Progressive and Persistent Downregulation of Surface CXCR4 in CD4⁺ T Cells Infected With Human Herpesvirus 7

THE 7-TRANSMEMBRANE G-protein–coupled CXC-chemokine receptor 4 (CXCR4) is known to be expressed in all hematopoietic cells, starting from immature CD34⁺hematopoietic progenitors and including CD4⁺ and CD8⁺ T-lymphocyte subsets.1-8 It has been shown that CXCR4 plays an essential role as a coreceptor for the T-cell line-adapted isolates of human immunodeficiency virus (HIV), which are highly cytopathic and typically emerge in the late stages of HIV disease.9,10 Because in vitro pretreatment with the high-affinity ligand for CXCR4, stromal cell-derived factor-1α (SDF-1α), inhibits the entry of these viruses into CD4⁺ T cells,11-13 the issue of CXCR4 modulation and subcellular trafficking has recently raised considerable attention.

CXCR4 undergoes efficient but transient endocytosis after interaction with SDF-1α or treatment with phorbol esters.14-16 In CD4⁺ T lymphocytes, it appears to respond to similar modulatory signals as CD4,3,16 and it has been proposed that comodulation of CXCR4 and CD4 may play a role in regulating the activity of CD4⁺ T cells. However, the different constitutive endocytosis rates of CD4 and CXCR4, expressed on the SupT1 CD4⁺ T-lymphoblastoid cell line, and the selective SDF-1α–induced downmodulation of CXCR4 but not of CD4,14rather suggest that these two proteins do not normally form stable associations. A complex of CD4 and CXCR4 is likely to be induced by the presence of HIV envelope gp120 protein.17,18 

Because human herpesvirus 7 (HHV-7) shows a selective tropism for CD4⁺ T lymphocytes19,20 and induces an abrupt downregulation of CD4 antigen,20-22 we investigated whether CXCR4 expression could be affected by HHV-7.

The CD4⁺ human T-cell line SupT1 (AIDS Research and Reference Reagent Program, Manassas, VA) and a CD4⁻derivative of SupT1 called BC7 (generously provided by Dr J.A. Hoxie, University of Pennsylvania, Philadelphia, PA)23 were routinely cultured in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) containing 10% fetal calf serum (GIBCO BRL). Primary CD8-depleted and/or -enriched CD4⁺ T cells were derived from the peripheral blood of healthy blood donors, by negative immunomagnetic selection, and phytohemagglutinin (PHA)/interleukin-2 (IL-2)–activated as previously described.24 

The HHV-7 isolates and the preparation procedures of viral stocks have been previously described.20,25 Briefly, HHV-7 infections of both SupT1 and primary cells were performed by incubation with 0.45-μm–filtered infectious supernatants obtained from HHV-7–infected SupT1 cells. Supernatants from uninfected SupT1 cells were used for mock infections. For inhibition assays, target cells were preincubated for 30 minutes at room temperature with SDF-1α (2.5 μg/mL), 12G5 anti-CXCR4 monoclonal antibody (MoAb; 25 μg/mL), or control mouse IgG2a (25 μg/mL) (all from Pharmingen, San Diego, CA). After the addition of HHV-7 inocula (MOI = 0.01), cells were maintained in the presence of SDF-1α or of the indicated antibodies for an additional 40 hours, at which time cells were harvested and used for RNA extraction (see below).

The occurrence of a productive HHV-7 infection was monitored by (1) indirect immunofluorescence staining on acetone-fixed cells by using a specific HHV-7 MoAb (5E1 MoAb; generously provided by Prof E. Campadelli-Fiume, University of Bologna, Bologna, Italy),26,27 as previously described24; and (2) HHV-7 reverse transcriptase-polymerase chain reaction (RT-PCR), by using H7 and H8 primers28 that amplify the HHV-7 U10 ORF.

Infection of SupT1 cells with HIV-1(IIIB) was performed as described20 by using viral inocula derived from HIV-1(IIIB)–infected H9 T-cells. HIV-1 infection was monitored by serial determinations of HIV-1 p24 antigen released into the culture supernatant (p24 ELISA Kit; DuPont-Merck Pharmaceutical Co, Wilmington, DE) and visual inspection for syncytia formation.

Aliquots of 1.5 × 10⁶ cells were subjected to single- or multiple-label staining to examine the presence of surface and/or intracellular Ags, as described previously.22 CXCR4 expression was analyzed either by indirect staining using 12G5 anti-CXCR4 MoAb (Pharmingen) followed by fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG or by using the phycoerythrin (PE)-conjugated anti-CXCR4 MoAb (Pharmingen). The expression of CD4 was analyzed by using either FITC-conjugated (Becton Dickinson, San Jose, CA) or PE-conjugated anti-CD4 MoAb (Pharmingen). CD3 expression was analyzed by using the Cy5-PE-conjugated MoAb (Becton Dickinson). Briefly, surface staining was performed in 200 μL of phosphate-buffered saline containing 1% fetal calf serum at 4°C for 30 minutes.

For the specific detection of intracellular CXCR4, SupT1 cells were pretreated with 20 μL of unconjugated anti-CXCR4 MoAb for 1 hour at 4°C to saturate surface CXCR4 before the fixing procedure. On the other hand, for the simultaneous detection of surface CXCR4 and intracellular CXCR4, SupT1 cells were first incubated with the anti-CXCR4 MoAb followed by incubation with FITC-goat antimouse IgG. Cells were then fixed in 2% paraformaldehyde and permeabilized with 0.2% Triton X100, as described previously.22 Intracellular staining was then performed by using the PE-conjugated anti-CXCR4 MoAb. Nonspecific fluorescence was assessed by using irrelevant isotype-matched MoAbs (Becton Dickinson and/or Pharmingen). After staining procedures, samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson).

Agonist-dependent increase in cytoplasmic Ca²⁺ was determined as described.29,30 Briefly, aliquots of 10⁶ cells were loaded with Fluo-3 reconstituted with Pluronic F-127 (Molecular Probes, Eugene, OR) for 20 minutes at 37°C. After incubation, the samples were washed once with RPMI and resuspended in 1 mL of the same medium. Data were acquired with a FACSCalibur, with excitation at 488 nm. Cells were gated based on forward and side scatter properties. After 20 seconds, SDF-1α (Pharmingen) or ionomycin (Sigma Chemicals, St Louis, MO) was added by using a magnetic device, and calcium mobilization was determined by a two-parameter density-plot measuring linear emission at 550 nm in the FL-1 window for the gated cell population over time.

Cell migration assays were performed as described.11Briefly, uninfected and HHV-7–infected cells were resuspended in RPMI 1640 medium plus bovine serum albumin (1 mg/mL). The cell density was adjusted to 5 × 10⁶ cells/mL and 100 μL of the cell suspension was added to the top chamber of a 24-Transwell apparatus (5-μm pore size; Costar, Cambridge, MA). RPMI 1640/bovine serum albumin containing various concentrations of the recombinant SDF-1α was added to the bottom chamber of the Transwell. After 3 hours of incubation at 37°C, the cell numbers in the bottom chamber were counted in a FACSCalibur, and percentages of the transmigration were determined for each concentration of SDF-1α, after subtraction of the background (absence of SDF-1α) transmigration.

RNA purification from uninfected and HHV-7–infected cells was performed using the SV total RNA isolation system (Promega, Madison, WI) following the manufacturer’s protocol. Synthesis of first strand cDNA and amplification were performed using the Access RT-PCR system (Promega) following the manufacturer’s protocol. As a control for DNA contamination, equal amounts of RNA were used for PCR without template retrotranscription. The resulting PCR products were separated on 2% Seakem GTG agarose gel (FMC BioProducts, Rockland, ME). Primers and reaction conditions for the amplification of HHV-7 U10 ORF28 (PCR product, 186 bp), CXCR430 (PCR product, 430 bp), and β-actin (PCR product, 661 bp; Stratagene, San Diego, CA) were as described previously.

The levels of surface CXCR4 in the SupT1 lymphoblastoid CD4⁺ T-cell line (Fig 1A and B) and in primary T lymphocytes (Fig 1C) were evaluated by flow cytometry at various time points after HHV-7–infection, using the 12G5 anti-CXCR4 MoAb, either alone (Fig 1A) or in combination with anti-CD4 MoAb (Fig 1B and C). 12G5 binding was progressively reduced in HHV-7–infected SupT1 cells compared with uninfected controls (Fig 1A). Moreover, double-staining performed with anti-CXCR4 plus anti-CD4 MoAb showed the presence of four distinct cell populations (CXCR4⁺/CD4⁺, CXCR4⁺/CD4⁻, CXCR4⁻/CD4⁺, and CXCR4⁻/CD4⁻; Fig 1B), clearly indicating that the two antigens were downmodulated by HHV-7 independently. On the other hand, CD11 and CD45LCA surface markers were unaffected by HHV-7 infection (data not shown). A marked decrease of both CXCR4 and CD4 surface antigens, but not of CD3, was also observed in CD8-depleted primary T lymphocytes after infection with HHV-7 (Fig1C).

In contrast, infection of SupT1 cells with HIV-1(strain IIIB) resulted in drastic downregulation of CD4, comparable to that observed in HHV-7–infected cells, but with no significant reduction in 12G5 reactivity (Fig 1D).

Because the experiments described above could not distinguish between loss or surface CXCR4 and blocking of the 12G5 epitope, we next evaluated the intracellular Ca²⁺ flux and chemotaxis in response to SDF-1α. To avoid the possibility that dead cells and cell debris could interfere with these biological assays in both uninfected and HHV-7–infected cultures, analysis was performed on viable cells gated on the basis of forward and side scatter properties. At day 4 postinfection (PI), HHV-7–infected SupT1 cells showed a reduction in the Ca²⁺ flux mediated by SDF-1α with respect to uninfected cells (Fig 2A). On the other hand, both uninfected and HHV-7–infected SupT1 cells responded in a similar fashion to a nonspecific agonist, such as the Ca²⁺ionophore ionomycin (Fig 2A), thus confirming that the reduced Ca²⁺ flux observed in HHV-7–infected cells in response to SDF-1 was specific.

In a chemotactic assay, HHV-7–infected SupT1 cultures showed a cell migration rate significantly (P < .01) lower than those uninfected (Fig 2B).

Having previously demonstrated that HHV-7 affects the expression of its primary receptor, CD4, via multiple mechanisms,22 including reduction of the de novo CD4 synthesis, we next evaluated the intracellular levels of CXCR4 in HHV-7–infected and uninfected cells by indirect immunofluorescence shown by flow cytometry (Fig 3A). At day 8 PI, when greater than 50% of surface CXCR4 was already lost in HHV-7–infected cells, all cells still stained positively for intracellular CXCR4 and the fluorescence intensity of intracellular CXCR4 was only modestly decreased in comparison to uninfected controls. Moreover, analysis of CXCR4 mRNA by semiquantitative RT-PCR unequivocally demonstrated that HHV-7 infection did not affect the levels of CXCR4 mRNA (Fig 3B).

Cellular receptors for enveloped viruses are characteristically downregulated from the cell surface after productive infection, rendering infected cells resistant to superinfection by viruses that use the same receptor.31 Indeed, the observation that CD4 was selectively downregulated on HIV-1–infected cells as well as on HHV-7–infected cells provided the initial evidence that CD4 was a receptor for these viruses.20,32 Given the evidence that CXCR4 was downregulated during HHV-7 infection, we used SDF-1α to determine if CXCR4 was also serving as a component of the receptor for HHV-7. As shown in Fig 4A, preincubation with SDF-1α (2.5 μg/mL) led to a drastic reduction of surface CXCR4 associated with a complete block of expression of specific HHV-7 mRNA transcripts, evaluated 40 hours PI. On the other hand, preincubation with 12G5 MoAb (25 μg/mL) or control IgG2a (25 μg/mL) failed to inhibit infection, indicating that the epitope recognized by 12G5 MoAb is not essential for HHV-7 entry. Similar results have been previously reported for several HIV-1 and HIV-2 isolates.33 

Although the downregulation of surface CXCR4 by HHV-7 and the inhibitory effects of SDF-1α on HHV-7 infection are consistent with a role of CXCR4 as a coreceptor for HHV-7, the final proof of this hypothesis would be to show that noninfectable cells that are CD4 and CXCR4 negative are rendered infectable by HHV-7 only when CD4 and CXCR4 are both expressed.

The possibility that CXCR4 might represent an alternative receptor for HHV-7 in the absence of CD4 was ruled out in infection experiments performed on the BC7 cells, a derivative line from SupT1 cells that expresses CXCR4 but not CD4.23 As shown in Fig 4B, BC7 cells were not permissive for HHV-7 infection.

Although CXCR4 functions in association with CD4 to permit the entry of different HIV isolates,9,10 the precise mechanism of action of this protein as coreceptor has not yet been fully elucidated. Irrespective of the mechanism of action of CXCR4 in mediating HIV entry, it has been clearly established that CXCR4 is downregulated only by a few HIV-2 isolates, which use CXCR4 as the primary receptor in the absence of CD4.23 On the other hand, CXCR4 is either unaffected or minimally downregulated by HIV-1 strains (Fig 2) or by those HIV-2 strains that still require CD4 for infection and cell fusion.23 

We have shown here that CXCR4 interacts with HHV-7 in a unique manner compared with HIV. In fact, both CD4 and CXCR4 are downregulated from the surface of HHV-7–infected CD4⁺ T cells, although with different kinetics and probably through different mechanisms. After HHV-7 infection, CD4 loss is more rapid and involves both transcriptional and posttranscriptional mechanisms,22whereas downregulation of surface CXCR4 does not depend on a block of transcription.

Although initial studies have suggested that chemokine receptor internalization is not required to block HIV entry, more recent data indicate that SDF-1α is a more effective inhibitor of T-cell line-adapted HIV isolates on cells expressing endocytosis-competent CXCR4.14,15 This suggests that SDF-1α–mediated CXCR4 endocytosis may make a significant contribution to chemokine protection from HIV entry.

Although the mechanisms of HHV-7–mediated downregulation of CXCR4 remain to be elucidated, the possibility that HHV-7 induced CXCR4 downregulation via an autocrine production of SDF-1α was ruled out, because SDF-1α mRNA was undetectable by RT-PCR in both HHV-7–infected and uninfected SupT1 cells (data not shown). It is also particularly remarkable that, whereas SDF-1α induces a rapid but transient (<1 hour) downregulation of surface CXCR4, HHV-7 infection produces a persistent loss of surface CXCR4.

Thus, beside the relevance of these finding for HHV-7 pathogenesis, the identification of the HHV-7 gene product(s) modulating CXCR4 may allow the design of novel strategies to inactivate CXCR4 by blocking its surface expression.34 

The authors are very grateful to J.A. Hoxie and to G. Campadelli-Fiume for providing the BC7 cell line and the 5E1 MoAb, respectively.