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RAPID COMMUNICATION
Human Immunodeficiency Virus (HIV)-Resistant CD4 " UT-7 Megakaryocytic
Human Cell Line Becomes Highly HIV-1 and HIV-2 Susceptible Upon
CXCR4 Transfection: Induction of Cell Differentiation by HIV-1 Infection
By Marta Baiocchi, Eleonora Olivetta, Cristiana Chelucci, Anna Claudia Santarcangelo, Roberta Bona, Paola d’Aloja,
Ugo Testa, Norio Komatsu, Paola Verani, and Maurizio Federico
Recent findings have shown that the expression of the seven
trans-membrane G-protein–coupled CXCR4 (the receptor for
the stromal cell-derived factor [SDF]-1 chemokine) is necessary for the entry of T-lymphotropic human immunodeficiency virus (HIV) strains, acting as a coreceptor of the CD4
molecule. In the human system, the role of CXCR4 in HIV
infection has been determined through env-mediated cell
fusion assays and confirmed by blocking viral entry in CD4"/
CXCR4" cells by SDF-1 pretreatment. We observed that the
human megakaryoblastic CD4" UT-7 cell line fails to express
CXCR4 RNA and is fully resistant to HIV entry. Transfection
of an expression vector containing the CXCR4 c-DNA ren-
dered UT-7 cells readily infectable by different T-lymphotropic syncytium-inducing HIV-1 and HIV-2 isolates. Interestingly, HIV-1 infection of CXCR4 expressing UT-7 cells (named
UT-7/fus) induces the formation of polynucleated cells
through a process highly reminiscent of megakaryocytic differentiation and maturation. On the contrary, no morphologic changes were observed in HIV-2–infected UT-7/fus
cells. These findings further strengthen the role of CXCR4
as a molecule necessary for the replication of T-lymphotropic
HIV-1 and HIV-2 isolates and provide a useful model to study
the functional role of CD4 coreceptors in HIV infection.
q 1997 by The American Society of Hematology.
T
such as glycoprotein (GP) IIb and GPIIIa as well as the
erythroid marker glycophorin A. These features, together
with the possibility of inducing UT-7 cells to differentiate
toward erythroid (by Epo treatment)14 or megakaryocytic (by
phorbol myristate acetate induction)13 differentiation, indicate that UT-7 cells are phenotypically related to a common
hematopoietic progenitor pertaining both to the megakaryocytic and erythroid cell lineages.15
In this report we show that, despite the exposure of the
CD4 receptor, UT-7 cells are totally resistant to HIV infection. The inability of HIV to entry and replicate directly
correlates with the lack of CXCR4 gene expression. In fact,
transfection of a vector expressing the CXCR4 gene renders
UT-7 cells highly susceptible to infection by different SI Tlymphotropic HIV-1 and HIV-2 strains. In addition, HIV-1
infection of UT-7 cells stably expressing CXCR4 c-DNA
(named UT-7/fus cells) induces morphologic changes highly
reminiscent of the megakaryocytic differentiation process.
HE BINDING OF CD4 receptor with viral gp120 envelope protein is the first step in human immunodeficiency virus (HIV) infection. However, as widely shown in
human and nonhuman cells, CD4 exposition is not per se
sufficient to allow viral entry.1,2 Recent studies have demonstrated the role that CXCR4 (also called LESTR, HUMSTR,
or fusin)3 and CC-CKR54 play as coreceptors of syncytiuminducing (SI) T-lymphotropic5 and non–syncytium-inducing
(NSI) macrophage (M)-tropic6-10 HIV strains, respectively.
Both molecules are receptors for chemokines; CXCR4 is
the stromal cell-derived factor (SDF)-111,12 receptor, whereas
MIP-1a, MIP-1b, and RANTES bind CC-CKR5 receptor.4
These pioneristic data were obtained by transiently transfecting target cells with different molecular constructs and
measuring the activity of reporter genes (ie, luciferase, cloramphenicol-acetyl-transferase, or b-galactosidase) in env-mediated cell fusion experiments or inserted in HIV env-defective
molecular clones in env-complementation assays.
UT-7 is a cell line established from a patient with acute
megakaryoblastic leukemia and is strictly dependent for
growth on either granulocyte-macrophage colony-stimulating factor (GM-CSF), interlukine-3 (IL-3), or erythropoietin
(Epo).13 These cells express specific megakaryocytic markers
From the Laboratories of Virology and Hematology-Oncology,
Istituto Superiore di Sanità, Rome, Italy; and the Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan.
Submitted December 12, 1996; accepted February 5, 1997.
Supported by grants from AIDS Projects of the Ministry of Health,
Rome, Italy.
Address reprint requests to Maurizio Federico, PhD, Laboratory
of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299,
00161 Rome, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8908-0053$3.00/0
MATERIALS AND METHODS
Cell cultures. UT-7 cells were grown in Iscove’s modified Dulbecco’s medium (GIBCO, Grand Island, NY) supplemented with
10% decomplemented fetal calf serum (FCS) and 1 ng/mL of recombinant human GM-CSF (Boehringer Mannheim, Mannheim, Germany) or IL-3 (Genetics Istitute, Cambridge, MA). UT-7/fus cells
were cultivated in the same medium supplemented with 1 mg/mL of
G418 antibiotic (GIBCO-BRL, Gaithersburg, MD; 70% of activity).
CEMss and C8166 cells were maintained in RPMI 1640 medium
supplemented with 10% decomplemented FCS.
Construction of CXCR4-expressing vector and transfection. Total RNA was extracted from quiescent human peripheral blood lymphocytes according to the manufacturer’s instructions (RNA-FastI
kit; Molecular System, San Diego, CA). Five micrograms of RNA
was reverse transcribed in a final volume of 50 mL using oligodT
as primer. Ten milliliters of reverse transcribed RNA was then subjected to polymerase chain reaction (PCR) using oligoprimers
(whose sequence was reported by Dragic et al9) overlapping the start
and stop codons of the CXCR4 open reading frame and carrying
HindIII (oligo forward) and EcoRI (oligo reverse) restriction sites
at the respective 5* ends. The reaction was performed as previously
described16 in a total volume of 50 mL. The product of amplification
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HIV INFECTION AND DIFFERENTIATION OF UT-7 CELLS UPON CXCR4 TRANSFECTION
was digested with HindIII and EcoRI enzymes and inserted in
pcDNA3 (Invitrogen, San Diego, CA) expression vector previously
digested with the same restriction enzymes.
UT-7 cells were transfected by the electroporation method by adding
10 mg of linearized plasmidic DNA at 5 1 106 cells pulsed at 200 V,
950 mF through a Gene Pulser apparatus (BioRad, Richmond, CA).
HIV infection and detection. Culture supernatants from acutely
infected CEMss cells were used as a source of HIV (NL4-3, LAV1, NDK, and HIV-2ROD ) preparations. Titers of SI strains (ranging
from 3 1 106 to 107 50% tissue culture infective dose [TCID50] per
milliliter) were measured as previously described17 by scoring the
syncytia number on C8166 cells 5 days after infection with serially
diluted virus preparations. HIV Bal-1 virus preparations were obtained as described18 and titrated as picograms per milliliter of p24
protein through quantitative enzyme-linked immunosorbent assay
(Abbott, North Chicago, IL). Infections were performed by adsorbing the virus for 1 hour at 377C. After infection, cells were
washed and fed. HIV release was monitored by reverse transcriptase
(RT) assay on culture supernatants.19
In experiments of PCR on infected UT-7 cells, only HIV strains
(ie, NL4-3 and HIV-2ROD ) free of intravirionic DNA detectable by
PCR were used.
DNA and RNA analyses. Early intracellular HIV retrotranscription in infected UT-7 cells was assessed by DNA-PCR using primers
specific for the long terminal repeat (LTR) region of both HIV-1
and HIV-2 already described.16 Viral preparations were treated with
DNAase (Boehringer, GmbH, Mannheim, Germany) for 2 hours at
room temperature (rt) in the presence of 1 mmol/L of MgCl2 . Samples were prepared by lysing 105 cells in 50 mL of tridistillated
DNA-free water at 957C for 5 minutes and subjected to PCR as
described.16 Amplified products were run on 1.8% agarose gel electrophoresis, transferred onto nitrocellulose membrane, and hybridized using a 32P end-labeled specific oligonucleotides16 as probe.
Cell lysates were contemporary amplified by using primers specific
for the human b-globin.17
RT-PCR for the detection of CXCR4 expression was performed
by using the same oligoprimers employed for molecular cloning (see
above) and probed with an oligoprimer internal of the CXCR4 cDNA (from nt 961 to nt 989).3 RNA were normalized by using
primers specific for the human b-2 microglobulin gene.20
Northern blot was performed as previously described.21 Filters
were hybridized with 32P random primed labeled CXCR4 c-DNA or
a 316-bp fragment of the human glyceraldehyde-3-phosphodehydrogenase (GAPDH) c-DNA (Ambion, Austin, TX) derived from exons
5 through 8.
Amplified CXCR4 c-DNA used for the UT-7 transfection was
sequenced by the dideoxy chain termination method using the SequenaseII kit (US Biochemical Corp, Cleveland, OH).
Gp120 competition assay and antigenic and morphologic analyses. Gp120 competition assay was performed by incubating 5 1
105 cells with 2 mg of LAV-1 gp120 recombinant protein (Intracel,
Cambridge, MA) for 1 hour at 47C. Cells were washed three times
with phosphate-buffered saline (PBS) and then incubated with 1:20
diluted phycoerythrine (PE) Leu3a monoclonal antibody (MoAb;
Becton Dickinson, Mountain View, CA) for 1 additional hour at
47C and finally analyzed with a cytofluorimeter (FAC-scan; Becton
Dickinson).
PE-conjugated MoAbs to CD4 (Leu3a), CD31, CD41a, and glycophorin A (Becton Dickinson) were used at appropriate dilutions.
After incubation at 47C for 1 hour, cells were washed and analyzed
by a cytofluorimeter. HIV gag-related proteins were detected by
intracellular direct immunofluorescence. Cells (3 1 105) were
washed three times with PBS, resuspended in 200 mL of Permafix
(Ortho Diagnostics, Raritan, NJ), and incubated for 30 minutes at
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Fig 1. HIV-1 gp120 competition assay. Both CEMss and UT-7 cells
were labeled with (I) PE-conjugated unspecific mouse IgG; (II) Leu3A
anti-CD4 MoAb; and (III) Leu3A MoAb after pretreatment with saturating amounts of HIV-1 recombinant gp120 protein. M1, marker 1.
rt. After two additional washes, cells were incubated 1 hour at rt
with 1:20 diluted KC57-RD1 (Coulter Corp, Hialeah, FL) anti-p24
HIV MoAb conjugated with PE, washed three times with PBS, and
finally analyzed by a cytofluorimeter.
Uninfected and HIV infected cells cytocentrifuged onto glass
slides were stained with May-Grünwald Giemsa and morphologically analyzed.
RESULTS
HIV does not penetrate into UT-7 cells, despite the exposure
of CD4 receptors able to bind HIV gp120. In a first set of
experiments we observed that, although UT-7 cells homogenously expose the CD4 receptors, infection with different HIV1 (either SI or NSI) or HIV-2 strains repeatedly failed. Thus,
to assess whether CD4 receptors were able to efficiently bind
the HIV env glycoprotein, gp120/CD4 competition assay was
performed. As shown in Fig 1, gp120 HIV protein efficiently
competes for the binding with the Leu3A MoAb, suggesting
that the CD4 molecules exposed on UT-7 cell membrane are
able to bind HIV virions. In this assay, CEMss, a T-lymphoblastoid human cell line exposing high levels of CD4 receptors,
was used as a positive control. To determine at which step the
HIV infection is blocked, we analyzed the earliest events of
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BAIOCCHI ET AL
Fig 2. PCR analysis of HIV-1– or HIV-2–infected UT-7 cells. Both
UT-7 and CEMss cells were infected with (A) NL4-3 HIV-1 or (B) HIV2ROD strains (moi 1); cells were collected 8, 24, and 48 hours postinfection; and cell lysates were analyzed by PCR. Lysates from CEMss
cells 5 days after infection with NL4-3 HIV-1 or HIV-2ROD were used
as positive controls (c"). Cell lysates were also amplified with bglobin–specific primers as control.
retrotransciption. DNA-PCR analysis on infected cell lysates
was performed by using oligonucleotides specific for HIV-LTR
sequences immediately 5* to primer binding-site16 that is the
genomic region firstly retrotranscribed. As shown in Fig 2, no
signals were detected in UT-7 cells at any time after infection
with HIV-1 or HIV-2. These data showed that the block of
HIV infection occurs in a step between viral binding and RNA
retrotranscription.
Transfection of the UT-7 cells with CXCR4 expressing
vector: characterization of UT-7/fus cells. We first examined whether the inability of HIV to penetrate into UT-7
cells may be correlated to the lack of expression of CXCR4,
the coreceptor for T-lymphotropic SI HIV strains. Both
Northern blot and RT-PCR analyses showed that UT-7 cells
do not express CXCR4 mRNA (Fig 3). We then transfected
UT-7 cells with an expression vector containing the human
CXCR4 c-DNA under the control of the immediate-early
cytomegalovirus promoter.
The G418-resistant cell population (UT-7/fus cells) was
characterized in terms of CXCR4 RNA expression, growth,
morphology, and antigenic phenotype. As expected, UT-7/
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fus cells expressed high levels of CXCR4 mRNA (Fig 3).
No significant differences in the rate of cell growth between
UT-7 and UT-7/fus cells were detected in the presence of
IL-3 or GM-CSF (not shown). Similarly, the analysis of
membrane antigens (CD4, CD31, CD41a, and glycophorin
A) failed to evidentiate any modification in UT-7/fus with
respect to UT-7 cells (not shown). However, a slight increase
in polyploidy was detectable in UT-7/fus with respect to
parental cells (see below). As a control, we used parental or
UT-7 cells transfected with empty pcDNA3 expressing vector, whose presence did not modify the morphology, the
antigen expression, and cell growth.
UT-7/fus cells are highly susceptible to HIV infection.
To assess whether the expression of the transfected CXCR4
gene was sufficient to render UT-7 cells susceptible to HIV
infection, UT-7/fus cells were challenged with the NL4-3
strain at different multiplicity of infection (moi; ie, 0.1, 0.4,
and 2) and the presence of virions in culture supernatants
was tested by RT activity. The results shown in Fig 4 indicate
that UT-7/fus cells were readily infectable at any moi used
and that the kinetics of viral release strictly depends on viral
load. In addition, the values of RT activity were as high as
those detected in highly HIV-sensitive CEMss cells (not
shown). Conversely, no virus release was observed after
infection of either UT-7– (Fig 4) or pcDNA3-transfected
UT-7 cells (not shown). Similar results were obtained on
UT-7 and UT-7/fus cells infected with LAV-1, NDK (not
shown), or HIV-2ROD strains (Fig 4).
To evaluate the proportion of HIV susceptible UT-7/fus
cells, immunofluorescence analysis for the production of intracellular p24 protein was performed 7 days after HIV-1
infection (moi, 0.4). Figure 5 shows that virtually all UT-7/
fus cells were HIV/. We also observed that, at different days
after infection, depending on the moi used (ie, 10 to 14 days
after infection with the higher moi), UT-7/fus cells died.
In contrast to HIV-1, HIV-2 infection of UT-7/fus cells
did not lead to any evident morphologic alterations, even
though the virus was able to enter and replicate, as shown
in Fig 4. Furthermore, upon HIV-2 infection, chronically
infected UT-7/fus cells could be readily obtained. As expected, UT-7/fus cells remain resistant to infection with Bal1, a M-tropic NSI HIV strain (data not shown).
HIV infection induces modifications in UT-7/fus cell morphology and phenotype. As reported above, in UT-7/fus
cells a slight increase of polyployidy with respect to the
parental cells was observed (Fig 6A). However, upon HIV-1
infection, UT-7/fus cells greatly modified their morphology,
progressively involving the entire cell population. Morphological analysis (Fig 6B) shows (1) a generalized and homogeneous enlargement of cell size, characteristic of differentiation process in normal megakaryocytes. This is also
detectable in dot plots (Fig 7), where a clear difference between UT-7/fus (R1) and HIV-infected UT-7/fus (R2) cell
populations could be observed. Also shown by morphological analysis are (2) an increase of polyploidy as shown by
the presence of cells with up to 8 nuclei (as also shown in
Fig 6A); and (3) a cytoplasm less azzurophilic than parental
UT-7 or UT-7/fus cells and some large eosinophilic areas
containing several purple punctuations.
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HIV INFECTION AND DIFFERENTIATION OF UT-7 CELLS UPON CXCR4 TRANSFECTION
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Fig 3. Analysis of CXCR4 expression in UT-7 and UT-7/fus
cells. Northern blot (A and B) and
RT-PCR (C and D) were performed on total RNA obtained
from human peripheral blood
lymphocytes and parental UT-7
and UT-7/fus cells. Northern blot
was hybridized with CXCR4 cDNA (A) or GAPDH (B) probes.
RT-PCR products were hybridized with an end-labeled oligoprimer internal to CXCR4 c-DNA
(C). As control, RT-PCR for b2microglobulin19 was performed
(D).
We next characterized HIV-infected UT-7/fus cells in
terms of CD4 (the HIV receptor), CD31 (the platelet endothelial cell adhesion molecule [PECAM-1] expressed on
platelets, but also on monocytes, granulocytes, and endothelial and T cells),22 CD41a (the GPIIb and GPIIIa complex,
specifically expressed on megakaryocytes and platelets),23
and glycophorin A (an erythroid marker) membrane antigen
exposure. When the majority of UT-7/fus cells was infected
by HIV-1 (ie, 7 days after infection with moi 0.4), a clear
increase of both CD31 and CD41a was detected with respect
to uninfected UT-7/fus cells (Fig 7). At the same time, a
general decrease in both glycophorin A (Fig 7) and CD4
Fig 4. HIV-1 and HIV-2 infection of UT-7 and UT-7/fus cells. Cells were infected with NL4-3 HIV-1 or with HIV-2ROD strains and RT activity
in supernatant cultures was assayed at different time points. RT activity below the background was detected in any sample derived from UT7 infected supernatant, regardless of moi or HIV strain used.
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Fig 5. FACS analysis for the
presence of intracytoplasmic
HIV-1 gag protein. Both UT-7
and UT-7/fus cells were labeled
with PE-conjugated unspecific
mouse IgG (A) or PE-conjugated
anti–HIV-1 p24 MoAb (B) 7 days
after infection (moi 0.4). M1,
marker 1.
exposure (not shown) was observed. Conversely, no variations in CD4, CD31, CD41a, and glycophorin A were detected in UT-7 cells challenged with HIV-1 or HIV-2 (not
shown). Similarly, in HIV-2 chronically infected UT-7/fus
cells, the exposure of these antigens was not modified, except
for CD4, which, as expected, was fully downmodulated as
in HIV-1–infected UT-7/fus cells (data not shown).
DISCUSSION
The demonstration that the pretreatment of CD4/ cells
with certain b-chemokines induces inhibition of HIV replication24 has been rapidly followed by the identification of
chemokine receptors as molecules involved in HIV entry1,6-10 and by the demonstration of their indispensable role
as coreceptors for HIV entry. In particular, it has recently
been shown that NSI M-tropic HIV strains (whose replication could be inhibited by pretreatment with chemokine MIP1a, MIP-1b, and RANTES)24 use CC-CKR56-10 and, in a
lesser extent, CC-CKR36,7 and CC-CKR2b6 as coreceptors.
Conversely, SI T-lymphotropic HIV strains need the presence of CXCR4 (the receptor for the SDF-1 chemokine) to
enter the cell.5 The CXCR4 molecule is a seven-transmembrane G-protein–coupled receptor, exhibiting approximately
30% homology with members of both a- and b-chemokine
receptor families.4 The CXCR4 ligand (SDF-1) has chemoattractive activity for hematopoietic stem cells, monocytes, B
lymphocytes, and, in a stronger fashion, T lymphocytes.11
Furthermore, it has proved to be a pre-B lymphocyte growthstimulating factor.25 CXCR4 mRNA is widely expressed in
hematopoietic and nonhematopoietic tissues.26,27 Thus, the
lack of CXCR4 expression in the UT-7 cell line renders a
unique model of human CD4/ hematopoietic cells that has
no counterparts thus far.
We show here that, in UT-7 cells, the presence of CD4
receptors fully active in binding the viral gp120 is not sufficient to allow the entrance of HIV-1 or HIV-2 SI isolates,
as suggested by the DNA-PCR analysis performed shortly
after HIV challenge. We showed that resistance to HIV is
due to the lack of CXCR4 expression. In fact, transfection
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of a vector expressing the human CXCR4 c-DNA renders
UT-7 cells highly susceptible to infection by SI HIV-1 isolates. We also show that the CXCR4 molecule is functional
as a coreceptor for HIV-2. The observation that UT-7/fus
cells are fully resistant to infection with M-tropic NSI HIV
strains was in line with previous studies on the inability of
CXCR4 to allow the fusion and entry of M-tropic isolates.5
It has been reported that normal human megakaryocytes
express CD4 on their surface and represent one of the targets
of in vivo viral infection.28-30 Previous in vitro studies on
megakaryocytic cell lines such as CMK and DAMI cells
showed that HIV infection does not induce modifications on
cell morphology. However, productive infection of CMK
cells was obtained only with the HIV-2ROD strain,31 whereas
DAMI cells, although fully CD40, could be productively
infected by the HTLV-IIIB HIV-1 strain.32 Nevertheless, the
viral outcome appeared very delayed (considering that, in
the most efficient of 26 different infection experiments and
at the highest moi, the earliest peak of viral production was
detected only 30 days postinfection) and levels of viral production appeared somewhat inconsistent. In addition, a relevant fraction of cells (from 40% to 80%) remained uninfected even after months of culture. Thus, the interaction
between DAMI cells and the T-tropic, syncytium-inducing
HTLV-IIIB strain led to a chronic, slow-low infective cycle.
It is conceivable that, in infected DAMI cultures, an HIV
quasispecies able to replicate in CD40 cells had been selected
(HIV strains able to infect CD40 cells have already been
reported)33 whose expression level, however, was not strong
enough to induce the overcoming of the differentiative block
in DAMI cells. In addition, the more advanced stage of
differentiation at which DAMI cells appeared blocked34 with
respect to UT-7 cells may render more difficult the evaluation of a possibly small differentiation progress that HIV
expression could induce on infected cells. Thus, considering
that both the CD4 receptor28-30 and the CXCR-4 HIV coreceptor (C. Chelucci, unpublished data) are expressed by a
consistent fraction of normal human cells of megakaryocytic
lineage, UT-7/fus cells represent the model that could better
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HIV INFECTION AND DIFFERENTIATION OF UT-7 CELLS UPON CXCR4 TRANSFECTION
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Fig 6. Morphologic analysis
of UT-7, UT-7/fus, and HIV-infected UT-7/fus cells. (A) Analysis of UT-7, UT-7/fus, and NL4-3
or NDK HIV-1–infected UT-7/fus
cells on the basis of nuclei number (N) as evaluated by optical
microscopy. Results from a representative
experiment
are
shown. (B) Cytospin of uninfected UT-7 and UT-7/fus cells
compared with UT-7/fus cells infected with NL4-3 or NDK HIV1 strains. (Original magnification
Ì 400.)
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Fig 7. Antigenic analysis of uninfected or HIV-1–infected UT-7/
fus cells. Cells were analyzed for the presence of CD41a, glycophorin
A, and CD31 antigens. In each panel, slopes from uninfected and
HIV-1–infected UT-7/fus cells are compared. The mean fluorescence
intensity of both uninfected and HIV-1–infected UT-7/fus cells labeled with unspecific mouse IgG was always less than 5. In addition,
on the top, the dot plots of each cell population are shown. The
uninfected and HIV-1–infected UT-7/fus cell populations analyzed
are, respectively, included in R1 and R2 areas.
reproduce the interactions occurring in vivo between HIV
and partially or fully differentiated megakaryocytic cells.
Two additional human megakaryocytic cell lines (MEG01 and CHFR-288) were shown to be both CD4/ and HIV1 susceptible,35,36 but no detailed data on the cellular response
after infection were reported. Conversely, we show that infection of UT-7/fus cells with SI HIV-1 isolates induces
evident morphologic alterations and, finally, cell death.
These morphologic changes (ie, increase in cell size, induction of polyploidy, and appearance in cytoplasm of eosinophilic areas containing multiple purple granules) are compatible with those described for normal in vitro megakaryocytic
differentiation.37 This evidence was also supported by the
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increase in HIV-1–infected UT-7/fus cells of a specific megakaryocytic marker as GpIIb/IIIa complex and the contemporary increase of PECAM-1, coupled with a clear decrease
in the exposure of the glycophorin A, a marker specific
for erythroid differentiation. Taken together, these findings
strongly suggest that a progression of UT-7/fus cells toward
megakaryocytic differentiation could be achieved through
the expression of HIV-1. Conversely, no morphologic modifications were observed in HIV-1–infected UT-7 cells (thus
excluding any unspecific effect due to contaminants possibly
present in HIV-1 preparations) or after HIV-2 infection of
UT-7/fus cells. The evidence that HIV-2–infected UT-7/fus
cells were fully CD4 downregulated strongly suggests that
a large majority of them was infected. Thus, the lack of
any evident multinuclearization among the HIV-2–infected
cultures could not be considered as the consequence of the
infection occurring in only few UT-7/fus cells.
The evidence that differentiation was induced by HIV-1
and not by HIV-2 infection suggests that the intracellular
expression of some HIV-1 proteins, rather than a possible
CXCR4 stimulation induced after viral binding, may be directly or indirectly involved in the induction of megakaryocytic differentiation. It has been shown that TE761 human
rhabdomyosarcoma cells can be induced to differentiate after
transfection with an HIV-1 molecular clone or with the vpr
gene alone.38 Similarly, it is conceivable that the expression
of the vpr gene (or eventually other HIV-1 genes) could
induce UT-7/fus cells to escape the block of differentiation
typical of UT-7 cells. Recently, the possibility to partially
overcome the differentiation block in UT-7 cells has been
demonstrated both in a thrombopoietin (TPO)-dependent
subclone of UT-7 cells39 and in UT-7 cells transfected with
mouse TPO receptor (mpl) and treated with TPO.40
There is no reason to believe that the polynucleated HIVinfected UT-7/fus cells are derived from the formation of
HIV-induced syncytia, because several morphologic and antigenic features suggesting megakaryocytic maturation were
displayed by multinucleated cells. Furthermore, syncytia formation has never been described in both productively HIVinfected CD4 positive megakaryocytic cell lines31,32 or in
CD34-derived human megakaryocytes infected with SI HIV
strains.41 The inability of UT-7/fus cells to form syncytia
was also deduced from the lack of any polynucleated cells
in cultures infected with HIV-2ROD , a viral strain able to
induce large syncytia in any CD4- and CXCR4-coexpressing
cells, such as T lymphocytes or HeLaCD4 cells. Finally, the
evidence that all the HIV-1–infected UT-7/fus cells appear
homogenously enlarged is a strong argument against the
virus-induced formation of syncytia as the cause of UT-7/
fus multinuclearization.
To our knowledge, UT-7 cells represent the first reported
example of a naturally CD4/ human cell line that is not HIV
infectable. The uniqueness of UT-7 cells under this aspect
may be coherent with the wide expression of CXCR4 among
different cell types. In this context, it is tempting to suggest
that the UT-7 cells may be blocked at an early differentiative
stage in which CXCR4 is not yet expressed and that this
phenomenon may be related to an event physiologically oc-
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HIV INFECTION AND DIFFERENTIATION OF UT-7 CELLS UPON CXCR4 TRANSFECTION
curring during megakaryocyte differentiation. Experiments
are in progress to evaluate this hypothesis in unilineage cultures of human megakaryocytic cells derived from highly
purified hematopoietic progenitors.
Furthermore, we believe that UT-7 and UT-7/fus cells
represent a unique tool in the study of early events of viral
infection, such as binding, envelope fusion with the target
membrane, and viral penetration. Comparative studies between UT-7 and UT-7/fus cells may also provide new insights into the role of G protein activation in HIV entry and
in HIV-induced cell pathogenesis.
ACKNOWLEDGMENT
We thank Dr A.R. Migliaccio for critical reading of the manuscript
and A. Lippa for editorial assistance.
REFERENCES
1. Maddon PJ, Dalgleish AG, McDougal JS, Clapham PR, Weiss
RA, Axel R: The T4 gene encodes the AIDS virus receptor and is
expressed in the immune system and the brain. Cell 47:333, 1986
2. Ashom PA, Berger EA, Moss B: Human immunodeficiency
virus envelope glycoprotein/CD4-mediated fusion of nonprimate
cells with human cells. J Virol 64:2149, 1990
3. Loetscher M, Geiser T, O’Reilly T, Zwahlen R, Baggiolini M,
Moser B: Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol Chem
289:232, 1994
4. Samson M, Labbe O, Mollerau C, Vassart G, Parmentier M:
Molecular cloning and functional expression of a new human CCchemokine receptor gene. Biochemistry 35:3362, 1996
5. Feng Y, Broder CC, Kennedy P, Berger E: HIV-1 entry cofactor:functional cDNA cloning of a seven-transmembrane, G proteincoupled receptor. Science 272:872, 1996
6. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC,
Parmentier M, Collman RG, Doms RW: A dual-tropic primary HIV1 isolate that uses fusin and the b-chemokine receptors CKR-5,
CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149, 1996
7. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD,
Lijun W, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C,
Sodroski J: The b-chemokine receptors CCR3 and CCR5 facilitate
infection by primary HIV-1 isolates. Cell 2:1135, 1996
8. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE,
Murphy PM, Berger EA: CC CKR5: A RANTES, MIP-1a, MIP-1b
receptor as a fusion cofactor for macrophage-tropic HIV-1. Science
272:1955, 1996
9. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton
WA: HIV-1 entry into CD4/ cells is mediated by the chemokine
receptor CC-CKR-5. Nature 381:667, 1996
10. Deng R, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart
M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper
SC, Schall TJ, Littman DR, Landau NR: Identification of a major
co-receptor for primary isolates of HIV-1. Nature 381:661, 1996
11. Oberlin E, Amara A, Bacheleire F, Bassia C, Virelizier J-L,
Arenzana-Seisdedos F, Schwartz O, Heard J-M, Clark-Lewis I,
Legler DF, Loetscher M, Baggiolini M, Moser B: The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by
T-cell-line-adapted HIV-1. Nature 382:833, 1996
12. Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA: The lymphocyte chemoattractant SDF-1 is a
ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829,
1996
AID
Blood 0059
/
5h33$$1161
2677
13. Komatsu N, Hiromitsu N, Miwa A, Ishihara T, Eguchi M,
Moroi M, Okada M, Sato Y, Wada H, Yawata Y, Suda T, Miura
Y: Establishment and characterization of a human leukemic cell line
with megakaryocytic features: Dependency on granulocyte-macrophage colony-stimulating factor, interlukin 3, or erythropoietin for
growth and survival. Cancer Res 51:341, 1991
14. Komatsu N, Yamamoto M, Fujita H, Miwa A, Hatake K,
Endo T, Okano H, Katsube T, Fukumaki Y, Sassa S, Miura Y:
Establishment and characterization of an erythropoietin-dependent
subline, UT-7/Epo, derived from human leukemia cell line, UT-7.
Blood 82:456, 1993
15. Debili N, Coulombel L, Croisille L, Katz A, Guichard J,
Breton-Gorins J, Vainchenker W: Characterization of a bipotent
erythro-megakaryocytic progenitor in human bone morrow. Blood
88:1284, 1996
16. Taddeo B, Federico M, Titti F, Rossi GB, Verani P: Homologous superinfection of both producer and nonproducer HIV-infected
cells is blocked at a late retrotranscription step. Virology 194:441,
1993
17. Federico M, Taddeo B, Carlini F, Nappi F, Verani P, Rossi
GB: A recombinant retrovirus carrying a nonproducer human immuno deficiency virus (HIV) type 1 variant induces resistance to
superinfecting HIV. J Gen Virol 74:2099, 1993
18. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo
RC, Papovic P: The role of mononuclear phagocytes in HTLV-III/
LAV infection. Science 233:215, 1986
19. Rossi GB, Verani P, Macchi B, Federico M, Orecchia A,
Nicoletti L, Buttò S, Lazzarin A, Mariani G, Ippolito G, Manzari
V: Recovery of HIV-related retroviruses from Italian patients with
AIDS or AIDS-related complex and from asymptomatic at-risk individuals. Ann NY Acad Sci 511:390, 1987
20. Chelucci C, Hassan HJ, Locardi C, Bulgarini D, Pelosi E,
Mariani G, Testa U, Federico M, Valtieri M, Peschle C: In vitro
human immunodeficiency virus-1 infection of purified progenitors
in single-cell culture. Blood 85:1181, 1995
21. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor
Laboratory, 1989
22. Newman PJ, Berndt MC, Gorski J, White GC, Lyman S,
Paddock C, Muller WA: PECAM-1 (CD31) cloning and relation to
adhesion molecules of the immunoglobulin gene superfamily. Science 247:1219, 1990
23. Phillips DR, Charo IF, Scarborough RM: GPIIb-IIIa: The
responsive integrin. Cell 65:359, 1991
24. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo
RC, Lusso P: Identification of RANTES, MIP-1a, and MIP-1b as
a the major HIV-suppressive factors produced by CD8/ T cells.
Science 270:1811, 1995
25. Nagasawa T, Kikutani H, Kishimoto T: Molecular cloning
and structure of a pre-B-cell growth-stimulating factor. Proc Natl
Acad Sci USA 91:2305, 1994
26. Rimland J, Xin W, Sweetnam P, Saijoh K, Nestler E, Duman
RS: Sequence and expression of a neuropeptide Y receptor cDNA.
Mol Pharmacol 40:869, 1991
27. Federsppiel B, Melhado IG, Duncan AM, Delaney A, Schappert K, Clark-Lewis J, Jirik FR: Molecular cloning of the cDNA
and chromosomal localization of the gene for a putative seventransmembrane segment (7-TMS) receptor isolated from human
spleen. Genomics 16:707, 1993
28. Kouri YH, Borkowsky W, Nardi M, Karpatkin S, Basch RS:
Human magakaryocytes have a CD4 molecule capable of binding
human immunodeficiency virus-1. Blood 81:2664, 1993
29. Zucker-Franklin D, Seremetis S, Zheng ZY: Internalization
03-13-97 22:05:25
bldas
WBS: Blood
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2678
BAIOCCHI ET AL
of human immunodeficiency virus type 1 and other retroviruses by
megakaryocytes and platelets. Blood 75:1920, 1990
30. Zucker-Franklin D, Cao Y: Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA. Proc Natl
Acad Sci USA 86:5595, 1989
31. Sakaguchi M, Sato T, Groopman JE: Human immunodeficiency virus infection of megakaryocytic cells. Blood 77:481, 1991
32. Monté D, Groux H, Raharinivo B, Plouvier B, deWulf J,
Clavel T, Grangette C, Torpier G, Auriault C, Capron A, Ameisen
JC: Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-a. Blood
79:2670, 1992
33. Clapham PR, McKnight A, Weiss RA: Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell
lines: Induction and enhancement by soluble CD4. J Virol 66:3531,
1992
34. Greenberg SM, Rosenthal DS, Greeley TA, Tantravahi R,
Handin RI: Characterization of a new megakaryocytic cell line: The
dami cell. Blood 72:1968, 1988
35. Matsuo Y, Kamiya T, Nakamura S, Yamamoto N, Uno F,
Yoshida M, Nii S, Kurimoto M, Minowada J: Characterization of
17 human immunodeficiency virus-1 carrier cell lines with T cell,
myelomonocyte, or megakaryocyte lineages. Proc Soc Exp Biol Med
202:271, 1993
36. Kouri YH, Borkowsky W, Nardi M, Karpatkin S, Basch RS:
AID
Blood 0059
/
5h33$$1161
Human megakaryocytes have a CD4 molecule capable of binding
human immunodeficiency virus-1. Blood 81:2664, 1993
37. Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro
E, Macioce G, Pace A, Ziegler B, Hassan HJ, Peschle C: Unilineage
megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture. Blood 86:3725, 1995
38. Levy DN, Fernandes LS, Williams WV, Weiner DB: Induction of cell differentiation by human immunodeficiency virus 1 vpr.
Cell 72:541, 1993
39. Komatsu N, Kunitama M, Yamada M, Hagiwara T, Kato T,
Miyazaki H, Eguchi M, Yamamoto M, Miura Y: Establishment and
characterization of the thrombopoietin-dependent megakaryocytic
cell line, UT-7/TPO. Blood 87:4552, 1996
40. Porteu F, Rouyez M-C, Cocault L, Benit L, Charon M, Picard
F, Gisselbrecht S, Souyri M, Dusanter-Fourt I: Functional regions
of the mouse thrombopoietin receptor cytoplasmic domain: Evidence
for a critical region which is involved in differentiation and can be
complemented by erythropoietin. Mol Cell Biol 16:2473, 1996
41. Chelucci C, Federico M, Guerriero R, Pelosi E, Testa U,
Hassan HJ, Peschle C: Productive human immunodeficiency virus
infection of highly purified megakaryocytic precursors and mature
megakaryocytes, in Sussel R, Guillet V, Ruedy N (eds): XI International Conference on AIDS. Vancouver, British Columbia, Canada,
Transcontinental Printing Inc, 1996, p 2043
03-13-97 22:05:25
bldas
WBS: Blood
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
1997 89: 2670-2678
Human Immunodeficiency Virus (HIV)-Resistant CD4+ UT-7 Megakaryocytic
Human Cell Line Becomes Highly HIV-1 and HIV-2 Susceptible Upon
CXCR4 Transfection: Induction of Cell Differentiation by HIV-1 Infection
Marta Baiocchi, Eleonora Olivetta, Cristiana Chelucci, Anna Claudia Santarcangelo, Roberta Bona, Paola
d'Aloja, Ugo Testa, Norio Komatsu, Paola Verani and Maurizio Federico
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