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J Virol, August 1998, p. 6389-6397, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Cytomegalovirus-Encoded Chemokine Receptor US28 Can
Enhance Cell-Cell Fusion Mediated by Different Viral
Proteins
Olivier
Pleskoff,
Carole
Tréboute, and
Marc
Alizon*
INSERM U.332, Institut Cochin de
Génétique Moléculaire, 75014 Paris, France
Received 11 February 1998/Accepted 28 April 1998
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ABSTRACT |
The human cytomegalovirus (CMV) US28 gene encodes a
functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the
actual role of the protein in the CMV life cycle. Expression of US28
allows human immunodeficiency virus type 1 (HIV-1) entry into certain
CD4+ cells and their fusion with cells expressing HIV-1
envelope (Env) proteins. Such properties were initially reported for
the cellular chemokine receptors CCR5 and CXCR4, which behave as
CD4-associated HIV-1 coreceptors. We found that coexpression of
US28 and either CXCR4 or CCR5 in CD4+ cells resulted
in enhanced synctium formation with HIV-1 Env+ cells. This
positive effect of US28 on cell fusion seems to be distinct from its
HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also
observed when US28 was expressed on the HIV-1 Env+ cells
instead of an CD4+ target cells. Furthermore, US28
could enhance cell fusion mediated by other viral proteins, in
particular, the G protein of vesicular stomatitis virus (VSV-G). The
HIV-1 coreceptor and fusion-enhancing activities could be affected by
mutations in different domains of US28. The fusion-enhancing activity
of US28 seems to be cell type dependent. Indeed, cells
coexpressing VSV-G and US28 fused more efficiently with human, simian,
or feline target cells, while US28 had no apparent effect on fusion
with the three mouse or rat cell lines tested. The positive effect of
US28 on cell fusion might therefore require its interaction with a
cell-specific factor. We discuss a possible role for US28 in the fusion
of the CMV envelope with target cells and CMV entry.
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INTRODUCTION |
G protein-coupled receptors (GCRs)
form an extremely large family of signal-transducing proteins involved
in numerous biological functions (50). Viral proteins with
seven predicted membrane-spanning domains and other features
indicating their homology with cellular GCRs were identified
initially in human cytomegalovirus (CMV), as the products of the
US27, US28, and UL33 genes
(9), and later in herpesvirus saimiri (37),
equine herpesvirus (51), mouse CMV (MCMV) (44),
and human herpesviruses 6 (23), 7 (36), and 8 (known as Kaposi's sarcoma-associated herpesvirus [KSHV]) (8). Some of these viral proteins are only putative GCRs, as their ligands are unknown (orphan receptors). Functionality in terms of
ligand binding and signal transduction has been shown for the US28
protein of CMV (22, 35), the ECRF3 protein of herpesvirus
saimiri (2), and the product of KSHV open reading frame 74 (3). Their ligands belong to the family of cellular chemokines, which are small soluble proteins (60 to 80 amino acids) involved in leukocyte chemotaxis (4). Chemokines from the CC subgroup activate US28, while ECRF3 is activated by interleukin-8 and
other CXC chemokines. The KSHV-encoded GCR can also bind interleukin-8, but it is constitutively activated, which may play a role in its oncogenic and angiogenic properties (5).
The role played by these GCRs or putative GCRs in the viral life cycle
is unknown. Expression of the M33 protein of MCMV was shown to be
necessary for virus dissemination in vivo but not in tissue culture
(14). Virally encoded GCRs might confer on infected cells
responsiveness to cellular chemokines, or to other ligands in the case
of orphan receptors. However, it cannot be ascertained that the GCR
activity observed in cells transfected to express these viral proteins
is relevant to their role in vivo. The US27,
US28, and UL33 genes are transcribed late after
CMV infection (53), which suggests that they encode
structural proteins rather than regulating proteins. The presence of
the UL33 protein in CMV particles was indeed demonstrated
(29).
Expression of the CMV-encoded chemokine receptor US28 in
CD4+ cells could allow their infection by human
immunodeficiency virus type 1 (HIV-1) or 2 (HIV-2) or their fusion with
cells expressing HIV-1 or HIV-2 envelope (Env) proteins
(42). Therefore, US28 apparently shares properties with the
cellular chemokine receptors CCR5 and CXCR4, which behave as
CD4-associated coreceptors for HIV-1 or HIV-2 (17,
33). Here we show that US28 can enhance cell-cell fusion by
a mechanism apparently distinct from HIV coreceptor activity, which
leads us to discuss a possible role in CMV entry.
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MATERIALS AND METHODS |
Cell lines.
The U373MG-CD4 (24), U87MG-CD4
(10), and HeLa P4 (CD4+) (12) cell
lines and the HeLa P4 CCR5+ derivative HeLa P5 cell line
(42) are stably transfected with Escherichia coli
lacZ under transcriptional control of the HIV-1 long terminal
repeat (LTRlacZ). The HeLa-Env/LAI (46) and HeLa-Env/ADA (42) cell lines stably express Env from HIV-1LAI
and HIV-1ADA, respectively. Cell lines expressing HIV-1 Tat
derived from HeLa (46), NIH 3T3 (18), and Dunni
and XC (15) cells have been described previously. The
Tat+ derivative of the B5 rhesus macaque cell line (ATCC
CL-160) was obtained from M. Sitbon (Centre National de la Recherche
Scientifique, Montpellier, France). The cat cell line CrFK
(38) was obtained from J. Richardson (Institut Cochin de
Génétique Moléculaire). All cell lines were
propagated in Dulbecco's modified Eagle's medium (Gibco) supplemented
with antibiotics (60 µg of penicillin per ml and 100 µg of
streptomycin per ml) and 10% fetal calf serum (or 10% newborn calf
serum in the case of NIH 3T3 cells).
Expression vectors.
The HIV-1LAI Env expression
vector was pMA243, a 
gag-pol HIV-1LAI
provirus also expressing Tat and Rev (46). The pCEL
(15) and pCMV-G (58) vectors allow expression of
human T-cell leukemia virus type 1 strain CR (HTLV-1CR) Env
and vesicular stomatitis virus G protein (VSV-G), respectively, from
the CMV immediate-early promoter. Chemokine receptors and GCR cDNAs
were subcloned in Rc/CMV (InVitrogen, La Jolla, Calif.) or pCDNA-3
(Clontech, Palo Alto, Calif.) downstream from the CMV immediate-early
promoter. The expression vectors for CXCR4 (41), CCR3
(48), and CCR5, US28, and MYC-tagged US28 (42)
are described elsewhere. Expression vectors for CCR1, CCR4, CXCR1, and
CXCR2 were obtained from N. Sol (Institut Cochin de
Génétique Moléculaire). The corresponding open
reading frames were PCR amplified from HeLa cells DNA and subcloned in
Rc/CMV. The deduced amino acid sequences of these GCRs were identical
to those reported previously (34, 35, 43). The US27 and UL33
open reading frames were PCR amplified from fibroblasts infected with
the CMV AD169 strain and subcloned in Rc/CMV. The US27 and UL33
sequences were identical to those reported previously
(9). The pCDNA/M33 expression vector (14) was
obtained from N. Davis-Poynter (University of Western Australia, Nedlands, Australia). A pCDNA.3 vector expressing KSHV GCR
(3) was obtained from M. N. Gershengorn (Cornell
University, New York, N.Y.). The US28 mutants listed in Table 1 were
obtained by oligonucleotide-directed mutagenesis (sequences of
oligonucleotides are available upon request). Mutants were screened for
the creation of restriction enzyme sites and checked by nucleotide
sequencing.
Transfection of cells and syncytium formation assays.
About
105 cells per well were seeded in six-well trays and
incubated overnight at 37°C in complete medium. The medium was
replaced 2 to 4 h before addition of the DNA-calcium phosphate
precipitate (4 µg of DNA per well) and after overnight incubation
with the precipitate. Cocultures were initiated 24 h after
transfection by adding fusion partner cells or by detaching transfected
cells with trypsin, mixing with fusion partner cells, and seeding a 12-well tray. In all cases, the fusion partner cells were in a 1:1
ratio. Cocultures were ended after 24 h by fixing cells with 0.5%
glutaraldehyde and staining with the 
-galactosidase substrate X-Gal
(5-bromo-4-chloro-3-indoyl--D-galactopyranoside) as
described previously (19). Blue foci were scored at a
magnification of ×20. Numbers of >200 were extrapolated from randomly
selected fields.
Flow cytometry.
HeLa P4 cells were cotransfected with
EGFP-N1 (Clontech), a green fluorescent protein (GFP) expression
vector, and with one or two chemokine receptor expression vectors or
Rc/CMV (controls). The weight ratio of EGFP-N1 to the other plasmids
was 1:6 or 1:4. Cells were detached 36 h after transfection with
phosphate-buffered saline containing 1 mM EDTA, stained with antibodies
in phosphate-buffered saline containing 1% fetal calf serum, fixed,
and analyzed by flow cytometry as described previously (42).
The following monoclonal antibodies were used: Leu3A (Becton Dickinson,
San Jose, Calif.), anti-CD4, 0.5 µg/ml; 12G5 (20)
anti-CXCR4, 6 µg/ml; 2D7 (6) anti-CCR5, 0.8 µg/ml; 9E10
(Boerhinger, Mannheim, Germany), anti-MYC, 0.4 µg/ml; and W6/32
(Dako, Glostrub, Denmark), anti-major histocompatibility complex class
I (MHC-I), 3.5 µg/ml. 12G5 and 2D7 were obtained from the National
Institutes of Health AIDS Reagent Program. Only Leu3A was directly
coupled to phycoerythrin (PE). In the other cases, cells were stained
with a secondary PE-coupled goat antimouse antiserum (Dako) used at 16 µg/ml.
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RESULTS |
US28 enhances HIV-1 fusion.
We have developed simple and
sensitive assays allowing detection and quantification of the fusion of
cells to form syncytia. These assays are based on the presence of the
HIV-1 transactivating protein (Tat) in one cell type and of a
Tat-inducible reporter gene, such as the E. coli
-galactosidase gene lacZ linked to the HIV-1 long
terminal repeat (LTRlacZ), in the other. Upon fusion of these cell
types and cytoplasm mixing, Tat gains access to the nucleus and
activates lacZ transcription. The high level of -galactosidase activity in syncytia allows their detection by histochemical staining (19). This technique can be used to
test the activity of candidate HIV-1 coreceptors. For example, HeLa P4
cells (LTRlacZ CD4+) naturally express CXCR4 and can form
syncytia with cells expressing Env from a cell line-adapted HIV-1
strain (HeLa-Env/LAI cells) but not from a macrophage (M)-tropic
HIV-1 strain (HeLa-Env/ADA cells). Fusion with HeLa-Env/ADA
cells can be observed when HeLa P4 cells are transfected with vectors
allowing expression of CCR5 or US28 (42).
Transfection of HeLa P4 cells with a US28 expression vector increased
the number of syncytia detected in cocultures with HeLa-Env/LAI cells
(Fig. 1A). This effect was not observed
when HeLa P4 cells were transfected with a CCR5 expression vector, as
expected, or with a CXCR4 expression vector. The coreceptor activity of
US28 toward cell line-adapted HIV-1 is relatively low (42)
and was unlikely to explain the markedly increased number of syncytia observed in this experiment. By measuring the -galactosidase activity in HeLa P4 cells after transfection with the US28 expression vector or with US28 and Tat expression vectors, we ruled out a direct
effect of US28 on LTRlacZ expression or Tat-mediated transactivation (data not shown).
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FIG. 1.
Enhancement of HIV-1 Env-mediated cell-cell fusion by
US28. HeLa- CD4-LTRlacZ cells (A and B) or U373MG-CD4-LTRlacZ cells (C
and D) were transfected with Rc/CMV (Mock) or with CXCR4, CCR5, and
US28 expression vectors, as indicated. Cocultures were performed with
HeLa cells stably expressing Tat and Env from cell line-adapted
HIV-1LAI (A and C) or from M-tropic HIV-1ADA (B
and D). Transfections were performed in six-well trays with 4 µg of
vector, or with 2 µg of each vector when chemokine receptors were
coexpressed. Cells from a subconfluent well were detached with trypsin
24 h posttransfection. Half of them were seeded with an equivalent
number of Env+ cells in one well from a 12-well tray. Cells
were fixed and stained with X-Gal after a 24-h coculture. Bars
represent mean numbers (with standard deviations) of blue-stained foci,
indicating cell fusion events, in duplicate wells.
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To look for a possible effect of US28 on cell fusion mediated by Env
from HIV-1
ADA, HeLa P4 cells were transfected with 4
µg of the CCR5 or the US28 vector or with 2 µg of each vector,
and
cocultures were performed with HeLa-Env/ADA cells. Higher
numbers of
syncytia were detected when HeLa P4 cells were cotransfected
with the
CCR5 and US28 vectors (Fig.
1B). As expected, transfection
of a CXCR4
expression vector did not allow fusion with HeLa-Env/ADA
cells.
Therefore, cell fusion mediated by an M-tropic Env seemed
to be more
efficient when target cells coexpressed the CCR5 coreceptor
and US28.
Positive effects of US28 on syncytium formation were also observed with
the LTRlacZ CD4
+ derivative of the U373MG astroglioma
cell line, which is naturally
resistant to infection by both M-tropic
and cell line-adapted
HIV-1 (
24,
42). Higher
numbers of syncytia were formed with
Env
+ cells when
U373MG-CD4 cells were cotransfected with the US28
and CXCR4
vectors or with the US28 and CCR5 vectors than when
parallel
transfections with the same quantity of DNA from each
of these vectors
were performed (Fig.
1C and D). These experiments
showed that cell
fusion mediated by the HIV-1 envelope proteins
was enhanced when
CD4
+ cells target cells coexpressed the US28 chemokine
receptor and
a HIV-1 coreceptor, either CXCR4 or CCR5.
The surface expression of CD4, MHC-I, and CXCR4 was assessed by flow
cytometry after transfection of HeLa P4 cells with US28,
CCR5, or CCR1
vectors, or with Rc/CMV (control cells), and a GFP
expression vector.
Surface expression of CD4, MHC-1, and CXCR4
among GFP-positive cells,
considered to represent the fraction
of HeLa P4 cells expressing
transfected DNA, was measured. The
presence of US28, CCR5, or
CCR1 had no apparent effect on the
surface expression of CD4 and MHC-I
(Fig.
2A). The surface expression
of
CXCR4 was downregulated in cells transfected with the US28
vector, in
comparison with control cells, or with cells transfected
with the CCR5
or CCR1 vector. In a similar experiment, we found
that CCR5 surface
expression was slightly lower in cells cotransfected
with CCR5 and US28
vectors than in cells cotransfected with CCR5
and CXCR4 vectors (Fig.
2B). The mechanism by which US28 influenced
the surface expression of
CXCR4 or CCR5 was not further explored
in this study. These experiments
ruled out the possibility that
the positive effects of US28 on cell
fusion were due to an increase
in the surface expression of CD4 or the
HIV-1 coreceptors.
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FIG. 2.
Flow cytometry analysis of cell surface markers in HeLa
P4 cells transfected with different chemokine receptor expression
vectors. (A) Surface expression of CD4, MHC-1, or CXCR4 after
transfection with two vectors, i.e., EGFP-N1 (GFP expression vector)
and either Rc/CMV-US28, Rc/CMV-CCR5, Rc/CMV-CCR1, or Rc/CMV (control),
in a 1:6 ratio. (B) Surface expression of CCR5 after transfection with
three vectors, i.e., EGFP-N1, Rc/CMV-CCR5, and either Rc/CMV-US28,
Rc/CMV-CXCR4, or Rc/CMV (control), in a 1:2:2 ratio. Cells were stained
with PE-coupled antibodies (red fluorescence) 36 h after
transfection and analyzed as indicated in Materials and Methods. The
graphs show red fluorescence intensity (x axis, arbitrary
units, log scale) and numbers of cells (y axis) among
GFP-positive cells (transfected cells). Thick lines, transfections with
chemokine receptor expression vectors; thin lines and gray areas,
control transfections with Rc/CMV.
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Coexpression of US28 and viral fusiogenic proteins.
We next
asked if US28 could enhance syncytium formation when it was expressed
in the Env+ cells instead of the target cells. A
Tat+ HeLa cell line was cotransfected to express
HIV-1LAI Env and either US28 or CCR5, and cocultures were
performed with HeLa P4 cells. The number of syncytia was markedly
higher when cells expressed US28 (Fig.
3A). Similar numbers of syncytia were
detected when cells were transfected with the CCR5 vector or with
Rc/CMV. As expected, there was no detectable fusion when
Tat+ HeLa cells expressed US28 (or CCR5) in the absence of
Env (data not shown). A positive effect on cell fusion was also
observed when US28 was expressed in HeLa-Env/ADA cells and coculture
was performed with HeLa P5, a CCR5+ cell line derived from
HeLa P4 (Fig. 3B). In these experiments, the positive effects on US28
cell fusion could not be explained by its HIV-1 coreceptor activity or
by the modulation of CD4, CXCR4, or CCR5 surface expression.
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FIG. 3.
Coexpression of US28 and viral fusiogenic proteins.
HeLa-Tat cells were cotransfected with expression vector for
HIV-1LAI Env (A), HTLV-1 Env (C), or VSV-G (D) and with
either Rc/CMV (mock), Rc/CMV-CCR5, or Rc/CMV-US28, as indicated. Each
of these Rc/CMV vectors was also transfected in cells stably expressing
Env from HIV-1ADA (B). Cocultures (six-well trays) were
initiated 24 h later by adding an equivalent number of HeLa P4
cells (A, C, and D) or their CCR5+ derivatives, HeLa P5
cells (B). Cells were fixed and stained with X-Gal after a 24-h
coculture. Bars represent mean numbers (with standard deviations) of
blue-stained foci in triplicate wells.
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We next looked for an effect of US28 on cell-cell fusion mediated by
the gp46 and gp21 envelope proteins of another retrovirus,
HTLV-1, and
by the rhabdovirus protein VSV-G. The ability of gp46
and gp21 to
mediate syncytium formation among cells from different
mammalian
species and tissue origins is well documented (
15,
49). As
expected, expression of gp46 and gp21 in Tat
+ HeLa cells
allowed their fusion with HeLa P4 cells (Fig.
3C).
VSV-G was shown to
induce syncytium formation only after exposure
of cells to a mildly
acidic pH (
55), in agreement with the pH-dependent
entry of
VSV (
30). However, we found that transfection of a
VSV-G
expression vector into Tat
+ HeLa cells allowed their fusion
with HeLa P4 cells under normal
tissue culture pH conditions (Fig.
3D).
The reason for this apparent
discrepancy is unknown. The sensitivity of
our cell fusion assay
might be higher, allowing detection of activity
of VSV-G under
suboptimal conditions. Alternatively, the activity of
VSV-G might
be different in certain cell types, in particular when
expressed
by transfection. Similar discrepancies are known for some
murine
leukemia virus strains, which also apparently infect cells by
a
pH-dependent pathway yet induce syncytium formation in certain
cell
types or under certain experimental conditions (
25,
56).
Cocultures were performed with HeLa P4 cells and Tat
+ HeLa
cells cotransfected to express HTLV-1 gp46-gp21 or VSV-G and either
CCR5 or US28. Higher numbers of syncytia were detected when cells
were
transfected with the US28 vector (Fig.
3C and D). Flow cytometry
experiments showed no effect of US28 on the surface expression
of gp46
(data not shown). The surface expression of HIV-1 Env
or VSV-G has not
been analyzed.
Effects of other chemokine receptors on cell-cell fusion.
Although CCR5 or CXCR4 had no apparent effect on cell-cell
fusion, besides their HIV-1 coreceptor activity, a panel of
GCRs was tested by coexpression with VSV-G in Tat+ HeLa
cells and coculture with HeLa P4 cells. Among cellular chemokine receptors, only CCR1 had a modest positive effect on cell fusion (Fig.
4), which was not seen in other
experiments (see Fig. 5; other data not shown). Notably, flow cytometry
analysis of cells transfected with epitope-tagged chemokine receptors
suggests that the surface expression is high in the case of CCR1
(11) and relatively low in the case of US28 (42).
The KSHV-encoded chemokine receptor and the putative GCRs encoded by
the US27 and UL33 genes of human CMV or by the
M33 gene of MCMV did not seem to enhance cell fusion in a
way comparable to that for US28 (Fig. 4).
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FIG. 4.
Coexpression of VSV-G and chemokine receptors or virally
encoded GCRs. Transfections of HeLa-Tat cells with expression vectors
for VSV-G and for the indicated GCRs and cocultures with HeLa P4 cells
were performed as described for Fig. 3. US27 and UL33 are putative GCRs
encoded by human CMV; M33 is a putative GCR from MCMV. Bars represent
mean numbers (with standard deviations) of blue-stained foci in
triplicate wells.
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Properties of mutant US28.
A series of US28 mutants was tested
for their HIV-1 coreceptor activity by transfection of HeLa P4 cells
and coculture with HeLa-Env/ADA cells and for their fusion-enhancing
activity by coexpression with VSV-G in Tat+ HeLa cells and
coculture with the human astroglioma cell line U87MG-CD4-LTRlacZ. The
results of these experiments are summarized in Table
1. The insertion of a 15-amino-acid (aa)
sequence from the human c-MYC oncoprotein at the amino terminus of US28
allows its detection at the cell surface by staining with the 9E10
antibody (42). When this mutant (MYC-tagged US28) was
expressed in HeLa P4 cells, the number of syncytia formed with
HeLa-Env/ADA cells was about fourfold lower than that with wild-type
(WT) US28. In contrast, the positive effect of MYC-tagged US28 on
cell-cell fusion mediated by the VSV-G was higher. The coreceptor and
fusion-enhancing activities could require different amounts of US28 at
the cell surface or could be mediated by distinct domains. The
importance of the amino-terminal (NT) extracellular domain for the
HIV-1 coreceptor activity is shown by the phenotype of the 2-22 US28 mutant, in which most of the 33-aa-long NT domain is deleted. This
mutant US28 had no detectable HIV-1 coreceptor activity, while it could
enhance cell-cell fusion in a way comparable to that for WT US28.
Truncation of the carboxy-terminal (CT) cytoplasmic domain of US28 (aa
296 to 355) beyond aa 317 had minor effects on HIV-1
coreceptor
activity and apparently increased fusion-enhancing
effects. Flow
cytometry analysis of cells transfected with MYC-tagged
forms of WT and
mutant US28 suggests that the truncation of the
CT domain results in a
higher level of cell surface expression
(Table
2).
Point mutations were created in the third and fourth extracellular
domains of US28, corresponding to the second and third
extracellular
loops (ECL), respectively. The replacement of two
basic residues
(lysine) by neutral residues (valine) at positions
158 and 159 in ECL2
had no apparent effect on the HIV-1 coreceptor
activity of US28 but
abolished its fusion-enhancing activity.
Opposite results were observed
with two mutants corresponding
to amino acid substitutions in ECL3.
Both had fusion-enhancing
activities comparable to that of WT US28,
while their coreceptor
activities were markedly reduced (in the case of
the K257V mutant)
or null (in the case of the E266V R267V mutant). The
surface expression
of MYC-tagged forms of these mutants was reduced by
comparison
with that of WT US28 (Table
2). The amount of US28 available
at the cell surface might be more important for the fusion-enhancing
activity than for the HIV-1 coreceptor activity, or these properties
might require interactions with different domains of US28.
Effect of US28 on different cell types.
In all previous
experiments, US28 enhanced syncytium formation between fusion effector
and target cells that were both of human origin (HeLa, U373MG, or U87MG
cell lines). Since the VSV-G protein does not require a cell-specific
receptor to mediate cell fusion, it was possible to test the activity
of US28 with target cells from different species in the same
experimental setting. HeLa P4 cells were cotransfected with expression
vectors for VSV-G and for either US28, CCR1, or CCR5, and cocultures
were performed with cell lines from human, simian, feline, or murine
origin, stably or transiently expressing Tat. For all target cells
tested, similar numbers of syncytia were detected when HeLa P4 cells
were transfected with the CCR5 or CCR1 vector (Fig.
5) or with Rc/CMV (data not shown),
confirming that CCR5 or CCR1 did not enhance cell fusion mediated by
VSV-G. The numbers of fusion events that were detected varied
according to the target cell type, which might reflect differences in
fusion efficiency but also in expression of Tat and/or efficiency of
LTRlacZ transactivation. The lower numbers of syncytia detected with
CrFK cells are probably due to their transient transfection to
express Tat. Transfection of the US28 vector resulted in markedly
increased numbers of syncytia formed with human HeLa cells, as
expected, or with macaque B5 cells or feline CrFK cells (Fig. 5),
indicating that the fusion-enhancing activity of US28 was not
restricted to cells of human origin. In contrast, US28 had no apparent
effect on VSV-G-mediated fusion with mouse NIH 3T3 and Dunni cells or
with rat XC cells, which suggests that its fusion-enhancing activity is
dependent on target cells.
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FIG. 5.
Cell type restriction of the fusion-enhancing activity
of US28. HeLa P4 cells were cotransfected in six-well trays with
expression vectors for VSV-G and for either US28, CCR5, or CCR1. An
equivalent number of the indicated target cells, stably expressing
HIV-1 Tat or transiently transfected with Rc/CMV-Tat (CrFK), was added
24 h later. Cells were fixed and stained with X-Gal after a 24-h
coculture. Bars represent mean numbers (with standard deviations) of
blue-stained foci in triplicate wells.
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DISCUSSION |
Expression of the CMV-encoded chemokine receptor US28 in
CD4+ cell lines allows their infection by HIV-1 and their
fusion with cells expressing HIV-1 envelope proteins (Env+
cells) (42). Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4 (17, 33), which were later shown to interact with the gp120 envelope protein and to
behave as CD4-associated HIV-1 coreceptors (27, 52, 57). The
interaction of gp120 with two cellular components, CD4 and a
coreceptor, is thought to trigger conformational changes that eventually activate the fusiogenic properties of the transmembrane Env
subunit gp41.
Coreceptor activity of US28.
Besides CCR5 and CXCR4, several
chemokine receptors and related orphan GCRs have been found to be able
to mediate CD4-dependent HIV-1 entry, with various efficacies (11,
16, 21, 45). Although their interaction with gp120 was not
formally established, these GCRs were inferred to behave as HIV-1
coreceptors, like CXCR4 or CCR5. In the case of US28, it can be
wondered if the promiscuous fusion-enhancing activity might not account
for its HIV-1 coreceptor activity. In other terms, could US28 allow
HIV-1 entry by a mechanism different from that of CXCR4 or CCR5? It might be envisioned that the CD4+ cells used to test
candidate HIV-1 coreceptors (e.g., HeLa or U373MG cells) have an
intrinsic ability to fuse with Env+ cells, too low to be
detected by current assays but revealed by the promiscuous activity of
US28 on cell-cell fusion. We found that a mutation in the third ECL of
US28 or a deletion in the NT domain abolished its HIV-1 coreceptor
activity, while these changes had no apparent effect on the
fusion-enhancing activity. Moreover, opposite effects resulted from
mutations in the second ECL. These results indicate that the
promiscuous effect of US28 on cell-cell fusion is not required for its
HIV-1 coreceptor activity. The mechanism by which US28 and other GCRs
allow infection of CD4+ cells by HIV-1 or their fusion with
Env+ cells is probably similar to the coreceptor activity
of CCR5 or CXCR4.
Mechanism of fusion enhancement.
The expression of US28
enhanced the efficiency of cell-cell fusion mediated by envelope
proteins from three different viruses, HIV-1, HTLV-1, and VSV. This
effect was observed when US28 was expressed in target cells or in cells
bearing the viral fusiogenic proteins. These elements strongly suggest
that the effect of US28 on syncytium formation is not due to a direct
interaction with the fusiogenic proteins or their cellular receptors.
The mechanism by which viral proteins mediate virus entry, or syncytium
formation, is not known in its molecular details.
The energy stored in
their conformation is used to overcome repulsive
hydration forces
between membranes in order to allow their close
apposition. The next
steps seem to be the formation of a fusion
pore, generally viewed as a
proteinaceous structure forming a
bridge between membranes, and its
dilatation (
28,
32,
54).
Physicochemical features of the
membranes, such as charge and
lipid composition, influence their
ability to engage in fusion.
For example, compounds decreasing the
surface potential of membranes,
such as polyethylene glycol, are well
known to induce cell-cell
fusion. High cholesterol concentrations in
target membranes can
enhance the efficiency of fusion mediated by viral
proteins (
26).
The positive effect of amphotericin B and
related polyene macrolides
on cell-cell fusion induced by HIV-1 Env or
by other viral proteins
(
39,
40) could be related to their
ability to interact with
membrane cholesterol (
7).
High local concentrations of a protein with multiple membrane-spanning
domains, such as US28, might affect the fluidity or
other physical
properties of membranes in a way favorable to fusion.
However, positive
effects on cell-cell fusion were not observed,
or were considerably
less efficient, for proteins sharing the
membrane topology of US28 and
apparently expressed at a higher
level, for example, CCR1. A major
argument against a direct effect
of US28 on membranes was its lack of
fusion-enhancing activity
in cocultures with murine target cells.
Indeed, US28 could enhance
VSV-G-mediated fusion with three human cell
lines (HeLa, U87MG,
and U373MG), the simian cell line B5, and the cat
cell line CrFK
but not that with NIH 3T3 or Dunni mouse cells or rat XC
cells.
In this experiment, the fusion-enhancing activity of US28
depended
upon the target cell and probably upon the presence or absence
of a component of its plasma membrane. The most likely hypothesis
seems
to be that the fusion-enhancing activity requires the interaction
of
US28 with a membrane component. Such a putative US28 ligand
would be
present in human cell lines of different tissue origin
but also in
simian and feline cells, suggesting a certain degree
of conservation
among species. On the other hand, it should either
be absent in the rat
or mouse cells that we have tested or be
too different in these species
to be functional. Analysis of other
cell types from different species
is necessary to confirm these
views and evaluate the biochemical nature
of this factor. Its
interaction with US28 might either promote
cell-cell contact or
result in an indirect effect on the
US28-expressing cell promoting
membrane fusion. It would be of
interest to test the effect on
fusion of a mutant US28 devoid of cell
signalling activity.
Possible role of US28 in CMV entry.
It is often envisioned
that US28 confers on CMV-infected cells responsiveness to CC
chemokines, thereby modulating viral gene expression or contributing to
establishment of latent infection (22). Changes in the
intracellular concentrations of calcium, diacylglycerol, and other
second messengers were indeed reported for CMV-infected cells
(1), but it cannot be ascertained that these phenomena are
mediated by US28. The US28, US27, and and UL33 genes were found to be transcribed with late CMV genes
(53), which generally encode structural proteins. According
to that study, US28, US27, or UL33 would be expressed soon before virus release and cell death, which seems to disfavor a possible role in the
regulation of CMV gene expression. It remains possible that smaller
amounts of US28 are expressed earlier in infected cells (31)
or that the pattern of expression is different in other cell types or
in vivo. Alternatively, it can be envisioned that the main function of
US28 is not to regulate CMV expression in response to CC chemokines
through its GCR activity.
The pattern of expression of US28 and its membrane topology are
compatible with its association with the lipidic envelope
of virions.
The putative GCR encoded by the
UL33 gene was indeed
shown
to be associated with the CMV envelope (
29). If US28 is
also
expressed in the CMV envelope, its role could be to direct
virions to
sites of inflammation, where high concentrations of
CC chemokines are
found. However, the nature of the viral and
cellular proteins involved
in CMV entry is still debated (
13,
47), and it can be
envisioned that US28 has a direct role in
CMV entry, through
its ability to enhance membrane fusion. This
hypothesis could be
addressed by testing the replicative ability
and efficiency of cell
entry of a US28-defective CMV. We did not
observe enhancement of HIV-1
infectivity when US28 was expressed
in HIV-1-producing cells or in
CD4
+ target cells (besides HIV-1 coreceptor activity).
However, the
experimental systems used did not ensure that US28 was
actually
borne by HIV-1 particles, and the stable expression of US28 at
a high level in target cells could not be achieved. Further experiments
are therefore necessary to define whether US28 can activate virus-cell
fusion and play a role in CMV entry.
Although numerous herpesvirus proteins display features of GCRs, few
were shown to have signal-transducing activity. Some
might be activated
through interaction with unknown ligands. Others
might be devoid of GCR
activity, in particular if cellular GCRs
were captured by viruses for
their ability to bind certain ligands
or for other properties, such as
their transmembrane topology.
Our results with US28 suggest that these
possibilities deserve
further investigation.
| |
ACKNOWLEDGMENTS |
We thank N. Davis-Poynter, M. N. Gershengorn, J. Richardson, O. Schwartz, M. Sitbon, and N. Sol for generous
gifts of reagents; I. Bouchaert and F. Letourneur for help with
flow cytometry and sequencing; and L. Picard and N. Heveker for
critical reading of the manuscript.
This work was supported by the Agence Nationale de Recherches sur le
SIDA and by a fellowship to O.P. from Ensemble contre le SIDA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U.332,
Institut Cochin de Génétique Moléculaire, 22 rue
Méchain, 75014 Paris, France. Phone: 33-1-40 51 64 86. Fax:
33-1-40 51 77 49. E-mail: alizon{at}cochin.inserm.fr.
Present address: Laboratoire de Virologie, Ecole Normale
Supérieure, Lyon, France.
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Abstract
Introduction
