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*
First Department of Internal Medicine and
Department of Pharmacology, Ehime University School of Medicine, Shigenobu, Ehime, Japan; and
Department of Microbiology, Kagoshima University School of Dentistry, Kagoshima City, Kagoshima, Japan
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Introduction |
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Recently, it has become apparent that the chemokine receptors, CXCR4 and CCR5, are the main coreceptors for T lymphocyte-tropic and macrophage-tropic HIV-1, respectively (19, 20, 21, 22, 23, 24). Evidence that HHV-6 and HIV-1 interact in CD4+ T lymphocytes and that HHV-7 and HIV-1 competitively infect CD4+ T lymphocytes led us to examine whether CXCR4 or CCR5 is also necessary for HHV-6 and HHV-7 infection. To clarify this, we examined whether these viruses can infect lymphocytes of individuals with CCR5 deficiency and whether anti-CXCR4 mAb inhibits infection with HHV-6 or HHV-7. Our results demonstrated that unlike HIV-1, HHV-6 and HHV-7 infections do not require the expression of CXCR4 or CCR5. Unexpectedly, however, it was found that dramatic down-regulation of CXCR4 at the transcriptional level is induced by both HHV-6 and HHV-7. Down-regulation of CXCR4 resulted in impairment of the functions of HHV-6- and HHV-7-infected lymphocytes, as determined by chemotaxis and elevation of the intracellular Ca2+ concentration ([Ca2+]i) in response to the natural CXCR4 ligand, stromal cell-derived factor-1 (SDF-1). The significance of down-regulation of the HIV-1 coreceptor induced by HHV-6 and HHV-7 infection was addressed.
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Materials and Methods |
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The U1102 strain of HHV-6 variant A (HHV-6A), the Z29 strain of HHV-6 variant B (HHV-6B), and the RK strain of HHV-7 were mainly used in the present study. They were grown in cord blood mononuclear cells that had been stimulated with PHA as described previously (2, 11). CD4+ T lymphocytes were enriched from PBMCs of healthy individuals using anti-CD4 mAb-coated magnetic beads (Dynal, Oslo, Norway). CD4+ T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FCS and PHA for 3 days. PHA-stimulated lymphocytes were then washed and suspended in RPMI 1640 medium supplemented with 10% FCS and inoculated with HHV-6A, HHV-6B, and HHV-7 at approximate multiplicities of infection of 1, 1, and 0.1 50% tissue culture infective doses (TCID50), respectively. HHV-6A-, HHV-6B-, and HHV-7-inoculated and mock-infected cells were cultured in a 5% CO2 incubator at 37°C until the cytopathic effect (CPE) became detectable. Maximal CPE was usually detected after 4 and 6 days of virus inoculation in HHV-6- and HHV-7-infected cells, respectively.
Detection of virus replication
Replication of HHV-6 and HHV-7 was determined by detection of typical CPE and by indirect immunofluorescence assays using HHV-6- and HHV-7-seropositive human serum as described previously (25). Briefly, virus-infected cord blood mononuclear cells were mounted on glass slides and fixed in cold acetone. Twentyfold diluted HHV-6- and HHV-7-seropositive human serum was applied to the slide, followed by incubation for 30 min at 37°C. After washing, FITC-conjugated goat anti-human IgG (Organon Teknika, Durham, NC) was added and incubated for 30 min at 37°C. After washing, the slides were examined using a fluorescence microscope.
The TCID50 was determined as follows. Virus-infected cells suspended in culture supernatant were frozen and thawed, then sonicated. PHA-stimulated cord blood lymphocytes (2 x 105) suspended in 0.1 ml of RPMI 1640 medium supplemented with 10% FCS were plated into a flat-bottom microtiter well, and then 0.1 ml of a 10-fold serially diluted sample was added to each well. Twelve wells were prepared for each dilution. The plates were incubated in an atmosphere of 5% CO2 at 37°C for >7 days, and the CPE was examined with an inverted microscope. The TCID50 was calculated by the method of Reed and Muench (26).
Effect of anti-CXCR4 mAb on virus infection
The inhibitory effect of anti-CXCR4 mAb on HHV-6 and HHV-7 infection was examined as reported previously (27). Briefly, PHA-stimulated lymphocytes were preincubated with sodium azide- and endotoxin-free anti-CXCR4 mAb, 12G5 (27) (PharMingen, San Diego, CA), at various concentrations for 1 h at room temperature, followed by inoculation with HHV-6A, HHV-6B, and HHV-7. The cells were maintained in the presence of anti-CXCR4 mAb at the same concentration for the duration of the experiment. To estimate the anti-HIV-1 activity of anti-CXCR4 mAb, a multinuclear activation of galactosidase indicator (MAGI) assay was performed as described previously (28). The MAGI cell line is a HeLa cell clone expressing human CD4 and HIV-1 long terminal repeat ß-galactosidase. MAGI-CCR5 cells are a clone of MAGI cells that express human CCR5 (29). These cells were placed in a 24-well plate at 4 x 104 cells/well in DMEM supplemented with 10% FCS. The cells were preincubated with or without anti-CXCR4 mAb, 12G5, for 1 h at room temperature and then inoculated with the IIIB strain of T lymphocyte-tropic HIV-1 or the JR-FL strain of macrophage-tropic HIV-1 at a multiplicity of infection of 0.2 in the presence of 20 µg/ml DEAE-dextran. Two days later, the medium was removed, and the monolayer was fixed with 1 ml of a solution of 1% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min at room temperature. The cells were then washed three times with PBS and incubated for 50 min at 37°C in 500 µl of a solution of 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2, and 400 µg/ml X-Gal (5-bromo-4-chloro-3-indolyl ß-D-galactoside). The reaction was stopped by removing the staining solution and washing the cells twice with PBS. Blue cells were then counted under a microscope.
Flow cytometric analysis of cell surface molecules
Expression of surface CD3, CD4, CXCR4, and CCR5 was examined by direct immunofluorescence using a flow cytometer. Cells were stained with FITC-conjugated anti-CD3 mAb, Leu 4 (Becton Dickinson, Mountain View, CA), FITC-conjugated anti-CD4 mAb, Leu 3a (Becton Dickinson), phycoerythrin-conjugated anti-CXCR4 mAb, 12G5 (PharMingen), and FITC-conjugated anti-CCR5 mAb, 45531.111 (R&D Systems, Minneapolis, MN). Cells to be used as unstained negative controls for CD3, CD4, CXCR4, and CCR5 were incubated with FITC- or phycoerythrin-conjugated monoclonal mouse IgG.
Northern blot analysis
Northern blot analysis of mRNA for CXCR4 was performed as follows. Total cellular RNA was extracted from virus-infected and mock-infected cells. From each RNA preparation, 15-µg portions were denatured with formaldehyde and size fractionated by electrophoresis on a 1% agarose gel. The RNAs were then transferred to a hybridization transfer membrane and hybridized with 32P-labeled CXCR4 cDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. To examine the posttranscriptional stability of CXCR4 mRNA, actinomycin D (Sigma, St. Louis, MO) was added to mock-infected and virus-infected cells at a concentration of 10 µg/ml to block transcription. Cells were collected after various incubation periods and were used for RNA preparation. Northern blot analysis was performed as described above. The half-life of CXCR4 mRNA was estimated by plotting the densitometric ratios of CXCR4 mRNA/GAPDH mRNA determined using a BAS 1000 bioimaging analyzer (Fujix, Tokyo, Japan).
Chemotaxis assays
Chemotaxis assays were performed using 5-µm-diameter pore
Transwell cell culture chambers (Costar, Boston, MA). Cells (4 x
105) suspended in 100 µl of RPMI 1640 medium supplemented
with 0.1% BSA (migration medium) were added to the upper chambers and
then carefully transferred to lower wells containing 500 µl of
migration medium with or without recombinant human SDF-1
(PeproTech,
London, U.K.) and EBI1-ligand chemokine (ELC) macrophage inflammatory
protein-3ß (MIP-3ß) (PeproTech) at various concentrations. The
plates were incubated at 37°C in 5% CO2 for 3 h,
then the upper chambers were carefully removed and the numbers of
viable cells were counted using trypan blue exclusion.
Calcium flux measurement
Calcium flux assays were performed by real time measurement of
[Ca2+]i changes using a F2000 spectrometer
(Hitachi, Tokyo, Japan). Briefly, cells were loaded with 2 µM
fura-2/AM (Dojin Chemical, Tokyo, Japan) at 37°C for 45 min in the
dark. The cells were then washed twice and resuspended at 1.5 x
106 cells/ml in phenol red-free RPMI 1640 medium
supplemented with 0.1% BSA. Cells suspended in 800 µl of assay
medium were placed in a stirred cuvette and excited sequentially at 340
and 380 nm. Fluorescence emission was monitored at 510 nm before and
after addition of SDF-1 at final concentrations of 100 and 500
ng/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
shows the replications of
HHV-6A, HHV-6B, and HHV-7 in PHA-stimulated CD4+ T
lymphocytes in the presence or the absence of anti-CXCR4 mAb. The
typical CPE of HHV-6 and HHV-7 was detectable in lymphocytes cultured
with anti-CXCR4 mAb. Similarly, indirect immunofluorescence using
HHV-6- and HHV-7-seropositive human serum demonstrated that HHV-6 and
HHV-7 Ags were present in lymphocytes cultured with anti-CXCR4 mAb
as well as in those cultured without the Ab. The titers of viruses that
grew in the cells cultured in the presence or the absence of
anti-CXCR4 mAb are shown in Table I.
The titers of HHV-6A, HHV-6B, and HHV-7 replicated in CD4+
T lymphocytes cultured in the absence and the presence of
anti-CXCR4 mAb at various concentrations were almost the same. On
the other hand, the concentrations of anti-CXCR4 mAb used in the
present study appeared to inhibit T lymphocyte-tropic, but not
macrophage-tropic, HIV-1 infection. These data demonstrate that the
expression of CXCR4 is not necessary for both HHV-6 and HHV-7
infection.
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Down-regulation of surface CXCR4 in HHV-6- and HHV-7-infected cells
It has been demonstrated that down-regulation of surface CXCR4 is
induced following infection with HIV-1 (27). Therefore, we next
examined whether such an alteration is also detectable in HHV-6- or
HHV-7-infected cells. As shown in Fig. 2,
marked down-regulation of surface CD3 and CD4 was detected in HHV-6A-
and HHV-7-infected cells, respectively, as reported previously by us
and other investigators (14, 15, 33). Whereas CCR5 expression was not
altered by HHV-6 or HHV-7 infection, the expression level of surface
CXCR4 appeared to decline dramatically after infection with HHV-6A,
HHV-6B, and HHV-7. Virus replication might be necessary to induce
down-regulation of CXCR4, since inoculation of viruses that had been
inactivated by UV irradiation had no effect on CXCR4 expression (data
not shown). The down-regulation of CXCR4 was also detected in
CD4+ T lymphocytes that were infected with two other
strains of HHV-6A and HHV-6B and two other strains of HHV-7 (data not
shown), suggesting that this phenomenon is commonly observed but is not
restricted to the strains of HHV-6 and HHV-7 we employed.
Down-regulation of CXCR4 was also observed in the virus-permissive cell
lines, i.e., the HHV-6A-infected JJHAN cell line, the HHV-6B-infected
MT-4 cell line, and the HHV-7-infected Sup-T1 cell line (data not
shown).
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To investigate the level at which the expression of surface CXCR4
was down-regulated in HHV-6- and HHV-7-infected cells, Northern blot
analysis of mRNA for CXCR4 was performed. As shown in Fig. 3, a markedly decreased level of mRNA for
CXCR4 was observed in CD4+ T lymphocytes after infection
with HHV-6A, HHV-6B, and HHV-7. Thus, it appeared that down-regulation
of CXCR4 induced by HHV-6 and HHV-7 occurs at the transcriptional
level.
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. The estimated half-lives of
CXCR4 mRNA from mock-infected and HHV-6A-infected CD4+ T
lymphocytes were 6.0 and 6.5 h, respectively, and those of
HHV-6B-infected and HHV-7-infected CD4+ T lymphocytes were
6.2 and 6.7 h, respectively (data not shown). These data indicate
that HHV-6 and HHV-7 infections do not affect significantly the
posttranscriptional stability of CXCR4 mRNA.
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It is known that addition of SDF-1 to CXCR4-positive cells results
in cell migration and elevation of [Ca2+]i.
In accordance with these recent findings, we compared the degrees of
migration and elevation of [Ca2+]i in
mock-infected cells and virus-infected cells following stimulation with
SDF-1. [Ca2+]i was elevated markedly in
mock-infected CD4+ T lymphocytes in response to SDF-1.
In contrast, no apparent elevation of [Ca2+]i
was detected in HHV-6- and HHV-7-infected cells (Fig. 5). Similarly, a significant difference
in the migration rate induced by SDF-1 between mock-infected and HHV-6-
or HHV-7-infected cells was detected. In contrast, the degrees of
migration in mock-infected cells and virus-infected cells stimulated
with a CC chemokine ELC/MIP-3ß were almost the same (Fig. 6). These data confirmed the functional
down-regulation of CXCR4 in HHV-6- and HHV-7-infected cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that HHV-6 alters the expression of various functional molecules in T lymphocytes. That is, down-regulation of the surface CD3/TCR complex occurs after infection with HHV-6 (14, 33). In addition, it has been reported that de novo expression of CD4 is induced in CD4- T lymphocytes and NK cells by HHV-6 infection (8, 9, 10). HHV-7 is also known to induce alteration of surface molecule expression and dysfunction of CD4+ T lymphocytes. In contrast to the case of HHV-6-infected cells, it has been reported that the expression level of surface CD3 is unchanged, but that of CD4 declines markedly in HHV-7-infected T lymphocytes (14, 15). The present study has shown that HHV-6 and HHV-7 also modulate the expression of the chemokine receptor as well as CD3 and CD4. Since chemokine receptors play an important role in cellular functions, such as directional migration, our data provide new insight into the pathogenesis of virus-induced immunodeficiency.
It has been shown that down-regulation of CXCR4 is also detectable in HIV-1-infected T lymphocytes (27). Since CXCR4 is the coreceptor for T lymphocyte-tropic HIV-1 (24), it has been suggested that the down-regulation of surface CXCR4 in HIV-1-infected cells is induced by the internalization resulting from direct binding with the HIV-1 component on the cell surface. Additionally, the present findings suggest that there is an other mechanism of CXCR4 down-regulation at the transcriptional level that may commonly occur in HHV-6A-, HHV-6B-, HHV-7-, and HIV-1-infected cells. Since the CXCR4 promoter sequence has been identified recently (34), further study focusing on the mechanisms of CXCR4 gene transcript repression in HHV-6- and HHV-7-infected cells remains to be performed.
The present study has also provided new insight into the roles of HHV-6 and HHV-7 in the pathogenesis of HIV-1 infection. Previous studies demonstrated that HHV-6 can coinfect and trans-activate the long terminal repeat of HIV-1 (5, 6, 7). In addition, it has been reported that CD4 expression is induced by HHV-6 infection of CD4- lymphocytes, rendering them susceptible to infection with HIV-1 (8, 9, 10). These data imply that HHV-6 infection results in the augmentation of HIV-1 replication. However, our present data demonstrating that HHV-6 induces down-regulation of the HIV-1 coreceptor, CXCR4, suggest a possibility opposite that proposed previously. Carrigan et al. demonstrated that HIV-1 replication was suppressed by HHV-6 (35). Their findings seemed to run contrary to previous reports, but might have resulted from down-regulation of the HIV-1 coreceptor by HHV-6 infection, as shown here. To clarify the importance of HHV-6-induced down-regulation of CXCR4 in HIV-1 infection, we investigated HIV-1 replication in T lymphocytes that had been infected with HHV-6. That is, CD4+ T lymphocytes were infected with HHV-6 and then inoculated with HIV-1 after 3 days, when CXCR4 expression declined. It was found that the degree of replication of HIV-1 decreased in HHV-6-infected cells compared with that in uninfected cells. However, we were unable to conclude that the inhibition of HIV-1 replication by HHV-6 was due solely to the disappearance of CXCR4 from the cell surface, since HHV-6 infection of CD4+ T lymphocytes resulted in the deterioration of cellular condition and the death of cells in long term culture as reported previously (36).
In contrast to HHV-6, HHV-7 has been considered an inhibitor of HIV-1 infection on the basis of the finding that HHV-7 and HIV-1 competitively infect CD4+ T lymphocytes and macrophages (15, 17). The previous studies demonstrated that the inhibition of HIV-1 infection by HHV-7 occurs at a very early stage, consistent with blocking at the level of virus attachment to cell surface CD4 molecules (15, 17). The present study suggests another mechanism of HHV-7-mediated HIV-1 inhibition at a late stage, when the level of CXCR4 expression has declined.
In summary, we have shown that CXCR4 and CCR5 are unnecessary for infection of T lymphocytes by HHV-6A, HHV-6B, and HHV-7 and also that the expression of CXCR4, which is the coreceptor for HIV-1, declines dramatically at the transcriptional level. Identification of the virus component that inhibits transcription of the CXCR4 gene and clarification of the detailed mechanism of CXCR4 down-regulation mediated by virus infection may shed light on the novel concept of prophylaxis against HIV-1 infection and the development of new agents for treatment of HIV-1 infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masaki Yasukawa, First Department of Internal Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan. E-mail address:
3 Abbreviations used in this paper: HHV, human herpesvirus; [Ca2+]i, intracellular Ca2+ concentration; SDF-1, stromal cell-derived factor-1; TCID50, 50% tissue culture infective doses; CPE, cytopathic effect; MAGI, multinuclear activation of galactosidase indicator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELC, EBI1-ligand chemokine; MIP-3ß, macrophage inflammatory protein-3ß.
Received for publication October 22, 1998. Accepted for publication February 10, 1999.
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References |
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T lymphocytes by human herpesvirus 6: transcriptional induction of CD4 and susceptibility to HIV infection. J. Exp. Med. 181:1303., MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955.[Abstract]
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