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Journal of Virology, May 2001, p. 4258-4267, Vol. 75, No. 9
Department of Hematology/Oncology, Graduate
School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Received 29 August 2000/Accepted 2 February 2001
Interaction between the human immunodeficiency virus type
1 (HIV-1) envelope and the relevant chemokine receptors is crucial for
subsequent membrane fusion and viral entry. Although the V3 region of
gp120 is known to determine the cell tropism as well as the coreceptor
usage, the significance of the binding of the V3 region to the
chemokine receptor has not been fully understood. To address this
issue, we adopted the pseudotyped virus infection assay in which the V3
region of the T-cell line-tropic (T-tropic) NL4-3 envelope was replaced
with a portion of stromal cell-derived factor 1 (SDF-1), the ligand of
CXCR4. The V3 region of the NL4-3 envelope expression vector was
replaced with three different stretches of SDF-1 cDNA. Expression of
each chimeric envelope protein was confirmed by immunoprecipitation and
Western blotting. Luciferase reporter viruses were prepared by
cotransfection of the pNL4-3.Luc.E Human immunodeficiency virus type 1 (HIV-1) enters target cells through the interaction between the viral
envelope glycoproteins and the cellular receptors, CD4 and
one of the coreceptors that are members of the seven-transmembrane
domain, G-protein-coupled receptor superfamily (1, 3, 10, 17, 18,
20, 22, 23, 30). The cell tropism of HIV-1 is thought to be
determined at the level of viral entry by the interaction of the viral
envelope with certain types of coreceptors that support either T-cell
line-tropic (T-tropic) or macrophage-tropic (M-tropic) HIV-1 infection
(13). It has been widely accepted that CXCR4 and CCR5 are
the major coreceptors for T- and M-tropic HIV-1 strains, respectively.
The viral envelope can be envisioned as a fusogenic apparatus,
catalyzing pH-independent fusion between the viral membrane displaying
the gp120-gp41 complex and the target cell membrane displaying CD4 plus
one of the coreceptors (2, 46). Numerous studies have
demonstrated that CD4 binding induces a conformational change in gp120
that exposes, creates, or stabilizes the coreceptor-binding determinants (42, 47, 54). The gp120 molecule contains
five variable loops designated V1 to V5 interspersed with five
relatively conserved regions designated C1 to C5 (54). It
has been speculated that subsequent to its binding to the CD4 molecule,
gp120 changes its conformation and exposes the cryptic V3 loop together
with the V1/V2 loop embedded in the gp120 molecule (39, 49,
53). Evidence has indicated that the V3 region contains a
critical determinant of envelope fusogenicity and tropism (2, 11, 45). Although it is known that the V3 region functions not alone but rather in concert with other gp120 regions, including V1, V2, and
C4, binding experiments using mutant gp120 molecules with a deletion at
the V3 region or anti-V3 loop neutralizing antibodies have strongly
suggested that the V3 region is crucial for interaction with the
relevant coreceptor (52). In accordance with this, we
previously reported that T-tropic HIV-1-derived V3 loop peptides directly bind to CXCR4 and inhibit T-tropic HIV-1 infection
(40). These results imply that the binding capability of
the V3 region to the relevant coreceptor rather than its
indigenous amino acid sequence is important for entry of HIV-1
into target cells. Thus, it remains to be tested whether a nonviral
amino acid sequence with an appropriate binding affinity to the
relevant coreceptor can replace the V3 region without losing viral infectivity.
In the present study, to evaluate the significance of binding between
the V3 region and the relevant coreceptor in viral entry, we prepared
pseudotyped virus whose V3 region of the T-tropic NL4-3 envelope was
replaced with three different fragments of stromal cell-derived factor
1 (SDF-1), the ligand of CXCR4, and carried out the pseudotyped virus
infection assay. Here, we show that pseudotyped HIV-1 with one of these
chimeric envelopes can infect CD4+ CXCR4+
target cells.
Cells and culture conditions.
HEK293T, originally
referred to as 293tsA1609neo, is a human embryonic kidney cell
line and was cultured in Dulbecco's modified Eagle's medium (DMEM)
(Life Technologies, Inc., Rockville, Md.) supplemented with 10% fetal
calf serum (FCS) (Life Technologies, Inc.) and
penicillin-streptomycin-glutamine (Life Technologies, Inc.)
(24, 36). U87MG is a human astroglial cell line
(38). Three stable transfectants of U87MG, U87.CD4
expressing CD4, U87.CD4.CXCR4 expressing CD4 as well as CXCR4,
and U87.CD4.CCR5 expressing CD4 as well as CCR5, were obtained
from the NIH AIDS Research and Reference Reagent Program (Rockville,
Md.) (4) and cultured in DMEM with 15% FCS. U87.CXCR4 and
U87.CCR5 cells were generated by transfection of CXCR4 and CCR5
expression vectors into U87MG cells in our laboratory.
Vectors.
The luciferase reporter HIV-1 clone
pNL4-3.Luc.E
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4258-4267.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Replacement of the V3 Region of gp120 with SDF-1 Preserves
the Infectivity of T-Cell Line-Tropic Human Immunodeficiency Virus
Type 1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
R vector
and each chimeric envelope expression vector, and the infection assay
was then carried out. We showed that pseudotyped viruses with one of
the chimeric envelopes, NL4-3/SDF1-51, could infect U87.CD4.CXCR4 but
not U87.CD4 or U87.CXCR4 cells and that this infection was inhibited by
the ligand of CXCR4, SDF-1
, by anti-human SDF-1 antibody, or by an
anti-CD4 antibody, Leu3a, in a dose-dependent manner. Furthermore,
chimeric NL4-3/SDF1-51 gp120 significantly inhibited binding of labeled
SDF-1 to CXCR4. It was suggested that replacement of the V3 region of
the NL4-3 envelope with SDF-1 preserved the CD4-dependent infectivity
of T-tropic HIV-1. These results indicate that binding between the V3
region and the relevant coreceptor is important for viral entry, whether its amino acid sequence is indigenous to the virus or not.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
R (8, 12, 25)
provided by the NIH AIDS Research and Reference Reagent Program was
used for the reporter gene assay of pseudotyped virus infection. pNL4-3.Luc.ER consists of pNL4-3
with the gene for the luciferase reporter fused in frame to
NotI and XhoI sites at the 5' end of the
nef coding region. In addition, a frameshift was introduced
near the 5' end of env to block production of gp160 and to
limit the virus to a single round of replication. We also used the
enhanced green fluorescence protein (EGFP) as a reporter gene
(14, 35). The coding region of EGFP cDNA was amplified
by PCR using primers containing a 5' NotI and a 3'
XhoI site from the pEGFP-C3 vector (Clontech, Palo Alto,
Calif.). The PCR product was then cleaved with NotI and
XhoI and inserted into
pNL4-3.Luc.ER at these sites. This resulted
in a replacement of the luciferase reporter gene with EGFP
(pNL4-3.EGFP.ER).
(MIP-1) cDNA encoding the full-length protein (70 amino acids) in the same way.
View larger version (47K):
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FIG. 1.
(A) Constructs of NL4-3 chimeric envelope expression
vectors. As a WT envelope expression vector, we used pME-NF-NL4-3env
with a FLAG tag at the 5' terminal end of the NL4-3 envelope gene. The
V3 region of the NL4-3 envelope was replaced with a fragment of SDF-1
by using PvuII and NheI restriction sites. (B)
Amino acid sequences of the V3 region of the NL4-3 envelope (NL4-3/V3)
and human SDF-1. Homology between NL4-3/V3 and SDF-1 is indicated as
follows: identical, asterisks; related, dots. Two different overlapping
portions of SDF-1 used for chimeric envelope expression vectors are
shown with three separate lines.
Immunoprecipitation and Western blotting. The chimeric envelope vectors were transfected into HEK293T cells with Lipofectamine (Life Technologies, Inc.). After 48 h, cells were washed once with phosphate-buffered saline (PBS) and lysed in lysis buffer (1% Triton X-100, 5 mM EDTA, 150 mM NaCl, 10 mM Tris-HCl, pH 7.6) at 4°C for 30 min with gentle mixing. Each cell lysate of HEK293T cells was subjected to immunoprecipitation by anti-FLAG monoclonal antibody (MAb) (M2) (Sigma Chemical Co., St. Louis, Mo.). The immunoprecipitates were fractionated on 7.5% polyacrylamide gels. The samples were transferred to a 0.45-µm-pore-size polyvinylidene difluoride (PVDF) membrane and immunoblotted with anti-SDF-1 polyclonal antibody (Ab) (R & D Systems, Minneapolis, Minn.), anti-gp120 (V3) MAb (MAb 902) (NIH AIDS Research and Reference Reagent Program) (9, 37), or anti-FLAG MAb (M2). Then the membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies and developed with the enhanced chemiluminescence system according to the supplier's instructions (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).
Purification of HIV-1 virions by sucrose density equilibrium gradients and analysis of the virion-associated proteins. To analyze the incorporation of HIV-1 envelope protein in the virion, HIV-1 pseudotyped virions were purified by sucrose density equilibrium gradients. Briefly, virions were pelleted by centrifugation, resuspended in 1 ml of PBS, layered on top of the sucrose gradient, prepared in PBS ranging from 20 to 60%, and centrifuged for 13 h at 30,000 rpm in an SW-41Ti rotor (Beckman, Palo Alto, Calif.). Gradient fractions were collected from the top of the gradient and pelleted by centrifugation. These samples were used for analyzing protein profiles of the virion by Western blotting. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.45-µm-pore-size PVDF, and immunoblotted with anti-gp120 (conserved region) MAb (ABI, Columbia, Md.) or anti-HIV-1 p24 MAb (ZeptoMetrix, Buffalo, N.Y.).
Pseudotyped virus and luciferase assay.
Pseudotyped
luciferase reporter viruses were prepared in HEK293T cells by the
contransfection of pNL4-3.Luc.ER
vector and the envelope expression vectors using the calcium phosphate
method. Briefly, HEK293T cells were seeded in 100-mm-diameter culture
dishes at a density of 106 cells per dish the day before
the transfection. The complexes of plasmid DNA and calcium chloride
were added to the cells. After changing to fresh medium the next day,
pseudotyped viruses in the supernatants were harvested after
48 h of transfection. Viral supernatants were filtered with a
0.45-µm-pore-size filter unit (Millipore, Bedford, Mass.). The titers
of pseudotyped viruses were measured by an enzyme-linked
immunosorbent assay kit for the p24 antigen (RETRO-TEK; ZeptoMetrix).
U87.CD4, U87.CXCR4, U87.CD4.CXCR4, and U87.CD4.CCR5 cells were
seeded at 8 × 104 cells/well in a six-well plate 1 day before infection. An adjusted amount of viruses was added to each
well. On day 3 postinfection, the cells were washed with PBS and lysed
in 500 µl of passive lysis buffer (Promega, Madison, Wis.). The
luciferase activity in 20 µl of the lysate was measured with a
luminometer (EG & G Berthold, Bad Wildbad, Germany) using the
commercially available substrate, Luciferase Assay Reagent (Promega).
Infection assay using EGFP reporter viruses.
The
pseudotyped EGFP reporter viruses were prepared in HEK293T
cells by the cotransfection of
pNL4-3.EGFP.ER vector and the envelope
expression vectors using the calcium phosphate method as described
above. As with the luciferase reporter viruses, an equal amount of
viruses was added to cells. On day 3 postinfection, the cells were
harvested, fixed with PBS containing 0.2% bovine serum albumin, 0.2 mM
EDTA, and 2% paraformaldehyde, and analyzed by flow cytometry using a
FACScan (Becton Dickinson, San Jose, Calif.).
Binding assays.
We prepared supernatants of HEK293T cells
which were transiently transfected with the envelope expression vectors
(pNL4-3env, pNL4-3env/SDF1-51, pYU2env, and mock control of pME18S).
Soluble gp120 proteins in the supernatants were concentrated 50-fold by the Ultrafree-15 centrifugal filter device (Millipore) and were subjected to Western blot analysis to measure and compare the amount of
gp120 proteins. U87.CD4.CXCR4 cells were incubated with the
supernatants containing equally adjusted amounts of gp120 proteins at
4°C for 1 h. Subsequently, 125I-SDF-1 (NEN Life
Science Products, Boston, Mass.) in binding buffer (DMEM containing 25 mM HEPES, 0.3% bovine serum albumin, and 0.05% NaN3) was
added to the cells. After incubation at 4°C for 1 h, cells were
washed twice with binding buffer and the cell-bound radioactivities
were counted in a gamma counter (Abbott Laboratories, Abbott Park,
Ill.).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of NL4-3/SDF-1 chimeric envelopes. We constructed chimeric envelope expression vectors based on T-tropic NL4-3 in which the V3 region of gp120 was replaced with portions of SDF-1, a ligand of CXCR4. We designed three kinds of cDNA fragments of SDF-1 to replace the V3 loop, namely the first 51 amino acids of SDF-1 (SDF1-51), the full-length protein (SDF1-67), and 43 amino acids (amino acids 11 to 53) which are weakly homologous to the V3 loop of NL4-3 (HIV-IIIB) (SDF1-V3) (Fig. 1).
We examined whether each chimeric envelope protein was expressed by immunoprecipitation and Western blotting. Four envelope expression vectors, namely pNL4-3env/SDF1-67, pNL4-3env/SDF1-51, pNL4-3env/SDF1-V3, and pNL4-3env/wild type (WT), and pME18S as a negative control were transfected into HEK293T cells by Lipofectamine transfection. Cell lysates of transiently transfected HEK293T cells were subjected to immunoprecipitation and immunoblotting. All envelope proteins were detected by anti-FLAG MAb (M2) except for the mock control pME18S vector. Anti-SDF-1 polyclonal Ab interacted with the NL4-3/SDF-1 chimeric envelopes but not with the WT, while MAb 902 reacted with the WT but not with the NL4-3/SDF-1 chimeric envelopes (Fig. 2A).
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Incorporation of the chimeric envelope proteins in the virion. Next, we analyzed whether the chimeric envelope proteins were incorporated into pseudotyped virions. We purified each pseudotyped virion by density equilibrium gradient analysis, and the fractions from the top of the gradient to the bottom were subjected to SDS-PAGE followed by Western blotting. As shown in Fig. 2B and C, the envelope proteins of NL4-3/WT were detected as a doublet of gp120 and gp160 and colocalized with HIV-1 Gag (p24) proteins. Similarly, the other chimeric envelope proteins were also demonstrated to be incorporated in the virions (Fig. 2D). It appeared that the incorporations of three NL4-3/SDF1 chimeric envelope proteins were comparable to that of NL4-3/WT except that the gp120/gp160 ratio was slightly lower than that of NL4-3/WT.
Infectivity of pseudotyped viruses with the NL4-3/SDF-1
chimeric envelope.
Luciferase reporter virus with each chimeric
envelope was prepared in HEK293T cells by the cotransfection of
pNL4-3.Luc.ER vector and one of the
envelope expression vectors. The titration of pseudotyped
viruses was performed with an enzyme-linked immunosorbent assay
of the p24 antigen. We used U87.CD4.CXCR4, U87.CD4,
U87.CD4.CCR5, and U87.CXCR4 cells as target cells to which each
pseudotyped virus, equalized by the amount of p24 antigen, was
added. Measurement of luciferase activities of the cell lysates showed
that pseudotyped viruses with NL4-3/SDF1-51 as well as NL4-3/WT
could infect U87.CD4.CXCR4 but not U87.CD4, U87.CXCR4, or U87.CD4.CCR5
cells (Fig. 3). Although the efficiency
of infection of the former was relatively low, significant levels of
infection were reproducibly observed. The pseudotyped viruses
with the NL4-3/SDF1-V3 and NL4-3/SDF1-67 chimeric envelopes could
infect U87.CD4.CXCR4 cells with somewhat lower efficiency than
NL4-3/SDF1-51. Four independent experiments gave similar results.
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Infectivity of pseudotyped viruses with the NL4-3/SDF1-51
chimeric envelope using EGFP reporter virus.
To confirm the
infectivity of HIV-1 with an SDF-1 chimeric envelope, we also examined
the infection assay using EGFP reporter viruses and U87.CD4.CXCR4
cells. On day 3 postinfection, we analyzed the infectivity by flow
cytometry. As shown in Fig. 4, many
bright EGFP+ cells were detected after infection of the
EGFP reporter pseudotyped viruses with the NL4-3/WT envelope.
Infection of the pseudotyped virus with the NL4-3/SDF1-51
chimeric envelope generated a significant number of dull
EGFP+ cells that were not present in infection with the
virus with NL4-3/no Env. These results provide more supportive evidence
that HIV-1 with the NL4-3/SDF1-51 chimeric envelope can infect
CD4+ CXCR4+ target cells.
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Effects of SDF-1 and anti-SDF-1 Ab on the infection of the
pseudotyped virus with the NL4-3/SDF1-51 chimeric
envelope.
Next, we examined whether the ligand of CXCR4, SDF-1,
could inhibit the infectivity of the pseudotyped virus with the
NL4-3/SDF1-51 chimeric envelope as well as the NL4-3/WT virus. Before
the addition of the pseudotyped virus, cells were incubated
with recombinant human SDF-1 (rhSDF-1) (R & D Systems) or
recombinant human MIP-1 (rhMIP-1) (R & D Systems) at 37°C for
30 min. As shown in Fig. 5A and B,
pretreatment with rhSDF-1 resulted in inhibition of the infectivity
of the pseudotyped virus with the NL4-3/SDF1-51 chimeric
envelope as well as with the NL4-3/WT virus in a dose-dependent manner,
while pretreatment with rhMIP-1 did not.
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Effect of anti-CD4 MAb Leu3a on the infectivity of the
pseudotyped virus with the NL4-3/SDF1-51 chimeric
envelope.
It is known that the infectivity of most strains of
HIV-1, including NL4-3, is CD4 dependent and can be inhibited by the
anti-CD4 MAb Leu3a (16, 41). So we next examined whether
anti-CD4 MAb could inhibit the infectivity of the pseudotyped
virus with the NL4-3/SDF1-51 chimeric envelope.
U87.CD4.CXCR4 cells were pretreated with an anti-CD4 MAb, Leu3a
(Becton Dickinson). After 1 h, the pseudotyped viruses
were added to the cells. As shown in Fig. 6, the preincubation with Leu3a strongly
inhibited the infectivity of pseudotyped viruses with the
NL4-3/SDF1-51 chimeric envelope as well as the NL4-3/WT virus. Together
with the difference in infectivity for U87.CD4.CXCR4 and U87.CXCR4
cells, this indicated that the infectivity of the pseudotyped
viruses with the NL4-3/SDF1-51 chimeric envelope was CD4 dependent.
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Effects of soluble gp120 on the binding of SDF-1 to CXCR4.
We showed that the NL4-3/SDF1-51 virus could infect U87.CD4.CXCR4 cells
but that its efficiency was much less than that of the NL4-3/WT virus.
To confirm the binding capability of the chimeric envelope proteins to
CXCR4, we examined whether these proteins could inhibit binding of
labeled SDF-1 to CXCR4. The gp120 proteins were prepared as the
supernatants of HEK293T cells transiently transfected with the envelope
vectors (pNL4-3env, pNL4-3env/SDF1-51, pYU2env, and the mock
control, pME18S). After concentration, the gp120 proteins
were visualized by Western blotting, and adjusted amounts were used
(data not shown). As shown in Fig. 7,
NL4-3/SDF1-51 gp120 could significantly inhibit binding of SDF-1
in a dose-dependent manner which was comparable to that of NL4-3/WT
gp120, while M-tropic YU2 gp120 as well as mock supernatants showed no
effects. These results strongly suggested that this chimeric envelope
could specifically bind to CXCR4.
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Infectivity of pseudotyped viruses with the NL4-3/MIP-1
chimeric envelope.
We subsequently examined whether the
replacement of the V3 region with MIP-1, a ligand for CCR5, resulted
in acquisition of infectivity of CCR5+ cells. We
constructed a chimeric envelope expression vector based on T-tropic
NL4-3 in which the V3 region of gp120 was replaced with the
full-length, 70-amino-acid MIP-1 protein (NL4-3/MIP1-70). As shown
in Fig. 8, the pseudotyped virus
with the NL4-3/MIP1-70 chimeric envelope could infect
U87.CD4.CCR5 cells. Although the efficiency was much lower than
that of pseudotyped viruses with the YU2 WT envelope the
infectivity of the NL4-3/MIP1-70 virus seems to be specific and CCR5
mediated, since it was inhibited by neutralizing anti-human MIP-1
MAb (R & D Systems) in a dose-dependent manner, while the infectivity
of the YU2 envelope virus (NL4-3/YU2) was not affected. This
virus did not infect U87.CD4 or U87.CCR5 cells but might have
slight infectivity for U87.CD4.CXCR4 cells (less than 10% of that for
U87.CD4.CCR5 cells; data not shown), which might be due to the backbone
structure of T-tropic NL4-3 gp120.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Entry of HIV-1 into the host cell begins with the binding of the gp120 envelope glycoprotein to CD4, which serves as the primary receptor. CD4 binding induces conformational changes in the gp120 glycoprotein, leading to the exposure and/or formation of a binding site for the relevant chemokine receptors (32, 42). These chemokine receptors, mainly CCR5 and CXCR4 for HIV-1, serve as second receptors for virus entry. Among the defined regions of gp120, the V3 loop region is the principal determinant of chemokine receptor specificity and has a close association with the cell tropism of HIV-1 (11, 45).
The V3 region of any strain of HIV-1 has a simple loop structure with
one disulfide bond. It was previously reported that the T-tropic
HIV-1-derived synthetic cyclized V3 peptides but not the linear or
M-tropic V3 peptides could directly bind to CXCR4 and inhibit T-tropic
HIV-1 infection (40). In addition to this, Murakami et al.
reported the inhibition of T-tropic HIV-1 infection by a small-molecule
CXCR4 antagonist, T22, which also has a simple loop structure with two
disulfide bonds, but not by a linear control peptide, 4-Ala-T-I
(33, 34). Thus, it appears that the loop structure is
important for the binding of the peptides to CXCR4, resulting in the
inhibition of HIV-1 infection. To test whether the entry and the
infection of HIV-1 depends on the loop structure and the binding
between the V3 region and the relevant coreceptor rather than on its
indigenous amino acid sequence, we examined the effect of the
replacement of the V3 region of gp120 with exogenous nonviral amino
acid sequences on viral infectivity. We prepared chimeric envelope
vectors in which the V3 region of the T-tropic NL4-3 envelope was
replaced with stretches of SDF-1, a ligand for CXCR4. SDF-1 was
originally described as a chemokine produced by a bone marrow stromal
cell line composed of two alternative splicing forms, SDF-1 (68 amino acid residues) and SDF-1 (72 amino acid residues) (44,
48). After processing at the C terminus, a form that is called
SDF-1 (67 amino acid residues) is generated from stromal cells
(5). SDF-1 adopts a chemokine-like fold consisting of
three antiparallel -strands and an overlying -helix (15). SDF-1 has two N-terminal sites for binding and
activating of its receptor, CXCR4. Receptor activation requires Lys-1
and Pro-2 within the N-terminal region. Amino acid residues 12 to 17 of
the loop region, the RFFESH site, are important for optimal binding.
The SDF-1 RFFESH loop binds to the N-terminal segment of CXCR4, but
inhibition of HIV-1 entry by SDF-1 does not require Lys-1 and Pro-2,
which can activate CXCR4, but does require the RFFESH loop. We prepared
three chimeric envelope vectors, with the first 51 amino acids of SDF-1
(SDF1-51), the full-length protein (SDF1-67), and 43 amino acids (amino
acids 11 to 53) which are weakly homologous to the V3 loop of NL4-3
(HIV-IIIB) (SDF1-V3), and carried out the pseudotyped virus
infection assay.
Among the pseudotyped viruses with these chimeric envelopes,
the NL4-3/SDF1-51 virus showed considerable and reproducible infectivity for U87.CD4.CXCR4 cells. The other chimeric envelope viruses, NL4-3/SDF1-V3 and NL4-3/SDF1-67, could also infect the cells,
but with somewhat lower efficiency. Since the incorporation of the
envelope proteins in the NL4-3/SDF1-V3 and NL4-3/SDF1-67 virions was
comparable to that in the NL4-3/SDF1-51 virus, the lower infectivity of
the former two viruses may be due to a subtle structural difference.
The infectivity of the NL4-3/SDF1-51 virus could be specifically
inhibited by rhSDF-1 as well as anti-human SDF-1 Ab. This suggests
that the interaction between a portion of SDF-1 of gp120 and the
coreceptor CXCR4 is essential for the infectivity of the NL4-3/SDF1-51
virus. Similarly, it was shown that pretreatment with anti-CD4 MAb
Leu3a inhibited the infectivity of the NL4-3/SDF1-51 virus, which
together with the difference in infectivities for U87.CD4.CXCR4
and U87.CXCR4 cells indicated that infection of this virus was CD4
dependent. Based on these findings, we presume the following steps:
first, the NL4-3/SDF1-51 virus binds to CD4 on the target cell;
second, the replaced portion of SDF1-51 in the V3 region of
the envelope becomes exposed by a conformational change of the
NL4-3/SDF1-51 envelope; and third, binding between the portion of
SDF1-51 and CXCR4 leads to infection with the virus.
To summarize, it is suggested that replacement of the V3 region of the NL4-3 envelope with SDF-1 preserves the infectivity of T-tropic HIV-1. We think that our data argue for the above-mentioned hypothesis that the binding between the V3 region and the corresponding coreceptor is important for viral entry, regardless of whether its amino acid sequence is virus derived or exogenous.
It was noted, however, that the infectivity of the pseudotyped
viruses with the SDF1-51 chimeric envelope was much less than that of
the WT virus. The reason for this may be a low binding affinity between
the V3 region of the NL4-3/SDF1-51 chimeric envelope and CXCR4 compared
with that of the NL4-3/WT envelope. With regard to this point, we could
not measure the direct binding of chimeric envelope gp120 to CXCR4 but
showed that chimeric NL4-3/SDF1-51 gp120 as well as NL4-3/WT gp120
could significantly inhibit binding of SDF-1 to CXCR4. Since the
regions of CXCR4 interacting with the HIV envelope are not the same as
the SDF-1-binding epitopes (21, 50), we could not compare
precisely the binding affinity of envelope proteins in this assay.
Nonetheless, it was suggested that this chimeric envelope protein
specifically bound to CXCR4 with such a level of affinity that it
could inhibit the ligand binding.
Accordingly, the following two possibilities should be taken into consideration as to the low infectivity of the NL4-3/SDF1-51 virus. One is somewhat poorer processing of the SDF-1 chimeric envelope in the virion than in the WT. It was noticed that the gp120/gp160 ratio of envelope in the NL4-3/SDF1-51 virus was slightly lower than that of the NL4-3/WT virus. In accordance with this, the amount of soluble gp120 of NL4-3/SDF1-51 in the supernatants was less than that of NL4-3/WT (data not shown). The other possibility is that a subtle structural difference is induced by the insertion of the portion of SDF-1. Kwong et al. and Wyatt et al. reported the crystal structure of the HIV gp120 envelope in complex with CD4 and a neutralizing antibody (27, 55). Based on this analysis, Rizzuto et al. suggested that a conserved gp120 structure adjacent to the V3 loop containing neutralizing epitopes induced by CD4 binding (CD4i epitope) is important for chemokine receptor binding (39). CD4 binding induces a conformational change in gp120 and results in movement of the V2 loop, which probably partially occludes the V3 loop and CD4i epitopes. Although the interaction between the V3 region and the other region of gp120 has not been fully understood, we speculate that the configuration of the portion of SDF-1 as well as the whole structure of the envelope might be changed by the V3 replacement, which could affect Env-coreceptor binding. Additionally, chemokine receptor binding may trigger a further conformational change in the envelope glycoprotein complex that ultimately leads to the fusion of the viral and target cell membrane. It is believed that some of these changes include exposure of the ectodomain of the gp41 transmembrane envelope glycoprotein (7, 19, 51). It is possible that the replacement of the V3 region with a certain exogenous peptide may affect this additional conformational change and also the noncovalent association of gp120 and gp41 subunits in the trimeric complex.
The systemic delivery of genes will open new applications for gene therapy (43). The development of retrovirus vectors is being considered for this purpose. Mammalian retrovirus vectors commonly used for gene transfer are classified on the basis of their host range as either ecotropic, which only infect murine cells, or amphotropic, which infect both murine and nonmurine cells. The host range of retrovirus vectors has become broad by using vesicular stomatitis virus glycoprotein as an envelope protein (6, 56). On the other hand, the modification of the retroviral envelope gene is becoming a promising strategy for targeting retroviral vectors (43). In the previous studies, the envelope genes of Moloney murine leukemic virus, spleen necrosis virus, and avian leukemia virus A were replaced with some fusion gene such as a single chain antibody and a ligand against a target receptor. For example, Kasahara et al. reported a tissue-specific targeting strategy by introducing the polypeptide hormone erythropoietin into the ecotropic Moloney murine leukemic virus envelope (26). There has been a report on retroviral targeting using a WT envelope of HIV (31) but not using the HIV chimeric envelope by replacing the V3 region with a certain exogenous peptide segment which binds to corresponding coreceptors. In the present study, we engineered pseudotyped viruses with chimeric envelopes in which the V3 region was replaced with a ligand for CXCR4, SDF-1, and demonstrated the infectivity of these viruses for CD4+ and CXCR4+ cells.
In addition to the replacement with SDF-1, we investigated whether the
replacement of the V3 region with another ligand besides CXCR4 endowed
the virus with infectivity for its cognate receptor-expressing cells.
We showed that the pseudotyped virus with a chimeric T-tropic HIV-1 envelope in which the V3 region was replaced with MIP-1 could
infect CCR5+ cells. Further studies are required to
generalize the concept that replacing the V3 region with a certain
exogenous peptide segment which can bind to the cognate receptor can
change and select the target cells. Our new strategy of replacement of
the V3 region with other ligand molecules may enable development of tissue- or cell-specific targeting of HIV-based vectors.
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ACKNOWLEDGMENTS |
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This study was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.
The following reagents were obtained through the AIDS Research
and Reference Reagent Program, Division of AIDS, NIAID, NIH: U87MG from Bruce Chesebro; U87.CD4, U87.CD4.CXCR4, and U87.CD4.CCR5 from HongKui Deng and Dan R. Littman;
pNL4-3.Luc.ER from Nathaniel Landau; pYU2
from Beatrice and George Shaw; and hybridoma 902 (anti-gp120)
from Bruce Chesebro. We thank K. Fukunaga for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3153. Fax: 81-75-751-4963. E-mail: thori{at}kuhp.kyoto-u.ac.jp.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, May 2001, p. 4258-4267, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4258-4267.2001
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