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. 1998 Mar;72(3):2097–2104. doi: 10.1128/jvi.72.3.2097-2104.1998

Modulation of Feline Immunodeficiency Virus Infection by Stromal Cell-Derived Factor

Margaret J Hosie 1,*, Nelleke Broere 1, Joseph Hesselgesser 2, Julie D Turner 3, James A Hoxie 3, James C Neil 1, Brian J Willett 1
PMCID: PMC109504  PMID: 9499065

Abstract

The α-chemokine receptor CXCR4 has recently been shown to support syncytium formation mediated by strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney cell line (CrFK-tropic virus). Given that both human and feline CXCR4 support syncytium formation mediated by FIV, we investigated whether human stromal cell-derived factor (SDF-1) would inhibit infection with FIV. Human SDF-1α and SDF-1β bound with a high affinity (KDs of 12.0 and 10.4 nM, respectively) to human cells stably expressing feline CXCR4, and treatment of CrFK cells with human SDF-1α resulted in a dose-dependent inhibition of infection by FIVPET. No inhibitory activity was detected when the interleukin-2 (IL-2)-dependent feline T-cell line Mya-1 was used in place of CrFK cells, suggesting the existence of a CXCR4-independent mechanism of infection. Furthermore, neither the human β-chemokines RANTES, MIP-1α, MIP-1β, and MCP-1 nor the α-chemokine IL-8 had an effect on infection of either CrFK or Mya-1 cells with CrFK-tropic virus. Envelope glycoprotein purified from CrFK-tropic virus competed specifically for binding of SDF-1α to feline CXCR4 and CXCR4 expression was reduced in FIV-infected cells, suggesting that the inhibitory activity of SDF-1α in CrFK cells may be the result of steric hindrance of the virus-receptor interaction following the interaction between SDF and CXCR4. Prolonged incubation of CrFK cells with SDF-1α led to an enhancement rather than an inhibition of infection. Flow cytometric analysis revealed that this effect may be due largely to up-regulation of CXCR4 expression by SDF-1α on CrFK cells, an effect mimicked by treatment of the cells with phorbol myristate acetate. The data suggest that infection of feline cells with FIV can be mediated by CXCR4 and that, depending on the assay conditions, infection can be either inhibited or enhanced by SDF-1α. Infection with FIV may therefore prove a valuable model in which to study the development of novel therapeutic interventions for the treatment of AIDS.

The initial stage in lentiviral infection involves the binding of the viral envelope glycoprotein (Env) to a molecule on the surface of the target cell. The primary high-affinity binding receptor for human immunodeficiency virus (HIV) is CD4 (9, 26), a member of the immunoglobulin supergene family of molecules. However, binding of the viral glycoprotein to CD4 is insufficient for infection to proceed (29); for virus-cell fusion to occur, the target cell must also express an accessory molecule or coreceptor. The principal coreceptors for HIV infection have now been identified as members of the seven-transmembrane domain (7TM) superfamily of molecules. Syncytium-inducing (SI) T-cell line-tropic strains of virus require coexpression of the α-chemokine receptor CXCR4 for infection (19), whereas non-syncytium-inducing (NSI) strains of virus require coexpression of the β-chemokine receptor CCR5 for infection (1, 6, 10, 13, 14). In addition, other chemokine receptors such as CCR2b and CCR3 (6, 13, 41, 48), the receptor encoded by human cytomegalovirus US28 (39, 41), and the orphan receptor STRL33 (28) can function as coreceptors for HIV infection. More recently, additional members of the 7TM superfamily have been identified as coreceptors for infection with simian immunodeficiency virus (SIV). Two of these receptors, termed Bonzo and BOB, support infection with not only SIV but also HIV type 2 (HIV-2) and macrophage-tropic or dualtropic (both macrophage- and T-cell-tropic) strains of HIV-1 (11). Bonzo has subsequently been identified as being identical to STRL33 (28), whereas BOB is identical to GPR15 (21). A subsequent study has demonstrated that an additional molecule, designated GPR1 (30), can function as a coreceptor for SIV (18). Thus, a diverse range of 7TM molecules which can support infection with primate lentiviruses have now been identified.

The selective usage of chemokine receptors as coreceptors for infection by HIV and SIV is borne out by the sensitivity of the viruses to inhibition by chemokines. Infection with viruses which use CCR5 can be inhibited by the β-chemokines RANTES, MIP-1α, and MIP-1β (7, 14), whereas those which use CXCR4 can be inhibited by stromal cell-derived factor (SDF-1) (3, 36). Although infection of primary macrophages by certain primary NSI viruses is not inhibited reproducibly by the β-chemokines RANTES, MIP-1α, and MIP-1β (14, 33, 44), analogs of the β-chemokines such as AOP-RANTES that inhibit HIV infection with an increased potency, inhibit infection of both peripheral blood mononuclear cells (PBMC) and primary macrophages, and do not trigger signalling via G proteins coupled to the chemokine receptor have been developed (47). Therefore, with the development of SDF-1 derivatives analogous to AOP-RANTES, it may be possible to generate therapeutic agents that are effective at inhibiting not only the NSI strains of HIV found in early infection but also the SI strains of virus which appear late in infection with the progression to AIDS.

Feline immunodeficiency virus (FIV) induces an AIDS-like illness in its natural host, the domestic cat (38). A proportion of primary isolates of FIV can be readily adapted to grow and form syncytia in the Crandell feline kidney (CrFK) cell line (45), analagous to the isolation of SI variants of HIV. Sequencing of the env gene from CrFK-tropic viruses would suggest that the principal determinant of CrFK tropism is an increase in charge of the V3 loop of the envelope glycoprotein (45, 51), further strengthening the analogy between CrFK-tropic strains of FIV and SI strains of HIV. While the primary high-affinity binding receptor for FIV remains elusive, recent studies have demonstrated a role for the feline homolog of CXCR4 in infection with CrFK-tropic strains of FIV (53, 56). Given that the appearance of CXCR4-dependent SI variants of HIV in the peripheral blood of HIV-infected individuals accompanies the progression to AIDS (8), the ability to study the role of such CXCR4-dependent strains of virus in disease pathogenesis is of obvious interest. Moreover, as it appears that several strains of SIV show preferential usage of CCR5 and not CXCR4 for infection (5, 11, 18), then FIV infection of the domestic cat is the only animal model described to date in which the contribution of CXCR4-dependent viruses to the pathogenesis of AIDS may be studied in the natural host of the virus.

In this study, we investigated the nature of the interaction between FIV and the chemokine receptor CXCR4. Given the high degree of amino acid sequence homology between human and feline CXCR4 (56), we examined the interaction between human SDF-1 and feline CXCR4. We have found that human SDF-1 binds specifically to feline CXCR4 and inhibits infection with FIV. We demonstrate that SDF-1 can upregulate CXCR4 expression with a corresponding enhancement of infection and that this effect can be mimicked by treatment of the cells with the phorbol ester phorbol myristate acetate (PMA). Moreover, infection of interleukin-2 (IL-2)-dependent T cells with FIV was resistant to the inhibitory effects of SDF-1, suggesting the existence of a CXCR4-independent mechanism of infection in these cells. These data suggest that the mechanism of infection with FIV bears striking similarities to infection with HIV and that the study of FIV infection of the domestic cat may provide a valuable insight into the pathogenesis of AIDS.

MATERIALS AND METHODS

Antibodies and reagents.

Recombinant human SDF-1, MCP-3, RANTES, MCP-1, MIP-1β, MIP-1α, and IL-8 were from PeproTech, Inc., Rocky Hill, N.J., or as described elsewhere (22). Synthetic human SDF-1 was obtained from I. Clarke-Lewis, University of British Columbia, Vancouver, British Columbia, Canada. Recombinant human RANTES, MIP-1α, and MIP-1β were kindly provided by T. Wells, Glaxo SA, Geneva, Switzerland. Anti-human CXCR4 monoclonal antibodies R&D 44702 and R&D 44717 were a generous gift from Monica Tsang, R&D Systems, Minneapolis, Minn. Phorbol myristate acetate (PMA) was obtained from Calbiochem, Nottingham, United Kingdom. Immunoaffinity-purified FIV envelope glycoprotein from the PET strain of FIV (FIVPET) was prepared as described previously (25). All culture media and supplements were obtained from Life Technologies, Paisley, United Kingdom. 125I-SDF-1α and -β (specific activity, 2,200 Ci/mol) were from Dupont NEN, Boston, Mass.

Cell lines and viruses.

U87 cells expressing feline CXCR4 stably from the pRep4 vector (U87-feCXCR4 cells) have been described previously (56). U87 cells expressing human CXCR4 (U87-huCXCR4 cells) were obtained from N. Landau, Aaron Diamond AIDS Research Center, New York, N.Y. U87-feCXCR4, AH927 (40), and CrFK cells were maintained in Dulbecco’s modification of minimal essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, sodium pyruvate (0.11 mg/ml), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (DMEM). F422 (42), T3 (35), Q201 (52), and Mya-1 (32) cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 5 × 10−5 M 2-mercaptoethanol (RPMI medium). Q201 and Mya-1 cells were maintained in RPMI medium supplemented with recombinant human IL-2 (100 IU/ml). The culture media for U87-feCXCR4 and U87-huCXCR4 were supplemented with hygromycin (10 μg/ml). The CrFK-tropic virus FIVPET was prepared from a culture of CrFK cells persistently infected with the F14 molecular clone of FIVPET (37).

Inhibition of FIV infection by chemokines.

CrFK cells were plated in 48-well tissue culture plates at 2 × 104 cells per well in DMEM and allowed to adhere overnight. Chemokines were diluted to working concentration in DMEM, added to the cells in a total volume of 100 μl per well, and incubated for 1 h at 37°C. Ten CrFK syncytium-forming units of virus in 50 μl was added per well and incubated for a further 1 h. The supernatant was then aspirated, the wells were washed twice with DMEM, and maintenance chemokine was added at the appropriate final concentration. The cells were cultured for 4 days and then fixed and stained, and the syncytia were enumerated by light microscopy. Supernatants were stored from each well for the detection of viral p24 by enzyme-linked immunosorbent assay (ELISA) (FIV antigen test kit; IDEXX, Portland, Maine). Blocking assays using the feline IL-2-dependent T-cell line Mya-1 were performed in 96-well round-bottom plates. Cells (105) were seeded into each well in 50 μl of RPMI. Chemokines were adjusted to double the final working concentration, added in 100 μl of RPMI medium to each well, and incubated for 1 h at 37°C. The final concentration of SDF-1α, RANTES, IL-8, MIP-1α, and MCP-1 was 1 μg/ml, while the MIP-1α–MIP-1β–RANTES mixture (MMR) consisted of 330 ng of each chemokine per ml. Virus was added in 50 μl per well and incubated for a further 1 h at 37°C. Finally, the cells were washed four times by centrifugation of the plate at 1,000 rpm followed by aspiration of the culture medium. The cells were then resuspended in fresh RPMI medium containing chemokines at the appropriate concentration and cultured at 37°C for 4 or 7 days, when samples of supernatants were collected for viral p24 ELISA.

Flow cytometry.

Flow cytometric analyses were performed essentially as described previously (55). Briefly, adherent cells were removed from the culture plastic by incubation with trypsin-EDTA, washed once by centrifugation through DMEM supplemented with 10% FBS, and resuspended in phosphate-buffered saline (PBS) supplemented with 1.0% bovine serum albumin and 0.1% sodium azide. The cells were then incubated with either anti-feline/human CXCR4 antibody 44717 or isotype-matched (immunoglobulin G2b) antibody 44702, which does not recognize feline CXCR4, for 30 min at 4°C, washed twice by centrifugation, and then incubated for a further 30 min with fluorescein isothiocyanate-conjugated F(ab′)2 fragment of goat anti-mouse immunoglobulin G. Samples were washed twice by centrifugation and then analyzed on a Coulter EPICS Elite flow cytometer, 5,000 events being acquired in LIST mode for each sample.

Binding studies.

The SDF-1α and SDF-1β binding studies were performed as described previously (22). SDF-1 was generated by peptide synthesis as described previously (22). U87-CXCR4 cells were seeded in 24-well plastic cell culture plates and grown until confluent. The culture medium was aspirated and replaced with PBS containing radiolabeled chemokine (500 pM) in the presence or absence of increasing concentrations of unlabeled chemokines or FIV envelope glycoprotein at room temperature for 30 min. The incubation was terminated by aspiration of the supernatant; the cells were then washed once with PBS and solubilized by the addition of 500 μl of 25% (wt/vol) sodium dodecyl sulfate. The lysate was then transferred to scintillation vials for counting in a gamma counter. Nonspecific binding was determined in the presence of 1 μM unlabeled chemokine, and each experiment was performed in duplicate. The binding data were curve fitted by using the IGOR software package (Wavemetrics Inc., Lake Oswego, Oreg.) to determine the binding affinity (KD), number of sites, and amount of nonspecific binding.

RESULTS

Binding of human SDF-1 to feline CXCR4.

The anti-human CXCR4 antibody 12G5 inhibits fusion between FIV-infected feline cells and human CXCR4-expressing cells but does not recognize feline CXCR4 (56). To examine the role of CXCR4 in infection of feline cells with FIV, we asked whether sufficient amino acid sequence homology existed between human and feline CXCR4 to permit SDF-1 binding to feline CXCR4. U87 cells stably transfected with feline CXCR4 were exposed to 125I-labeled human SDF-1α (Fig. 1a) or SDF-1β (Fig. 1b) in the presence of increasing concentrations of the unlabeled ligand. The cells were incubated for 1 h, and then free chemokine was aspirated. The cells were then lysed, and bound chemokine was measured in a gamma counter. Homologous competition binding studies followed by Scatchard analysis revealed that SDF-1α bound to the feline CXCR4-transfected cells, but not feline CD9-transfected cells, with a KD of 12.0 ± 5.3 nM and that SDF-1β bound with a KD of 10.4 ± 1.5 nM. Therefore, feline CXCR4 acts as a high-affinity binding receptor for human SDF-1α and SDF-1β.

FIG. 1.

FIG. 1

FIG. 1

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SDF-1α and -β binding to feline CXCR4 and FIV gp120 competition. (a) Homologous competition binding curve of 125I-SDF-1α to feline CXCR4 in U87 cells. The insert shows Scatchard analysis of binding (KD = 12.0 ± 5.2 nM; representative experiment [n = 3]). Total cpm = 92,000; background cpm = 37,000. (b) Homologous competition binding curve of 125I-SDF-1β to feline CXCR4 in U87 cells. The insert shows Scatchard analysis of binding (KD = 10.4 ± 1.5 nM; representative experiment [n = 2]). Total cpm = 37,000; background cpm = 13,500. (c) Heterologous competition binding of FIV gp120 and 125I-SDF-1α to feline CXCR4 in U87 cells. The insert shows Scatchard analysis of binding (KD = 1.06 ± 0.14 nM [n = 2]). Total cpm = 76,000; background cpm = 20,000.

Transfection of U87 cells with CXCR4 alone is sufficient to render the cells susceptible to infection with the CrFK-tropic strain FIVPET. Furthermore, infection of CrFK cells by FIV is inhibited in a dose-dependent fashion by SDF-1. We therefore asked whether the envelope glycoprotein (gp120) from FIVPET interacts directly with feline CXCR4. Feline CXCR4-transfected U87 cells were incubated with 125I-labeled SDF-1α, and the displacement of the chemokine by immuno-affinity-purified FIVPET gp120 was measured. (Fig. 1c). FIVPET gp120 displaced SDF-1α binding to U87 cells with a high affinity, Kd = 1.06 nM + 0.14. Binding of SDF and displacement by SDF and FIV gp120 further confirm the interaction of these proteins with feline CXCR4.

Inhibition of FIV infection by SDF-1α.

Having demonstrated that SDF-1α bound with high efficiency to feline CXCR4, we investigated whether infection of feline cells by FIV could be inhibited by SDF-1. CrFK cells were infected with FIVPET in the presence of increasing concentrations of either recombinant human SDF-1α (Fig. 2a) or synthetic human SDF-1α (Fig. 2b). Four days postinfection, the cells were fixed and stained and the number of syncytia per well was quantified by light microscopy. Increasing concentrations of either recombinant or synthetic SDF-1α led to a dose-dependent inhibition of syncytium formation, with approximately 50% inhibition at 12.5 ng/ml and 80 to 100% inhibition of infection being achieved at concentrations of 0.5 to 1.0 μg/ml. These concentrations correlate well with the effective concentrations observed for inhibition of HIV infection by SDF-1 (3, 36). Culture supernatants were collected from the cells treated with synthetic human SDF-1 and analyzed for viral p24 production by ELISA. SDF-1 treatment led to a dose-dependent decrease in p24 production (Fig. 2c), in good agreement with the assay for syncytium formation, and complete inhibition of p24 production was achieved at a concentration of 1 to 2 μg/ml. No inhibition of infection was observed in CrFK cells treated with human MCP-3, RANTES, MCP-1, MIP-1β, MIP-1α, or IL-8 at similar concentrations (not shown). Moreover, in a separate experiment we found that the inhibitory activity of SDF-1 against FIV infection could be partially neutralized by a polyclonal anti-SDF-1 antiserum (data not shown).

FIG. 2.

FIG. 2

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Inhibition of FIVPET infection of CrFK cells by SDF-1α. CrFK cells were cultured overnight in 48-well plates and incubated in the presence of increasing concentrations of either recombinant human SDF-1α (a) or synthetic human SDF-1α (b) for 1 h prior to infection with FIVPET. Four days postinfection, the cells were fixed and stained and the number of syncytia per well was determined. Results are the means of triplicate wells. Supernatants were collected from the cells treated with synthetic human SDF-1α and analyzed for viral p24 by ELISA (c). Incubation with increasing concentrations of SDF-1α led to a dose-dependent reduction in viral p24 release.

Having demonstrated that SDF-1 could inhibit infection of CrFK cells with the cell culture-adapted isolate FIVPET, we examined whether infection of IL-2-dependent T cells with FIV would be inhibited to a similar degree. Mya-1 cells (32) were incubated with either SDF-1α, RANTES, MCP-1, MIP-1α, or IL-8 or with MMR and then infected with either FIVPET or the primary isolate FIVGL8. Samples of culture supernatant were collected daily and analyzed for viral p24 by ELISA. No evidence of inhibitory activity by SDF-1 was observed in Mya-1 cells irrespective of the virus or chemokine concentration (data not shown). Furthermore, infection of Mya-1 cells was refractory to inhibition by human RANTES, MCP-1, MIP-1α, MMR, or IL-8. The data implicate the existence of a CXCR4-independent pathway of infection in feline T cells and suggest that the cell culture-adapted FIVPET isolate has the ability to utilize either CXCR4-dependent or -independent routes of infection, depending on the target cell type.

Modulation of feline CXCR4 expression by PMA and SDF-1α.

The interaction between SDF-1α and human CXCR4 leads to down-regulation of the receptor (2, 23, 46). Previous studies on the IL-8 and MCP-1 receptors have indicated that internalization of chemokine receptors can occur via endocytosis (17, 43), and colocalization of CXCR4 and transferrin in early endosomes (2) suggests that down-regulation of CXCR4 by SDF-1α is mediated by endocytosis. To determine whether a functional interaction occurred between human SDF-1 and feline CXCR4, we next investigated the down-modulation of feline CXCR4 by SDF-1 and PMA. Previously we demonstrated that the anti-human CXCR4 antibody 12G5 failed to recognize feline CXCR4 (56). We therefore screened a newly developed panel of anti-human CXCR4 antibodies for reactivity against feline CXCR4. We identified three monoclonal antibodies (R&D 44701, 44717, and 44718) that reacted with the feline T-lymphoma cell lines 3201 (97.3% positive), T3 (89.8% positive), and F422 (99.5% positive) and the adherent cell line CrFK (37.0% positive) (Fig. 3). The three antibodies were indistinguishable with respect to fluorescence intensity and percentage positive on each of the cell lines. Northern blotting analysis confirmed that each of these cell lines expressed high levels of CXCR4 mRNA (data not shown and previous results [56]). In contrast, the AH927 cell line did not appear to express CXCR4 (1.5%), in agreement with previous findings that these cells do not express CXCR4 mRNA (56). Surprisingly, the Mya-1 cell line was not recognized by any of the three monoclonal antibodies despite expressing significant levels of CXCR4 mRNA. Indeed, Mya-1 mRNA was used as the source of CXCR4 mRNA for the generation of the feline CXCR4 cDNA clone (56). Similar findings were observed with a second IL-2-dependent feline T-cell line, Q201. Given that these antibodies recognize cells transfected with feline CXCR4 (50), the data suggest either that feline CXCR4 is not expressed at the cell surface in these cell lines or that the epitope recognized by these antibodies is masked.

FIG. 3.

FIG. 3

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CXCR4 expression on feline cell lines. Flow cytometric analysis of CXCR4 expression on feline cells was performed with the anti-human CXCR4 antibody R&D 44718; 5,000 events were collected for each sample in LIST mode. Histograms illustrate percentage positive (open) and the isotype-matched control (filled) which was used to set an analysis gate in which <1.0% cells were positive.

The CXCR4-positive cell lines T3 and F422 and the adherent cell line CrFK were treated with SDF-1α (0.33 μg/ml) or PMA (10 mg/ml) and cultured for 24 h. The cells were then analyzed by flow cytometry for CXCR4 expression (Fig. 4) using antibody R&D 44717 (54). Overnight treatment with SDF-1α or PMA led to a marked down-regulation of CXCR4 expression on T3 cells (control, 98.5% positive; SDF-1α, 2.6% positive; PMA, 17.8% positive) and F422 cells (control, 99.1% positive; SDF-1α, 38.7% positive; PMA, 44.0% positive). These findings are in good agreement with previous studies on SDF-1 down-regulation of human CXCR4 on CEM cells, PBMC, or HeLa cells (2). However, SDF-1α treatment of CrFK cells for 24 h led to a significant increase in CXCR4 expression on CrFK cells (control, 19.4% positive; SDF-1α, 38.9% positive). The up-regulation of CXCR4 expression was more marked following PMA treatment of CrFK cells (69.5% positive). Given that incubation of CrFK cells with SDF-1α for 1 h prior to infection with FIV inhibits infection in a dose-dependent fashion (Fig. 2), the finding that SDF-1α does not down-regulate CXCR4 expression on CrFK cells would implicate steric hindrance of the gp120-CXCR4 interaction as the principal mechanism of inhibition of FIV infection by SDF-1α. Furthermore, if SDF-1α and PMA up-regulate CXCR4 expression on CrFK cells, we would predict that susceptibility to infection with FIV would increase accordingly. CrFK cells were treated overnight with either SDF-1α or PMA, washed, and then infected with FIVPET. Four days postinfection, supernatants were collected and assayed for viral p24 by ELISA, the cells were fixed and stained, and the syncytia were quantified. Overnight treatment of CrFK cells with either SDF or PMA enhanced FIV infection of CrFK cells (Fig. 5), a significant increase being observed in both the level of viral p24 in the culture supernatant (Fig. 5a) and the number of syncytia per field (Fig. 5b). As the number of syncytia reflects the number of successful entry events rather than enhanced viral replication, the data suggest that SDF-1α or PMA treatment enhanced viral entry. Thus, while treatment of CrFK cells with SDF-1α inhibits infection with a 1-h incubation, a 24-h incubation enhances infection. We next examined the kinetics of CXCR4 up-regulation by PMA. CrFK cells were plated in six 25-cm2 culture flasks. PMA was added at 10 ng/ml to one of the six flasks. Further additions were made to a fresh flask of cells after 24, 36, 45, and 47 h. One hour after PMA treatment of the fifth flask, all six flasks were subcultured with trypsin, and half of the cells in each flask were reseeded, cultured for 1 h, and then infected with FIV; the remaining cells were stained with anti-CXCR4 antibody and analyzed by flow cytometry. Thus, all cells were seeded at the same time and maintained in culture for 48 h and yet differed in the duration of exposure to PMA (0, 1, 3, 12, 24, and 48 h). Flow cytometric analysis revealed that 37.0% of the cells were CXCR4 positive in the control culture. After 1 h of exposure to PMA, a marginal (40.3%) up-regulation of CXCR4 was evident, confirming that CXCR4 expression was not down-regulated by SDF and ruling out receptor down-regulation as the principal antiviral mechanism in CrFK cells. In contrast, CXCR4 expression increased sharply in the cells exposed for 3 h or more, reaching a maximum of 73.6% at 12 h posttreatment (Fig. 6a). The FIV-infected cultures were fixed and stained at 4 days postinfection, and supernatants were collected for analysis of viral p24 levels. The number of syncytia per well correlated with the increase in CXCR4 expression detected by flow cytometry, a significant increase in the number of syncytia being observed in the cultures treated with PMA for 12 h or more (Fig. 6b). The increase in the number of syncytia was accompanied by an enhancement of p24 production as detected by ELISA (Fig. 6c), confirming that FIV infection was enhanced.

FIG. 4.

FIG. 4

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Modulation of CXCR4 expression on feline cell lines following treatment with SDF-1α or PMA on F422, T3, and Ho6T1 (derivative of CrFK cell line) cells. The feline T-lymphosarcoma cell lines T3 and F422 and the adherent cell line CrFK were exposed to SDF-1α (1 μg/ml) or PMA (10 ng/ml) overnight and then analyzed by flow cytometry for CXCR4 expression with R&D 44717. Histograms illustrate treated cells (open) relative to untreated cells (filled) stained with R&D 44717; 5,000 events were collected for each sample.

FIG. 5.

FIG. 5

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Enhancement of infection of CrFK cells following treatment with SDF or PMA. CrFK cells were incubated with SDF (1 μg/ml) or PMA (50 ng/ml) overnight, washed, and then infected with FIVPET. Four days postinfection, supernatants were collected and analyzed for viral p24 by ELISA (a). The cells were fixed and stained, and the number of syncytia per well was determined (b).

FIG. 6.

FIG. 6

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Time course of CXCR4 up-regulation on CrFK cells by PMA. CrFK cells were incubated with PMA (50 ng/ml) for 0, 1, 3, 12, 24, or 48 h. The cells were then subcultured; half were replated and infected with FIVPET, while the remainder were analyzed for CXCR4 expression by flow cytometry (a). The data represent mean number of positive cells relative to the isotype-matched control (○) and the mean fluorescence intensity (▿). Four days after infection with FIVPET, the cells were fixed and stained and the number of syncytia per well was determined (b). Supernatants were collected, and viral p24 was quantified by ELISA (c). Results represent a typical experiment and are the means of three estimations.

Loss of CXCR4 expression following FIV infection.

Infection with the CXCR4-dependent vcp strain of HIV-2 leads to down-regulation of surface expression of CXCR4 (16). We therefore investigated the effect of FIV infection on CXCR4 expression on CrFK. CrFK cells persistently infected with either FIVPET or FIVGL8, or uninfected control cells, were stained with the antibody R&D 44717 or an isotype-matched control antibody, and reactivity was analyzed by flow cytometry (Fig. 7). While 68.7% of the uninfected CrFK cells were positive for CXCR4 expression, CXCR4 expression was markedly reduced on the FIVGL8- and FIVPET-infected cells; in each case, only 2.0% of the cells were CXCR4 positive (isotype-matched control stained cells were 1.7 and 1.9% positive, respectively), suggesting either down-regulation of CXCR4 expression or elimination of CXCR4-expressing cells from the culture. The data provide further evidence for the interaction between FIV and CXCR4 in CrFK cells.

FIG. 7.

FIG. 7

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Down-regulation of CXCR4 in FIV-infected CrFK cells. CrFK cells persistently infected with a CrFK-adapted stock of either FIVGL8 (a) or FIVPET (b) were analyzed by flow cytometry for expression of CXCR4. Infected cells (open) are shown relative to uninfected CrFK cells (shaded) analyzed in parallel.

DISCUSSION

FIV can be selected for growth and syncytium formation in the feline cell line CrFK, and isolates which grow in this cell line have an extended cell tropism including nonfeline cells such as the human cell line HeLa. Previously, we found that syncytium formation by CrFK-tropic FIV was mediated by the chemokine receptor CXCR4. In this study, we extend our previous observations and demonstrate that human SDF-1α and SDF-1β bind with a high affinity to feline CXCR4. Furthermore, human SDF-1α inhibited FIV infection of CrFK cells efficiently, leading to a dose-dependent reduction in both the number of syncytia and viral p24 production. In contrast, the human β-chemokines RANTES, MIP-1α, MIP-1β, MCP-1, and MCP-3 or the α-chemokine IL-8 had no effect on FIV infection of CrFK cells. It is possible that the amino acid sequences of the human β-chemokines RANTES, MIP-1α, and MIP-1β are sufficiently divergent from their feline homologs to render them nonfunctional in assays using feline cells, and indeed preliminary reports suggest that the feline homologs of MIP-1α and MIP-1β display only 75.3 to 79.6% and 73.9 to 88.0% homology to those of other species (15). However, given that SDF-1α inhibited FIV infection of CrFK cells completely, the data suggest that CrFK-adapted strains of FIV are analogous to T-cell line-adapted strains of HIV such as LAI or NL4-3 and that CrFK cells can be considered to be analogous to HeLa-CD4 cells, where infection is mediated exclusively by CXCR4 and is inhibited completely by SDF-1α.

SDF-1α had no effect on infection of the IL-2-dependent T-cell line Mya-1 with CrFK-tropic FIV, implying the existence of a CXCR4-independent mechanism of infection and suggesting that CrFK-tropic viruses are in fact dualtropic. By analogy, infection of human PBMC with an HIV-1 recombinant pseudotyped with the envelope glycoprotein from an obligate CXCR4-dependent virus (HXBc2) was shown to be inhibited by SDF-1 (3); thus, if CrFK-tropic FIV was restricted to usage of CXCR4 alone (as with HIV HXBc2), we would expect to see inhibition of infection by SDF-1α irrespective of the cell type. In contrast, infection with a dualtropic virus such as 89.6 is inhibited by SDF-1 only when the target cell expresses CXCR4 alone; if the target cell expresses both CXCR4 and CCR5, then SDF-1 does not inhibit infection (12).

Intriguingly, CrFK cells were recognized by the cross-species-reactive anti-CXCR4 antibodies whereas Mya-1 cells were not, despite CXCR4 mRNA being abundant in both cell lines (56). Such anomalies have been described previously for HIV, where the anti-CXCR4 antibody 12G5 failed to block fusion mediated by LAI envelope in U87.CD4 cells transfected with CXCR4 (31). As recent studies have suggested that the epitope recognized by the monoclonal antibody 12G5 is masked in complexes between CD4 and CXCR4 but can be revealed by treatment of the cells with the anti-CD4 antibody Q4120 (34), the epitope recognized by the anti-feline CXCR4 antibodies may be masked on Mya-1 and Q201 cells. Alternatively, Mya-1 and Q201 cells may have a high turnover of CXCR4 at the cell surface, and thus although CXCR4 is expressed at the cell surface, it may be rapidly internalized. Recent studies have demonstrated that CXCR4 undergoes constitutive internalization in several human cell lines and that the rate of internalization is enhanced by the addition of phorbol ester and SDF-1 (46). Furthermore, greater than 90% of SDF-1α bound to the human cell line Jurkat is internalized within 2 h via CXCR4 (23). Future studies will analyze the kinetics of feline CXCR4 expression in order to ascertain the role of CXCR4 internalization in susceptibility and resistance to FIV infection.

FIV envelope glycoprotein competed with human SDF-1 for binding to feline CXCR4. Previous studies have suggested that CXCR4 and CD4 form a complex with envelope glycoprotein from T-tropic, SI strains of HIV (27), while CCR5 and CD4 form complexes with the envelope glycoprotein from macrophage-tropic, NSI strains of HIV (49, 57). In this study, we demonstrate that FIV envelope glycoprotein competes specifically with SDF for binding to feline CXCR4, confirming that there is a direct interaction between the virus and CXCR4. Furthermore, we have found that CXCR4 expression is down-regulated on FIV-infected CrFK cells. These findings parallel the CD4-independent binding of HIV T-tropic envelope glycoprotein to CXCR4 on hNT neurons (22), HIV-1 IIIB infection of hNT cells (20), and the usage of CXCR4 by CD4-independent strains of HIV-2, vcp and ROD-B (16). Of the coreceptors identified to date for HIV, only CXCR4 appears to be capable of supporting infection in a CD4-independent fashion (16). Recent studies have suggested that envelope glycoproteins from CCR5-dependent strains of HIV-2 and SIV can interact directly with CCR5 in a CD4-independent fashion, displacing the chemokine MIP-1α in the process (24). However, optimal binding and membrane fusion still require the formation of a trimolecular CCR5-CD4-gp120 complex (24, 49, 57). It has been proposed that Envs from HIV-2 and SIV may interact directly with chemokine receptors with a higher affinity than Envs from HIV-1 and that it is this reduced requirement for an interaction with CD4 that has enabled CD4-independent strains of HIV-2 to arise (24). FIV Env competed with SDF-1α for binding to CXCR4 with an efficiency similar to that for the competition between HIV-2 ST Env and MIP-1α for CCR5 (24). Thus, these data provide further support for CXCR4-dependent strains of FIV resembling CD4-independent infection with HIV and suggest a common mechanism of infection by the primate and feline lentiviruses.

Given that previous studies had demonstrated that CXCR4 down-regulation contributed to the antiviral activity of SDF-1 against HIV infection (2), we looked at the effects of SDF-1α and PMA on FIV infection of CrFK cells. Surprisingly, overnight treatment of CrFK cells with SDF-1α or PMA led to a marked up-regulation of CXCR4 expression and a concomitant increase in susceptibility to infection with FIV. CXCR4 up-regulation on human T cells has been observed following stimulation with either phytohemagglutinin or IL-2 (4). In this study, we have found that on CrFK cells, but not T3 or F422 cells, prolonged treatment with SDF-1α or PMA had a similar effect. Thus, FIV infection of CrFK cells can be either inhibited or enhanced by treatment with SDF-1α, depending on the incubation time. These findings provide a possible explanation for many of the conflicting data regarding the inhibition of HIV infection by chemokines; the inhibitory effects of chemokines on HIV infection may be entirely dependent on the target cell type, and indeed, in assay systems based on heterogeneous cell populations such as PBMC, it is likely that the net effect may be the result of conflicting inhibitory and enhancing actions of the chemokine. Accordingly, the inhibitory activity of SDF against FIV infection in CrFK cells may be the sum of steric hindrance of the interaction between FIVPET gp120 and CXCR4 and up-regulation of CXCR4 expression by SDF.

Our studies demonstrate that CXCR4 is the principal receptor for CrFK-tropic strains of FIV. Future studies will investigate the prevalence of such SI strains of FIV in infected cats and establish whether such viruses are more prevalent in cats which progress to AIDS. Understanding the contribution of these viruses to the immunodeficiency induced following FIV infection of the domestic cat may help to elucidate the pathogenesis of AIDS in HIV-infected individuals.

ACKNOWLEDGMENTS

We thank Monica Tsang, Ian Clarke-Lewis, and Timothy Wells for generous provision of reagents and Tom Dunsford for technical assistance. We thank Paul Clapham, Bob Doms, Dan Littman, John Moore, and Alexandra Trkola for helpful discussions.

This study was supported by the Medical Research Council (M.J.H.) and The Wellcome Trust (B.J.W.). N.B. was supported by a Leonardo Da Vinci grant and an award from the Stichting Bekker La Bastide Fonds, The Netherlands.

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