By
From the * Department of Immunology, Research Institute, Osaka Medical Center for Maternal and
Child Health, Osaka 590-02, Japan; Shionogi Institute for Medical Science, Osaka 566, Japan; § Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan;
Department of Molecular
Preventive Medicine, Division of Social Medicine, Graduate School of Medicine, University of Tokyo,
Tokyo 113, Japan; and ¶ Department of Medicine III, Osaka University Medical School, Osaka
565, Japan
Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.
Identifying mechanisms responsible for species specific
tropism of HIV-1 may be important to develop an animal model simulating HIV-1 infection as well as to unravel
cellular factors for viral infection. Mice are convenient, low
cost, well-characterized laboratory animals but are not a
natural target of HIV-1, and murine cells present some
blocks to viral infection. The first block is located at the
level of viral binding to murine cells. Human CD4 binds to
HIV-1 but its murine counterpart does not (1). Moreover, previous studies have revealed that when human CD4 is
expressed in vitro at the surface of some murine cells including T cell lines, binding of HIV occurs but entry does
not (2). This result demonstrated that murine cells expressing human CD4 do not support viral entry and suggested
that the defect is due to the absence of another human specific coreceptor required for membrane fusion.
HIV-1 strains exhibit marked differences in their abilities to
infect CD4-positive cells. Some strains infect monocytes, classified as monocyte- or macrophage-tropic strains (M-tropic)
and other strains infect T cell lines, classified as T cell line
tropic (T-tropic). As HIV-1 infection progresses, the initially
dominant M-tropic viruses are usually replaced by T-tropic
viruses (3, 4, 5). In 1996, CXCR4/fusin, a G protein coupled receptor with seven transmembrane helices, was shown
to be essential for entry of T-tropic HIV-1 into human CD4positive cells (6). The results prompted us to ask whether
the function of CXCR4 as a coreceptor of viral entry is
species specific. Recently, murine CXCR4 that was shown
to be a receptor for CXC chemokine PBSF/SDF-1 (7)
was isolated and has 90% amino acid identity with human CXCR4 (7).
In this study, we established cells transfected with both
human CD4 and murine CXCR4 and examined whether
HIV-1 coreceptor CXCR4 is an intrinsic barrier in mouse
cells for viral entry.
Cell Lines.
Mouse NIH3T3 cells, human small intestine epithelial cell-derived SW480 cells and human glioma cell lines
U87MG were cultured in DMEM with 10% FCS. Human HeLaS3
cells were cultured in RPMI1640 with 10% FCS. Human osteosarcoma-derived HOS cells were cultured in Eagle MEM with
1% Nonessential amino acid (GIBCO BRL, Gaithersburg, MD)
and 10% FCS. DW34 cells were provided by S.-I. Nishikawa (Kyoto University, Kyoto, Japan).
Viruses.
HIV-1 strain, NL432 was provided by A. Adachi
(University of Tokushima, Tokushima, Japan) (10). IIIB was provided by S. Harada (Kumamoto University, Kumamoto, Japan)
(11). SF162 was provided by J.A. Levy (University of California,
San Francisco) (12). HIV-1 chimeric clones, NL432env-162 and
NL432V3-162 were provided by Y. Isaka (Shionogi Institute). Recombinant Vaccinia viruses, Vac.Env (NL432 env), Vac.Env162
(SF162 env), and Vac T4 (CD4) were provided by T. Shioda
(University of Tokyo, Tokyo, Japan). LO-T7 (T7 polymerase) was
provided by M. Kohara (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan).
Transfection of Cell Lines.
NIH3T3 cells were plated overnight in 24-well plate at 5 × 104 cells per well, and transfected
with coreceptor in pBluescript by lipofectamine. After 4 h, cells
were washed by PBS, added by culture medium, incubated at
37°C overnight, and used for fusion assay. SW480 cells and HOS
cells were plated overnight in 6-cm dishes at 5 × 105 cells per
dish. SW480 cells were transiently transfected with 5 µg of coreceptor in pEF-BOS and 7.5 µg of T4-Neo and 2.5 µg of LTR
(EcoRV)- RT-PCR.
Total RNA was isolated and 3.0 µg was used for
cDNA synthesis using Ready to Go (Pharmacia, Uppsala, Sweden). One-thirtieth of this reaction was used as a template for
PCR amplification for 40 cycles at 92°C for 0.5 min, 55°C for 1 min, and 74°C for 2 min. The primer pairs were for murine glyceraldehyde 3-phosphate dehydrogenase (G3PDH; CLONTECH,
Palo Alto, CA) for murine CXCR4: 5 Measurements of Intracellular Ca2+ Response to Murine PBSF/
SDF-1.
Murine PBSF/SDF-1 was synthesized in the Peptide
Institute, Inc. (Minoo, Japan). Real-time measurement of Intracellular Ca2+ measurements in NIH3T3 cells and murine CXCR4transfected CHO cells loaded with fura-PE3 were done as described (7).
Cell Fusion Assays.
To quantitate cell-cell fusion events, we
used a modified fusion assay based on Infection Assays.
Human SW480 or HOS cell lines bearing
human CD4 and coreceptors were seeded as monolayers in 12well tissue culture plates. To each well, 200 µl of HIV-1 viruscontaining fluid (reverse transcriptase [RT] activity, 2 × 106 RT/
ml for SF162, NL432env162, NL432V3-162, 5 × 106 RT/ml for IIIB,
and 3 × 106 RT/ml for NL432) was added, and the plates were
incubated for 2 h at 37°C in 5% CO2 and 2.5 ml of culture medium was added. An aliquot was removed after 4 days of infection
and 400 µl of Reporter lysis buffer (Promega) was added and
freezed to First, to determine if murine CXCR4 allows HIV-1
env-mediated membrane fusion, we used the assay system
in which fusion between effector env-expressing cells and
target coreceptor- and CD4-expressing cells leads to activation of a reporter enzyme. In this assay, effector HeLaS3 cells
were infected with a recombinant vaccinia virus that expresses
Second, we examined whether murine CXCR4 also allows for virus infection to target cells. Since murine cells,
including NIH3T3 that expressed human CXCR4 and CD4
supported HIV-1 replication much less efficiently (data not
shown), we used three types of human cells, small intestine
epithelial cell-derived SW480 cells, osteosarcoma-derived
HOS cells and glioma cell-derived U87MG as the target
cells for viral infection. Cells were transfected with an integrated HIV-1 long terminal repeat (LTR)-driven reporter gene, lacZ. If viruses enter the cells, HIV-1 encoded transactivating protein Tat is expressed and induces the expression of lacZ. In addition, they were transfected with human
CD4 plus chemokine receptors. The cells were infected
with T-tropic virus strains, NL432 or IIIB, or an M-tropic
strain SF162 and harvested. As shown in Fig. 2 A, NL432
or IIIB entered SW480 that expressed both murine CXCR4
and human CD4 equally compared to the cells that expressed both human CXCR4 and human CD4, consistent
with the results of the env-mediated fusion assay described
above. Entry did not occur when human CCR2b or
CCR5 was expressed in place of CXCR4. SF162 entered
cells expressing both human CCR5 and CD4 but not cells expressing human or murine CXCR4 plus human CD4.
Similar results were obtained from using HOS cells (Fig. 2
B) or U87MG cells (Fig. 2 C) in place of SW480 cells.
Thus, murine CXCR4 supports entry of the T-tropic virus
strains into target cells and may not affect the synthesis, integration, and expression of proviral DNA in human cells.
Human CXCR4-mediated HIV-1 entry has been shown
to be inhibited by the monoclonal antibody directed against
the V3 loop (6). Then, to confirm that the function of murine CXCR4 can replace the function of human CXCR4
further, we investigated whether the V3 loop of envelope
gp120 is required for murine CXCR4-mediated HIV-1 entry. SW480 cells expressing human CD4 and chemokine receptors were infected with viral chimeric clones, NL432env-162
and NL432V3-162. As shown in Fig. 3 A, NL432env-162 was
constructed by substitution of an M-tropic strain SF162
env region into a T-tropic strain NL432 proviral clone.
NL432V3-162 was constructed by substitution of an SF162
V3 region into a NL432 proviral clone. Although NL432 entered SW480 cells expressing murine CXCR4 and human CD4, both NL432env-162 and NL432V3-162 failed to enter those cells. Both NL432env-162 and NL432V3-162 entered
SW480 cells expressing human CCR5 and human CD4.
These results revealed that the V3 region is required for viral entry supported by murine CXCR4 as well as human
CXCR4.
This study revealed that murine CXCR4 could support
T-tropic HIV-1 env-mediated membrane fusion and viral
entry, indicating that there is no species specific barrier at
CXCR4. Previous studies showed that when human CD4
is expressed in vitro at the surface of murine lymphoid or
non-lymphoid cells including NIH3T3 and T cell clone
3DT, binding of HIV-1 occurs but entry does not (2). One
explanation of the results is that the murine cells expressing human CD4 do not express CXCR4 at the cell surface. In
fact, CXCR4 mRNA and murine PBSF/SDF-1-responsive CXCR4 were not seen in NIH3T3 cells (Fig. 1, A and
B). However, murine CXCR4 is expressed in both double-positive (CD4+CD8+) and single positive (CD4+CD8 It has been reported that a murine counterpart of CCR5,
an entry coreceptor for M-tropic HIV-1 (17) does not
support viral entry (22), indicating that species specific limitation to a coreceptor function for M-tropic HIV-1 is distinct from that for T-tropic HIV-1. The difference may be
due to high amino acid sequence conservation of CXCR4
between species compared to other chemokine receptors,
including CCR5. The amino acid sequence of murine
CXCR4 has 90% identity with that of human CXCR4,
while CCR5 and CXCR2 are 82 and 71% identical between mouse and human. The strong conservation of
CXCR4 is consistent with the unique functions of its
ligand, PBSF/SDF-1 among chemokines, including MIP-1 The previous studies and the result that a murine cell line
NIH3T3 transfected with human CD4 and coreceptors supported HIV-1 entry but produced virus particles much less
efficiently than human cells (data not shown) suggest the
absence of intracellular molecules that are critical for viral
replication in mice (24). However, we could develop a
murine HIV-1 infection model by constructing the transgenic mice containing the human DNAs of the molecules responsible for species specific barriers. Our results indicate that we do not have to introduce human CXCR4 into
those mice and provide useful information to develop an
animal model to simulate all phases of infection, since the
physiological expression of CXCR4 may be involved in
initiation and exacerbation of the shift from M-tropic to
T-tropic HIV-1 strains leading to advanced clinical disease.
Gal-Neo by modified calcium phosphate method (13). HOS cells, stably expressing human CD4 and LTR-
Gal were
transiently transfected with 15 µg of coreceptor by the same
method. Cells were incubated at 35°C in 3% CO2 overnight,
washed with PBS (
), harvested with 0.5 mM EDTA/PBS (
),
passaged to 12-well plate and incubated at 37°C overnight. Next
day, cells were tested for infection assay.
-TAGCGGCCGCGTTGCCATGGAACCGAT-3
and 5
-GCGTCGACTTTGCATAAGGGTTAGCTG-3
.
-complementation of
-galactosidase (
-gal) (Shida, H., manuscript in preparation). In
brief,
subunit of
-gal and env proteins were introduced into
effector HeLaS3 cells (1 × 105 cells/well in 24-well plate) by infection of the recombinant vaccinia viruses. Human CD4,
subunit of
-gal and T7 RNA polymerase were introduced into target NIH3T3 cells (5 × 104 cells/well in 24-well plate) by the
vaccinia virus recombinants, and coreceptors were transfected using lipofectamine. 16 h later, effector and target cells were washed
with PBS containing 0.5 mM CaCl2, and treated with anti-vaccinia virus antibody, 2D5, provided by Y. Ichihashi (14), to inhibit vaccinia-induced cell fusion. The effector cells were suspended in Hanks' balanced salt solution containing 3 mM CaCl2,
pH 7.6, overlayed on the target cells in 24-well plates and centrifuged at 1,300 rpm for 5 min to initiate fusion. Cells were incubated for 12 h at 37°C in 5% CO2. If fusion occurred, the cytoplasmic contents of the fused cells lead to
-complementation
between
and
subunits of
-gal resulting in active enzyme.
Then, 200 µl per well of chlorophenolred-
-D-galactopyranoside containing solution, including 8 mM of chlorophenolred-
-
D-galactopyranoside (Boehringer Mannheim), 45 mM of 2-ME, 1 mM of MgCl2, 100 mM of Hepes, pH 8.0, 0.5% NP40, and 0.1 mg/ml of DNAse I was added and incubated for 30 min at 37°C.
Then 200 µl per well of 2% SDS was added to stop reaction and
assayed for
-gal activity at OD590.
80°C and thawed. Then samples were transferred to
eppendorf tubes, centrifuged at 12,000 rpm for 5 min at 4°C. The
supernatant was assayed for
-gal activity by Luminescent
-gal
Detection Kit (CLONTECH).
subunit of
-gal and HIV-1 env protein. We used
NIH3T3 cells as the target cells since no CXCR4 mRNA
was seen and 1,000 nM PBSF/SDF-1, a ligand for CXCR4
did not induce an increase in intracellular free Ca2+ in
NIH3T3 cells (Fig. 1, A and B). The target NIH3T3 cells
were infected with a recombinant vaccinia virus that expresses
-subunit of
-gal, T7 polymerase and human
CD4. Then NIH3T3 cells were transfected with plasmids
containing human CXCR4 or CCR5 or murine CXCR4.
Effector and target cells were mixed together and incubated. If fusion occurred, the cytoplasmic contents of the
fused cells lead to activation of
-gal. As shown in Fig. 1,
HeLaS3 cells expressing the env proteins derived from the
T-tropic HIV-1 strain NL432 readily fused with NIH 3T3
cells expressing human CXCR4 and human CD4, but not
with cells expressing human CCR5 and human CD4. Surprisingly, they also fused with cells expressing murine CXCR4 and human CD4. HeLaS3 cells expressing the
env proteins derived from the M-tropic strain SF162 fused
with cells expressing human CCR5 and human CD4, but
not with cells expressing murine or human CXCR4 in
conjunction with human CD4.
Fig. 1.
Murine CXCR4 supported membrane fusion mediated by T cell line-tropic HIV-1 env protein. (A) RT-PCR analysis of murine CXCR4 mRNA expression on NIH3T3 cells and DW34 cells. (B) Intracellular Ca2+ response of NIH3T3 cells and CHO cells transfected with murine CXCR4
in response to murine PBSF/SDF-1. Rise in intracellular Ca2+ concentration is represented by the increase in relative fluorescence. (C) Quantitation of fusion by -galactosidase activity assay. NIH3T3 target cells were infected with a recombinant vaccinia virus that expresses human CD4, T7 polymerase and
subunit of
-gal. Then the cells were transfected with murine CXCR4 or human CXCR4 or CCR5. HeLaS3 effector cells were infected with a recombinant vaccinia virus that expresses
subunit of
-gal and env proteins derived from HIV-1 strains NL 432 or SF162. Cells were allowed to fuse and
assayed for
-gal activity.
[View Larger Versions of these Images (17 + 28K GIF file)]
Fig. 2.
Murine CXCR4 supported infection of a T cell line-tropic HIV-1 virus. SW480 cells (A), HOS cells (B), or U87MG cells (C) were cotransfected with human CD4 plus chemokine receptors, and then infected with HIV-1 strains NL432, IIIB, or SF162. Cell lysates were assayed for -gal activity.
[View Larger Versions of these Images (26 + 29 + 25K GIF file)]
Fig. 3.
The V3 loop of envelope gp120 is required for murine CXCR4-mediated HIV-1
entry. (A) Structure of chimeric
proviral clones. The env protein
or V3 loop region of macrophage-tropic HIV-1 strain SF162
was introduced into a T cell linetropic strain NL432 proviral DNA. E, EcoRI; Ba, BamHI; St,
StuI; Nh, NheI. (B) SW480 cells expressing human CD4 and
listed chemokine receptors were
infected with the chimeric
proviral clones, NL432env-162 or
NL432v3-162.
[View Larger Versions of these Images (14 + 26K GIF file)]
,
CD4
CD8+) thymocytes (7). It is important to determine
whether 3DT cells express CXCR4. Recently, some murine cells that express human CD4 were shown to be infected by T-tropic HIV-1 strains (15), supporting our conclusions. In addition, the previous study showed that a rat
cell line can be infected by T-tropic HIV-1 strains (16). This
result, together with our results, suggest that rat CXCR4 is
also functional for HIV-1 entry.
,
MIP-
, and RANTES, ligands of CCR5. PBSF/SDF-1
has essential functions in development including hematopoiesis and cardiogenesis (23) while other chemokines are
thought to be involved in trafficking of leukocytes in inflammation.
Address correspondence to Takashi Nagasawa, Department of Immunology, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 590-02, Japan.
Received for publication 30 January 1997 and in revised form 6 March 1997.
Drs. Tachibana and Nakajima contributed equally to this work.We thank A. Adachi, S. Harada, J.A. Levy, T. Shioda, M. Kohara, Y. Ichihashi, S.-I. Nishikawa, and Y. Isaka for valuable reagents; S. Kawauchi, A. Suyama, and S. Miki for technical assistance.
This work was supported by grants from the Ministry of Education, Science, and Culture of Japan.
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