv-CCR3-AU1) decreased membrane CCR3 by 76 and 68 %, respectively, compared with their respective control vectors in U87-CD4-CCR3 cells (P<0.001) (Fig. 2). Most cells (80–90 %) were transduced as determined by AU1 immunopositivity (data not shown). Expression levels of CD4 were unchanged with the different transductions (Fig. 2). We then analysed downmodulation of CCR3 and CCR5 in brain microglial cells and macrophages (Fig. 3). CCR3 in microglial cells transduced with SV(R3-526)-siRNA and SV(SFv-CCR3-AU1) was specifically decreased by 60 and 36 %, respectively, (P<0.001 and P<0.01) with no change in CCR5 surface expression when analysed by FACS (Fig. 3). Fig. 3 represents average percentages of positive cells from three different experiments. A representative panel of FACS histograms is shown. Transduction with SV(RNAi-R5) in microglial cells led to decreased CCR5 expression by 89 %, without affecting CCR3 expression. These manipulations did not alter expression of cell surface CD4 (Fig. 3). Transduction of macrophages with SV(R3-526)-siRNA and SV(SFv-CCR3-AU1) decreased surface CCR3 by 73 and 64 % when analysed by FACS. SV(RNAi-CCR5) decreased cell surface CCR5 by approximately 77 % when analysed by FACS in macrophages. Both RNAi- and SFv-bearing constructs were specific for their targets: RNAi-R5 did not decrease cell membrane CCR3; neither SV(SFv-CCR3-AU1) nor SV(R3-526)-siRNA altered CCR5 (Figs 2 and 3).
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). SV(RNAi-R5), targeting CCR5, decreased p24 levels comparably for HIV-1 ADA(R5) and 96USSN20(R5/X4) (P<0.01) (Fig. 1b). SV(RNAi-R5) also significantly inhibited JR-FL(R5) and SF-2(R5/X4) (P<0.01) (Fig. 1b). When analysed by FACS histograms, both RNAi- and SFv-bearing constructs were specific for their targets (Fig. 3
). HIV-1 ADA(R5) and 96USSN20(R5/X4) were more sensitive to decreases in CCR3 in microglia than other HIV-1 isolates tested. HIV-1 JR-FL(R5) and SF-2(R5/X4) were sensitive to decreases in CCR5 but not CCR3 (Fig. 1a). Productive infection of LAV and HxB2, which are X4-tropic isolates, was not affected by transductions targeting CCR5 or CCR3 (data not shown). These data suggest that R5-tropic strains of HIV-1 may fall into two groups: some strains may require CCR3 in addition to CCR5 for entry into microglial cells, while others require CCR5 but not CCR3.
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All viruses replicated in microglia, with varying efficiency. Both CCR3 and CCR5 were efficiently utilized as co-receptors by NA176 (B93)(R3/R5) and NA353 (B27)(R3/R5). Transduction with SV(SFv-CCR3-AU1), SV(R3-526)-siRNA, SV(SFv-CCR5) or SV(RNAi-R5) significantly inhibited (P<0.05) replication of NA176 (B93)(R3/R5) and NA353 (B27)(R3/R5): HIV-1 replication was decreased by 80 % (Fig. 4) compared with controls, SV(HBS) for the SFvs and SV(Lamin-A/C-siRNA) for the RNAis.
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). Generally, the results of these studies paralleled those observed with gene delivery of RNAi and SFv targeting CCR3 and CCR5. That is, some strains of HIV-1, in particular NA176 (B93)(R3/R5), 96USHIPS7(R5) and ASJM108(R5), were largely inhibited by these external agents blocking either CCR3 or CCR5 (P at least <0.05). Infection with NA353 (B27)(R3/R5) was more strongly inhibited by TAK-779, compared with controls (P<0.001), than by any of the other inhibitors, although inhibition by the latter was also statistically significant (P<0.05). The two strains of HIV-1 that were only inhibited by targeting CCR5 in transduction studies [92US657(R5/X4) and 92US723(R5/X4)] were, similarly, only inhibited by TAK-779 (CCR5-blocking agent) and, to a lesser extent with the 2D7 anti-CCR5 antibody (Fig. 5).
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). Control transductions using both SV(HBS) and SV(Lamin-A/C-siRNA) also showed similar productive infection profiles in MDM.
HIV-1 strain ADA(R5) infection of MDM was comparably inhibited by targeting either CCR3 or CCR5 (Fig. 6) (P<0.05). Similar results were obtained when cultures were treated with either of the hybridoma antibodies to CCR3 or CCR5, or the respective inhibitors, eotaxin and TAK-779 (Fig. 7).
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). In parallel these isolates were inhibited in MDM after treatment with anti-CCR3 or anti-CCR5 antibodies, and TAK-779 or eotaxin (P<0.01) (Fig. 7).
Significant protection from infection with 96USHIPS7(R5) and ASJM108(R5) was seen in macrophages transduced with either of the anti-CCR3 constructs (P<0.05) (Fig. 6) and especially with SV(RNAi-R5) (P<0.01). Both sets of hybridoma antibodies and chemokine receptor ligands also blocked MDM infection by 96USHIPS7(R5) significantly (P<0.01) and ASJM108(R5) (P<0.05 for either of the hybridoma antibodies and eotaxin, and P<0.01 for TAK-779) (Figs 6 and 7).
None of the anti-CCR3 or anti-CCR5 transgenes, hybridoma antibodies or chemokine receptor ligands significantly reduced replication of 92US723(R5/X4) HIV-1 consistently (Figs 6 and 7).
Taken together, the data suggest that all R5-tropic strains of HIV-1 tested utilize CCR5 as a co-receptor for productive infection of primary microglia and MDM, but that some R5-tropic HIV-1 isolates also utilized CCR3 for viral entry into both of these primary cell types. The fact that inhibition of either CCR3 or CCR5 diminished productive infection by these latter HIV-1 isolates suggests that both chemokine receptors may act together as HIV-1 co-receptors.
CCR3 co-localizes with CD4 and CCR5
The possibility that some strains of HIV-1 may require both CCR3 and CCR5 co-receptors suggests that these two chemokine receptors may co-localize at the cell membrane, with each other and with CD4. We therefore studied the subcellular localization of CD4 and CCR3 on U87-CD4-CCR3 cells and of CCR3, CD4 and CCR5 on microglia using confocal microscopy. CCR3 co-localized with CD4 in both cell types (Fig. 8). CCR3 was more diffusely distributed on U87-CD4-CCR3 cells than was CD4 (Fig. 8a). The latter was more localized at the cell membrane and >90 % of CD4 co-localized with CCR3. In microglia, however, CD4 and CCR3 were distributed similarly at the cell surface and approximately 50 % of CD4 co-localized with CCR3 (Fig. 8c). In microglia and U87-CD4-CCR3 cells transduced with SV(SFv-CCR3), the anti-CCR3 SFv and its target, CCR3, co-localized mainly in the cytoplasm (Fig. 8d). In microglia, CCR5 co-localized virtually completely with CCR3 (Fig. 8e). The physical proximity of CCR3 and CCR5 may help explain the apparent requirement of some strains of HIV-1 for both CCR3 and CCR5 for productive infection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) using standard techniques, and delivered effectively to both a CCR3-expressing cell line and primary human brain microglia. We also identified an RNAi species that targeted the CCR3 transcript (R3-526) and decreased cell surface CCR3. In parallel, we used two previously reported vectors, one with an SFv and one with an RNAi, that reduce CCR5 to examine co-receptor usage by a series of M-tropic and neurotropic strains of HIV-1. The specificity of each vector for its respective chemokine receptor target was documented: vectors targeting CCR5 did not reduce cell membrane CCR3 and vice versa.
To deliver these transgenes, we used Tag-deleted SV40-derived gene transfer vectors. rSV40s were used because of their high efficiency in transducing microglia, monocytes, MDM and dendritic cells (Cordelier et al., 2003). A C-terminal AU1 epitope was added to the anti-CCR3 SFv cDNA to facilitate detection. It was observed by FACS analysis that the SFv and RNAi decreased cell membrane CCR3 by 40–75 %. This is comparable in magnitude to the reduction of CCR5 achieved using the RNAi versus CCR5 (Cordelier et al., 2003).
Confocal microscopy showed almost complete co-localization of the anti-CCR3 SFv and cellular CCR3 mostly within the cytosol in SV(SFv-CCR3-AU1)-transduced microglia. Little CCR3 remained unbound, although some was present at the cell membrane, where it co-localized with the anti-CCR3 SFv. In U87-CD4-CCR3 cells, considerably more CCR3 did not co-localize with the SFv. This may be because these cells overexpressed CCR3 at levels exceeding the binding capacity of the SFv.
The quantitative functional effects of these transgenes on CCR3 activity exceeded their reduction in immunologically detectable cell membrane CCR3. Transduction with both transgenes completely blocked eotaxin-induced cellular calcium transients and chemotaxis (data not shown).
We then used these vectors, together with one carrying SFv against CCR5 and another with an RNAi to CCR5, to study co-receptor requirements among different strains of HIV-1. Antibodies against CCR3 and CCR5 and respective ligands, eotaxin and TAK-779, were also used to block these co-receptors. We primarily focused on brain microglia, as it has been suggested that CCR3 may play a role in CNS infection by HIV-1 (Choe et al., 1996). Since MDM also carry both CCR3 and CCR5, selective downregulation of each was also used to evaluate their respective contributions to HIV-1 infectivity. Replication of some strains of HIV-1 in microglia and MDM was not impaired by decreasing CCR3, but was inhibited by decreasing CCR5. These strains included HIV-1 SF-2(R5/X4), JR-FL and primary isolates, 92US657(R5/X4) and 92US723(R5/X4). The primary brain isolates, NA176 (B93)(R3/R5), NA353 (B27)(R3/R5) and isolates ADA(R5), 96USHIPS7(R5) and ASJM108(R5) showed a different phenotype. They were largely inhibited by prior treatment with vectors targeting either CCR3 or CCR5: SV(R3-526)-siRNA, SV(SFv-CCR3-AU1), SV(SFv-CCR5) or SV(RNAi-R5), suggesting that they utilize both simultaneously.
It should be noted that SV(RNAi-R5) and SV(SFv-CCR5), which decrease CCR5 comparably to the reductions in CCR3 seen with the anti-CCR3 SFv or RNAi, inhibit productive infection of microglia and MDM by R5-tropic strains of HIV-1 out of proportion to the magnitude of their downregulation of CCR5 (Cordelier et al., 2003). Also, unimpeded HIV-1 replication in the face of CCR3 or CCR5 downregulation does not necessarily imply that this strain of HIV-1 does not utilize, or even require, the chemokine receptor in question. It may mean simply that it can enter cells with lower co-receptor density.
Growth of HIV-1 strains HIV-1 ADA(R5), HIV-1 96USSN20(R5/X4), NA176 (B93)(R3/R5), NA353 (B27)(R3/R5), 96USHIPS7(R5) and ASJM108(R5) was inhibited comparably by both the anti-CCR3 and the anti-CCR5 antibodies and eotaxin. None of these strains was completely inhibited, but residual HIV-1 replication was comparable in CCR3-depleted and CCR5-depleted cells. These data again suggest that, at least for these strains of HIV-1, both chemokine receptors were needed for optimal effective infection. TAK-779 was a more potent inhibitor than eotaxin, the anti-CCR3 hybridoma, or even the 2D7 anti-CCR5 hybridoma for some strains of HIV-1 (e.g. NA353 in MDM), suggesting a strong preference for CCR5 by those strains and/or the possibility that TAK-779 is a more avid receptor blocker. The latter conclusion appears more likely as, even in strains strongly inhibited by TAK-779, substantial inhibition by eotaxin was also seen (e.g. NA176 in MDM).
If some strains of HIV-1 require (or prefer) both CCR5 and CCR3, one would expect that CD4, CCR5 and CCR3 would co-localize on the cell membrane in microglia. Confocal microscopy demonstrated that these three cell membrane proteins were distributed along the cell membrane virtually identically. Therefore, it is possible that these strains of HIV-1 demonstrate a complexity of co-receptor usage, at least in microglia that differs from what has been described previously. The use of CCR3 as a co-receptor for infection of microglial cells by some strains of HIV-1 has been reported (Heath et al., 1997). However, our data show that some strains of HIV-1 may be inhibited if either CCR5 or CCR3 is decreased, suggesting that both co-receptors may be needed, or preferred, by those isolates of HIV-1 in order to achieve optimal replication in microglia and macrophages. This phenotype is somewhat different from that observed with most dual-tropic strains of HIV-1. Dual-tropic strains may generally utilize one or another co-receptor (e.g. CXCR4 or CCR5) with comparable efficiency so that the virus replication occurs if either, or neither, co-receptor is blocked. It is possible, however, to see the apparent requirement or preference for both co-receptors on the part of some HIV-1 strains as representing a special case of dual tropism. It should be emphasized that this phenotype was observed when the target cells were of peripheral origin (MDM) as well as of CNS origin (microglia), and in HIV-1 strains isolated both from the CNS and from the periphery.
These data have important implications for therapeutics. If CCR3 is commonly involved as a co-receptor for HIV-1, perhaps in preference to CCR5 in some cases, therapeutic strategies focused on inhibiting viral entry may benefit by taking such tropism into account.
In conclusion, CCR3 may play an important role in the pathogenesis of HIV-1 infection in the CNS and in the periphery. Some strains of HIV-1 may use CCR3 together with CCR5. For other strains, the presence of CCR5 may be sufficient for cell entry. Reagents described here, viral vectors carrying SFv and RNAi that are effective in downregulating CCR3, may be useful in elucidating the role of the eotaxin receptor in HIV-1 infection.
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ACKNOWLEDGEMENTS |
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Received 1 August 2008;
accepted 24 November 2008.
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