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Journal of Virology, May 1999, p. 4062-4073, Vol. 73, No. 5
Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
191041; IRIBHN, Université Libre
de Bruxelles, Campus Erasme, B-1070 Brussels,
Belgium2; and Department of
Medicine, Northwestern University Medical School, Chicago, Illinois
606113
Received 26 October 1998/Accepted 1 February 1999
With rare exceptions, all simian immunodeficiency virus (SIV)
strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a
primary receptor to infect CD4-negative cells such as rhesus brain
capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low
levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely
dissecting interactions between Env and CCR5 which may otherwise be
masked due to the stabilization of these contacts by Env-CD4 binding.
Based on the ability of SIV Env to bind to and mediate infection of
cells expressing CCR5 chimeras and mutants, we identified the N
terminus of CCR5 as a critical domain for direct Env binding and for
supporting CD4-independent virus infection. However, the activity of
N-terminal domain CCR5 mutants could be rescued by the presence of CD4,
indicating that other regions of CCR5 are important for post-binding
events that lead to viral entry. Rhesus CCR5 supported CD4-independent
infection and direct Env binding more efficiently than did human CCR5
due to a single amino acid difference in the N terminus. Interestingly,
uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less
efficiently than did monomeric gp120. Finally, several mutations
present in chronically infected monkey populations are shown to
decrease the ability of CCR5 to serve as a primary viral receptor for
the SIV isolates examined.
Simian immunodeficiency virus (SIV)
is frequently used as a model system for human immunodeficiency virus
(HIV) infection because it causes a syndrome very similar to human AIDS
in several Asian macaque species. Like HIV, SIV uses chemokine
receptors in conjunction with CD4 for viral entry (2, 6, 11, 13, 17, 21, 22, 24, 31, 45). With rare exceptions, all SIV strains
identified to date use CCR5 for entry regardless of cellular tropism
(11, 24, 37, 45, 47). CXCR4 is used as a coreceptor only
very rarely by SIV isolates, although rhesus CXCR4 is functional for X4
HIV-1 strains and several recently described pathogenic SHIVs use CXCR4
as a coreceptor (24, 33, 36, 44, 47). Thus, SIV tropism does
not follow the R5/X4 designations given to HIV-1 (5).
Although both T- and M-tropic SIVs use CCR5, they may interact with
CCR5 differently, and there is evidence to suggest that only M-tropic
Env proteins signal through CCR5 (24, 62). SIV typically
displays a broader coreceptor use pattern than HIV-1, with most SIV
strains using GPR15/BOB and/or STRL33/Bonzo as coreceptors in addition
to CCR5 (18, 25, 29, 47). Use of CCR8, ChemR23, GPR1, CCR2b,
and APJ as coreceptors by SIV is more restricted (12, 25, 26, 29, 47, 54, 56).
Several lines of evidence support the hypothesis that chemokine
receptors actually represent the primordial primate lentivirus receptors. A number of HIV-2 strains, including some primary isolates, are able to infect cells which express CXCR4 or CCR5 but lack CD4
(10, 13a, 14, 28, 52). Laboratory-adapted isolates of feline
immunodeficiency virus also use CXCR4 as a receptor in the absence of
CD4 (51, 64). Furthermore, a significant number of SIV
isolates can use CCR5 as a primary receptor and can infect
CCR5-positive, CD4-negative primary cells such as rhesus brain
capillary endothelial cells (27). In keeping with their decreased dependence on CD4 for infection, HIV-2 and SIV envelope (Env)
proteins have a lower affinity for CD4 than does HIV-1 Env and are
more resistant to inhibition by soluble CD4 (sCD4) (8, 57,
58). In fact, low to moderate levels of sCD4 enhance both syncytium formation and infection by a variety of HIV-2 and SIVagm strains (4, 14, 63).
We have extended our studies of CD4-independent infection by SIV
strains to better understand the factors that govern this entry process
and to identify regions of CCR5 important for SIV Env binding in the
absence of CD4. We found that CD4-independent SIV infection
occurred over a broad range of CCR5 expression levels, including levels
barely detectable by fluorescence-activated cell sorting (FACS). The
addition of CD4 in soluble or membrane-bound form enhanced infection
for most CD4-independent viruses, suggesting that CD4 is required
for optimal entry efficiency. While both human CCR5 (Hu CCR5) and
rhesus CCR5 (Rh CCR5) functioned equally well as coreceptors in the
presence of CD4, in its absence Rh CCR5 supported infection more
efficiently and for a larger number of virus strains. The increase in
CD4-independent infection through Rh CCR5 mapped to the Asp at
position 13 in the N terminus and correlated with direct Env binding.
Several mutations in the CCR5 N-terminal domain were identified that
blocked virus infection in the absence (but not in the presence) of
CD4, suggesting that the N-terminal domain of CCR5 is more important
for Env binding than for triggering the conformational changes that
lead to membrane fusion. Interestingly, we found that the ability of
uncleaved Env to bind both CCR5 and CD4 was markedly reduced relative
to gp120. Finally, several mutations in CCR5 which exist as
polymorphisms in chronically infected monkey populations were
identified as limiting CD4-independent infection for the SIV strains tested.
Reporter virus infections.
All SIV Env proteins, CD4, and
CCR5 constructs were cloned into the pcDNA3 or pcDNA3.1 vectors
(Invitrogen). Luciferase reporter viruses were produced by
cotransfecting 293T cells with the env gene of interest
under the control of the cytomegalovirus promoter and with the
NL-Luc-R Flow cytometry.
293T cells were lifted with PBS, transferred
to FACS tubes, and washed once with FACS staining buffer (PBS plus 2%
FCS). The cells were incubated with the anti-CCR5 monoclonal antibody
CTC5 (Protein Design Labs, Mountain View, Calif.) at 10 µg/ml for 30 min at 4°C. They were then washed twice with FACS staining buffer and
incubated with phycoerythrin-conjugated horse anti-mouse antibody (Vector Laboratories) at 1 µg/ml for 30 min at 4°C in the dark. They were washed twice with FACS staining buffer and analyzed with a
Becton Dickinson FACScanner immediately following staining. Antibody
quantification was performed with a QuickCal kit (Sigma) and antibody
2D7 to CCR5 (Pharmigen). Hu CCR5 and RhCCR5 expression levels on
transfected 293T cells were assessed by FACS with two monoclonal
antibodies: 455519, a generous gift from R&D Systems, and mCR35.4,
kindly provided by Protein Design Labs. These antibodies bind to
regions in CCR5 that are identical between Rh CCR5 and Hu CCR5
(43).
Direct Env binding assay.
Secreted Env for the binding assay
was produced by infecting 293T cells with the recombinant vaccinia
virus vTF1.1 (which expresses T7 polymerase [1]) for
1 h in DMEM containing 2.5% FCS and then transfecting the cells
in DMEM-10% FCS with gp120 or gp140 constructs in pcDNA3 under the
control of the T7 promoter. At 4 h posttransfection, the cells
were washed twice with PBS and placed in serum-free DMEM. Approximately
24 h posttransfection, the medium was harvested and cleared by
centrifugation, and vaccinia virus inactivated by the addition of
Triton X-100 to a final concentration of 0.1% (wt/vol). Env was
purified from the medium by lectin chromatography (wheat germ
agglutinin coupled to agarose beads [Vector Laboratories]) and eluted
with 0.5 M N-acetyl-D-glucosamine (Sigma) in
PBS. Two buffer exchanges were performed in a 50-ml Amicon stir cell
with a YM30 membrane by using morpholineethanesulfonic acid (MES)
buffer, (pH 7.0). For the Env binding assay, 293T cells were
transfected with 3 µg of CCR5 construct or vector DNA per 24-well
plate as indicated and 24 h later were incubated in DMEM-10% FCS
containing 4 µg of soluble Env protein with or without 100 nM sCD4.
The cells were incubated for 2 h at 37°C, washed once with
1 ml of serum-free DMEM, lysed in 0.5% Triton X-100 in PBS containing
protease inhibitors, and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis followed by Western blotting
with an anti-gp120 monoclonal antibody and enhanced chemiluminescence
(Amersham) detection. Alternatively, Env was quantified in cell lysates
by an antibody capture enzyme-linked immunosorbent assay (ELISA). ELISA
plates were prepared by an overnight incubation at 4°C of 96-well
plates with capture buffer (20 mM Tris, 0.1 M NaCl, 0.05%
NaN3 [pH 8.0]) containing three monoclonal antibodies
generated against SIVmacCP-MAC gp140 and mapped to the SU subunit,
i.e., 36D5, 115G2, and 168B2. The plates were then washed with a plate
washer and ELISA wash solution (0.05% Tween 20 in PBS), blocked for 1 to 2 h at room temperature in BLOTTO (5% nonfat dry milk, 0.1%
Tween 20, and 0.1% NaN3 in PBS), washed, and incubated
with 100 µl of lysate/well for 2.5 h at room temperature. After
being washed, the plates were incubated with a 1:500 dilution of serum
from a macaque infected with SIVmac251 (from the AIDS Research
and Reference Reagent Program) for 1.5 h at room temperature,
washed, incubated with rabbit anti-monkey horseradish
peroxidase-conjugated secondary antibody (Sigma) at a 1:4,000 dilution
for 30 min, and given a final wash.
2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) tablets
(Pierce) were dissolved in substrate buffer (0.1 M sodium acetate)
supplemented with 0.05% H2O2, the solution was
added to the plate, and the absorbance was measured at 405 nm.
AOP-RANTES was a generous gift of Timothy Wells and Amanda Proudfoot
(Serono Pharmaceutical Research Institute) (59), and 12G5
(28) was kindly provided by Jim Hoxie.
CCR5 constructs.
The majority of the chimeras and N-terminal
truncations have been described previously (20, 55). The
additional chimeras 55(25)5 and 55(52)5 were constructed by replacing a
ClaI-EcoRI cassette in wild-type CCR5 with a
fragment generated by PCR as described previously for N-terminal domain
chimeras (55). The junction between CCR5 and CCR2 sequences
corresponded to the conserved cysteine (C178) that is presumably
involved in a disulfide bond with the first
extracellular loop. The mutant cassettes were transferred as
needed in other backgrounds, such as CCR2b or a (52)222 chimera. All
final constructs were verified by sequencing before being used in
transfection experiments. The N13D reciprocal point mutants were
constructed with a Quickchange mutagenesis kit (Stratagene). The
cloning of vervet CCR5 is described in detail elsewhere
(40). Briefly, total cellular DNA was isolated from
peripheral blood by using the Puregene DNA Isolation kit (Gentra
Systems, Inc.) and amplified with primers in the flanking untranslated
regions of the Hu CCR5 gene 5' (CCR5A;
5'-GGAGGGCAACTAAATACATTCTAGG-3') and 3'
(CCR5B; 5'-GACTGGTCACCAGCCCACTTGAGTCC-3'). PCR-products were re-solved on a 1.0% agarose gel by electrophoresis, and the appropriately sized
band was excised, extracted by the QIAquick gel extraction method, and
inserted into the pCR2.1 vector (Invitrogen) by the TA method. The
inserts were sequenced in both orientations by using ABI dye-terminator chemistry.
Effect of CCR5 expression levels on CD4-independent infection.
Previous studies have shown that multiple strains of SIV can use CCR5
as a receptor for viral infection in the absence of CD4 in both primary
and transiently transfected cells (27). Since surface levels
of CD4 and chemokine receptors can influence the efficiency of virus
entry (38, 50, 54), we performed linked pseudotyped virus
infection and FACS experiments on cells expressing decreasing amounts
of Rh CCR5 in the presence and absence of CD4. 293T cells were
transfected with variable amounts of Rh CCR5 plasmid and a constant
amount of CD4 or control vector plasmid. The next day, cells were
either infected with luciferase reporter virions bearing the SIV/17E-Fr
Env protein or analyzed by FACS for CCR5 and CD4 expression. Virions
containing the amphotropic murine leukemia virus Env protein
(which does not use either CD4 or CCR5 for infection) served as
positive controls.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Functional Dissection of CCR5 Coreceptor Function through the Use
of CD4-Independent Simian Immunodeficiency Virus Strains
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-E backbone plasmid in the presence
of 7.5 mM sodium butyrate (9, 15). Supernatant containing
virus was harvested 2 days posttransfection and used to infect 48-well
plates of 293T target cells transfected with 1 µg of CD4 and 0.5 µg
of CCR5 construct or vector as indicated. Infections were performed in
Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL) with 10% fetal
calf serum (FCS; Hyclone) and 1% penicillin-streptomycin (Gibco-BRL)
and supplemented with 8 µg of DEAE-dextran (Sigma) per ml, except for
infections with the alanine scan mutants, which were done in the
absence of DEAE. Cells were lysed in 0.5% Triton X-100 in
phosphate-buffered saline (PBS) 3 days postinfection, and luciferase
activity in the cell lysate was quantified in a luminometer. sCD4 was
kindly provided by Jim Hoxie, University of Pennsylvania.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (37K):
[in a new window]
FIG. 1.
Effect of CCR5 expression levels on pseudotyped virus
infection. 293T cells were transiently transfected with from 0.5 to
0.0005 µg of Rh CCR5 plasmid per 48-well and 1 µg of CD4 or vector
plasmid as indicated. The next day, cells were either evaluated for
CCR5 expression by FACS (a) or infected with luciferase
reporter viruses pseudotyped with the SIV/17E-Fr Env protein (b) as
described in Materials and Methods. Data from one representative
experiment of six are presented.
Rh CCR5 is a more efficient primary receptor for SIV than is Hu
CCR5.
Hu CCR5 and Rh CCR5 function equally well as SIV coreceptors
in the presence of CD4 (11, 24, 45). To determine if this is
the case for CD4-independent virus infection, we expressed Hu CCR5 or
Rh CCR5 in 293T cells with or without CD4 and infected them with a
panel of luciferase reporter viruses bearing different SIVmac or SIVsm
Env proteins (Fig. 2). Rh CCR5 and Hu
CCR5 were expressed at nearly identical levels as judged by FACS with
two different CCR5 monoclonal antibodies (data not shown). To compare data from different experiments, relative light unit values are presented as the percentage of the luciferase signal obtained with Rh
CCR5 in the presence of CD4. We found that Hu CCR5 and Rh CCR5
functioned equally well as SIV coreceptors in the presence of CD4 but
that Rh CCR5 functioned more efficiently in its absence. In fact, Envs
derived from SIVmac1A11 and SIVsm
B670 clone 12 mediated the
infection of cells expressing Rh CCR5 equally well in the presence and
absence of CD4. Interestingly, SIVmacCP-MAC Env displayed complete CD4
dependence with Hu CCR5 while efficient CD4-independent infection was
observed with Rh CCR5. Only the SIVmac239 Env protein required CD4 for
infection via both Hu and Rh CCR5. These findings indicate that
CD4-independent interactions between Rh CCR5 and SIV Env are more
efficient than those with Hu CCR5.
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Aspartic acid 13 is responsible for the increased efficiency of CD4-independent infection through Rh CCR5. Hu CCR5 and Rh CCR5 are highly homologous, with only three amino acid changes in the extracellular domains (I9T, N13D, and R171K) (Fig. 3a). The N13D change was shown by Martin et al. to be important for CD4-independent binding of SIVmac239 gp120 to CCR5 (46). To determine whether the increased ability of Rh CCR5 to support CD4-independent infection by SIVmacCP-MAC was associated with this change, we generated reciprocal point mutations at position 13 in Hu CCR5 and Rh CCR5 and performed luciferase virus infections, in the presence and absence of CD4 (Fig. 3b). In the presence of CD4, all receptor constructs served equally well as virus coreceptors. However in the absence of CD4, Hu CCR5(N13D) and Rh CCR5 functioned as efficient primary receptors whereas Hu CCR5 and Rh CCR5(D13N) did not. Thus, consistent with its proven role in the direct binding of SIVmac239 Env to CCR5 (46), Asp13 plays an important role in supporting CD4-independent infection by some SIV strains.
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Binding of oligomeric, non-cleaved Env gp140 to CCR5. The binding assays described above were performed with monomeric gp120. Since SIV Env is oligomeric, we sought to determine whether the Env quaternary structure affected CCR5 binding. To investigate this, we generated a soluble SIV/17E-Fr Env containing all of the gp120 and the gp41 ectodomain. The bulk of this protein is recovered in a noncleaved, oligomeric form (Fig. 5 and data not shown). The remainder is cleaved to generate gp120 and gp41 ectodomain subunits which exist either as monomers or unstable oligomers. This protein mixture was tested for the ability to bind to Hu CCR5 and Rh CCR5, as well as the N13D and D13N reciprocal mutants, in the presence and absence of sCD4. Both gp140 and gp120 bound directly to Rh CCR5 and Hu CCR5(N13D) in the absence of sCD4 (Fig. 5). However, gp120 bound far more efficiently to CCR5 than did uncleaved gp140, as shown by the inversion of the gp140/gp120 ratio in the Env-bound to CCR5 relative to the input mixture. It is unclear whether the bound, uncleaved gp140 truly interacted with CCR5 or was bound as a consequence of forming mixed oligomers with gp120. Addition of sCD4 enhanced the binding of gp120 and gp140 to all of the CCR5 constructs, with gp120 binding again exceeding that of gp140. The gp120 also bound membrane-anchored CD4 more efficiently than did gp140, which could partially account for differences in gp120 and gp140 binding to CCR5 in the presence of sCD4 (Fig. 5). Taken together, our results indicate that uncleaved, oligomeric SIV/17E Fr Env interacts much less efficiently than does gp120 with both CD4 and CCR5.
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CCR5 determinants for Env binding and conformational change
induction.
In the absence of CD4, CCR5 must bind Env and trigger
the conformational changes that lead to membrane fusion. In the
presence of CD4, high-affinity binding of Env to CCR5 may be less
important since CD4 may supply this function in trans. If
so, mutations in CCR5 which primarily affect Env binding should have a
greater effect on CD4-independent virus infection. To examine this, we selected two CD4-independent SIV Env proteins, SIV/17E-Fr and SIVsmB670 clone 3, for infection studies with receptor chimeras generated between Hu CCR5 and CCR2b in the presence or absence of
membrane-anchored CD4 or sCD4. As always with analyses of chimeric receptors, greater emphasis should be placed on chimeras which function
rather than those which do not, since loss of function could be due to
specific alteration of an important region or to nonspecific,
conformational effects.
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12
mutant. Clone 3 was able to use the 4 mutant at wild-type levels,
showed decreased ability to use 8, and did not use 12. These
findings underscore the critical importance of the N terminus for
direct binding of Env to Hu CCR5 and suggest that Env binding
determinants lie within the first 12 residues of CCR5.
Effects of sCD4 on virus infection.
The addition of sCD4
enhances SIV infection under some circumstances (4, 63). We
reasoned that conformational changes induced in Env by sCD4 might
rescue the ability of some receptor chimeras to serve as primary viral
receptors for infection, possibly by increasing the efficiency of the
interaction with CCR5. Indeed, we found that sCD4 enhanced the ability
of 5255, 5252 (chimeras which contain the CCR5 N terminus but do not
function CD4 independently), and 5552 to serve as primary receptors for
one or both of the viruses tested (Fig. 6a and b). The addition of sCD4
did not affect the use of chimeras lacking the CCR5 N-terminus or
ECL2, consistent with their critical roles in virus infection as
described above. However, sCD4 could rescue infection through
4 and 8 for SIV/17E-Fr but not through 12 or 16 for either
SIV/17E-Fr or SIVsmB670 clone 3. These results suggest that sCD4
enhances the affinity of Env for the N terminus of CCR5 and
that the N-terminal domain supporting direct Env binding is
disrupted in the 12 and 16 mutants. Because sCD4 enhancement
of SIV infection has been demonstrated with a variety of CCR5-negative
cell lines, we tested whether sCD4 could allow the use of
GPR15 or STRL33 as primary receptors. While GPR15 and STRL33
could serve as efficient coreceptors for infection by
SIV/17E-Fr and SIVsmB670 clone 3, they did not serve as
primary receptors even in the presence of sCD4 concentrations as high
as 400 nM (data not shown). In addition, direct Env binding to GPR15
and STRL33 could not be detected in the presence of 100 nM sCD4 (data
not shown).
Residues in the N terminus and ECL2 are critical for primary
receptor function.
To further map the regions important for
CD4-independent coreceptor function, we examined CCR5 point mutants in
which the charged residues in the extracellular domains were
individually changed to alanine (20). In the presence of
CD4, all of these mutants served as efficient coreceptors (Fig.
7a). However, in the absence of CD4, D2A
and, in particular, D11A showed decreased activity as primary receptors
(Fig. 7b). Binding of SIV/17E-Fr gp120 was not detected to D2A or
D11A with or without sCD4 (data not shown). These results are
consistent with the fact that coreceptor function declined beginning
with 4 for SIV-17E/Fr, which is sensitive to the D2A mutation (Fig.
6a), and that both viruses tested lost CD4-independent and sCD4-rescued
infection between 8 and 12 (Fig. 6a and b). Since these point
mutations were constructed in a background of Hu CCR5 (which
lacks Asp13), the effects of D2A and D11A may be rendered more
apparent. Furthermore, a point mutation which changes the Ser at
position 180 in ECL2 of Hu and Rh CCR5 to a Pro affected
CD4-independent but not CD4-dependent viral infection (Fig. 7). Since
this mutant functions at wild-type levels in the presence of CD4, it
suggests that ECL2 contributes in some way to the Env binding site.
Interestingly, the S180P mutation is present in the sooty mangabey
population (10). In addition, a CCR5 clone isolated from an
African green monkey (AGM) (vervet) containing the D13N mutation found
in the AGM population (39) was evaluated (40).
Vervet CCR5 failed to serve as a primary receptor for SIV/17E-Fr and
allowed only inefficient infection by SIVsmB670 clone 3, although it
functioned as an efficient coreceptor in the presence of CD4 for both
viruses (Fig. 7). These results are potentially interesting since sooty
mangabeys and African green monkeys become chronically infected with
SIV but do not progress to simian AIDS (3, 7, 49).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sequential interactions with CD4 and a coreceptor
ultimately result in conformational changes in HIV-1 Env that
lead to virus-mediated membrane fusion. In contrast to HIV-1, a number
of laboratory-adapted and primary HIV-2 and SIV strains have been
identified that short-circuit this normal entry process and
require only CCR5 or CXCR4 to infect cells (10, 14, 27,
28). CD4-independent infection may influence viral pathogenesis
in several ways. First, infection of CD4-negative, CCR5-positive cells
such as brain capillary endothelial cells may play a role in
neurotropism (27); B cells and cytotoxic T lymphocytes
represent other CCR5+ CD4 cell types which
might be infected by CD4-independent viruses in vivo (34,
66). Second, since multimeric CD4 interactions are thought to be
required for HIV-1 entry (42, 48), viruses that can enter
independently of CD4 may be able to broaden their cellular tropism by
more efficiently infecting cells that express low levels of CD4.
Third, shed gp120 may affect CD4-negative cell types which express
chemokine receptors by mechanisms such as Env signaling and receptor
down-regulation (16, 62). In addition, these viruses
represent a means of examining the interaction between Env and
chemokine receptors in the absence of CD4, potentially allowing the
identification of determinants in both Env and CCR5 necessary to
support high-affinity binding. In this study, we extended our previous
observations that many SIV strains can infect CCR5+
CD4 cells, explored the consequences of receptor
expression levels on CD4-independent infection, and used
CD4-independent virus strains to identify domains in CCR5 important for
supporting Env binding.
Coreceptor expression levels can influence the efficiency of virus entry (38, 50, 54). However, we found that CD4-dependent and -independent SIV infection pathways exhibited a similar dependence on CCR5 expression levels and that CD4-independent infection occurred even at CCR5 levels barely detectable by FACS (Fig. 1). Therefore, gross overexpression of CCR5 is not required for infection in the absence of CD4, at least not for infection by SIV/17E-Fr. This finding is consistent with our previous work, which showed that levels of CCR5 on brain capillary endothelial cells are sufficient to support CD4-independent virus entry (27).
In our previous study, we demonstrated that some SIV strains can infect cells that transiently express Hu CCR5 (27). Furthermore, we and others have found that Rh CCR5 and Hu CCR5 function equally well as SIV coreceptors in the presence of CD4 (11, 24, 45). However, we found that Rh CCR5 supported CD4-independent infection more efficiently and for a larger number of virus strains than did Hu CCR5 (Fig. 2). In fact, with the exception of SIVmac239, all SIV strains tested exhibited some degree of CD4 independence with cells transiently expressing Rh CCR5. We mapped the enhanced infection through Rh CCR5 in the absence of CD4 to residue 13 of the CCR5 N-terminal domain. These findings are consistent with a previous study by Martin et al. which showed that SIVmac239 gp120 can bind directly to Rh but not Hu CCR5 and that residue 13 was responsible for this phenotype (46). In summary, CD4 independence by SIV strains is more widespread than was first suspected due to the more efficient use of Rh CCR5 than Hu CCR5, and this difference maps to the CCR5 N terminus.
For most HIV-1 strains, CD4 binding to Env induces conformational changes in gp120 that confer the ability to interact with the coreceptor (41, 61, 65). These changes may include increased exposure of a highly conserved region in gp120 thought to play a critical role in coreceptor binding (53). Subsequent Env-coreceptor interactions are believed to trigger additional conformational changes in Env that lead to membrane fusion. It is likely that Env remains bound to CD4 when it associates with the chemokine receptor (41). Consequently, direct, high-affinity binding of gp120 to CCR5 might not be required in the presence of CD4 which could provide this function in trans. If true, mutations in CCR5 which predominantly affect Env binding should have an enhanced effect in the absence of CD4. Mutations which are defective in both the presence and absence of CD4 may not induce conformational changes in Env required for membrane fusion or may disrupt CCR5 binding more dramatically (46).
We found that in the presence of CD4, numerous single-amino-acid substitutions as well as truncations of the N-terminal domain of CCR5 were well tolerated. However, in the absence of CD4, many of these mutations abolished or strongly suppressed virus infection. Several previous studies have implicated the N terminus of CCR5, including residues D2 and D11, as important for interaction with HIV-1 Env and, in the case of D11, with SIVmac239 (20, 23, 30). Based on our findings with the N-terminal domain mutants and the ability of gp120 derived from SIV/17E-Fr to efficiently bind to Rh CCR5 in the absence of CD4, we conclude that Env proteins derived from CD4-independent SIV strains exist in a partially triggered conformation that allows them to interact directly with CCR5, with the N-terminal domain playing an important role. The enhancement of Env binding in the presence of sCD4 is probably due to conformational changes in Env that enable it to bind to CCR5 with even higher affinity.
The finding that membrane-anchored CD4 can rescue the activity of N-terminal CCR5 mutants, presumably by providing a high-affinity binding site in trans, but has no effect on the activity of chimeras lacking CCR5 ECL2 suggests that ECL2 may play a role in triggering membrane fusion subsequent to Env binding, although ECL2 could also affect Env-CCR5 binding. In fact, the S180P mutation in ECL2 affected CD4-independent but not CD4-dependent virus infection. In an effort to identify a CCR5 mutant which was able to support Env binding but not virus infection, we examined the ability of SIV/17E-Fr gp120 to bind 5525. Surprisingly, not only did Env fail to bind to 5525, but also binding could not be detected to 5552, a chimera which can serve as a primary receptor for infection even in the presence of sCD4. These results suggest that the Env binding site on CCR5 involves the N terminus but that binding is highly dependent on the overall conformation of CCR5 and other domains are probably involved.
It is important to note that we used monomeric gp120 to measure the ability of Env proteins to bind wild-type and mutant forms of CCR5. As a consequence, low-affinity gp120-CCR5 interactions may not have been detected by our assay. Since Env exists as an oligomer in the viral membrane, multimeric Env-CCR5 interactions could stabilize otherwise weak gp120-CCR5 binding events by increasing the avidity of the interaction. We attempted to address this point by using a secreted oligomeric form of SIV Env, gp140, that exists largely in an uncleaved state. Surprisingly, oligomeric gp140 interacted with CD4 and CCR5 much less efficiently than did gp120, suggesting that the chemokine receptor binding site in gp120 is affected by Env cleavage. This may have implications for vaccine development, since uncleaved Env proteins may not elicit neutralizing, broadly cross-reactive antibodies to the chemokine receptor binding site (60). If uncleaved forms of Env are less able to interact with coreceptors, this may also prevent nonproductive gp160-coreceptor interactions in the biosynthetic pathway.
For many years, it has been recognized that AGMs and sooty mangabey monkeys are chronically infected with SIV in the wild and, while these viruses cause no disease in their natural host, they produce an immunodeficiency syndrome when transmitted to rhesus or pigtailed macaques (3, 7, 19, 32, 35, 49). It is interesting that the AGM population harbors a CCR5 allele which contains the D13N mutation, which plays an important role in CD4-independent SIV infection (39). Since SIV is thought to have existed in the AGM population for many generations, it has been hypothesized that mutations in CCR5 are likely to occur in regions of CCR5 which limit SIV pathogenicity (39). Interestingly, a separate study found that in four of four sooty mangabeys examined, CCR5 contained the S180P mutation, which we also found to limit CD4-independent virus infection (10). It is tempting to hypothesize that in AGMs and sooty mangabey monkeys, host adaptation to limit viral pathogenesis includes the limitation of CD4-independent infection, and it will be important to evaluate primary AGM and sooty mangabey isolates for CD4 independence with AGM and sooty mangabey CCR5.
In conclusion, we found that multiple SIV strains can utilize Rh CCR5
independently of CD4 for virus infection, that some virus strains can
distinguish between CCR5 molecules that differ by only one amino acid
in the N terminus, and that the N-terminal domain of CCR5 plays an
important role in Env binding. In future studies, the CD4-independent
SIV phenotype can also be used to identify Env determinants that are
important for receptor interactions. By using closely related pairs of
virus strains, it will be possible to identify residues that are
involved in direct CCR5 interactions and in distinguishing Rh from Hu
CCR5. It will also be interesting to determine what role,
if any, CD4 independence plays in vivo through further
examination of SIV infection in AGMs and sooty mangabeys, evaluation of
the extent of infection of CCR5+ CD4 cells
such as B cells and cytotoxic T lymphocytes in vivo, and infection of
rhesus macaques with closely related SIV strains that differ only in
their dependence upon CD4.
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ACKNOWLEDGMENTS |
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We thank Trevor Hoffman, Joseph Rucker, and Benhur Lee for helpful discussions and advice. SIVmac251 antiserum was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. CCR5 monoclonal antibodies were generously provided by Protein Design Labs and R&D Systems. sCD4 and 12G5 were a kind gift of Jim "Arlo Guthrie" Hoxie.
A.L.E. was supported by MSTP grant 2T32GM07170. M.P. was supported by the Agence Nationale de Recherche sur le SIDA, an Action de Recherche Concertée of the Communauté Française de Belgique, and BIOMED EC grant BMH4-CT98-2343. C.B. is Aspirant of the Belgian Fonds National de la Recherche Scientifique. The work was supported by grant R01-40880 to R.W.D.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, University of Pennsylvania, 806 Abramson, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 898-0890. Fax: (215) 573-2883. E-mail: doms{at}mail.med.upenn.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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