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J Virol, March 1998, p. 1918-1924, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multiple Residues Contribute to the Inability of
Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human
Immunodeficiency Virus Type 1 Isolates
Ted M.
Ross,1
Paul D.
Bieniasz,1,2 and
Bryan R.
Cullen1,2,*
Department of
Genetics1 and
Howard Hughes Medical
Institute,2 Duke University Medical Center,
Durham, North Carolina 27710
Received 26 August 1997/Accepted 14 November 1997
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ABSTRACT |
Infection of CD4-positive cells by human immunodeficiency virus
type 1 (HIV-1) requires functional interaction of the viral envelope
protein with a coreceptor belonging to the chemokine receptor family of
seven-membrane-spanning receptors. For the majority of
macrophage-tropic HIV-1 isolates, the physiologically relevant
coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine
homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection,
chimeric hCCR-5/mCCR-5 molecules containing single extracellular
domains derived from hCCR-5 are effective coreceptors for certain
macrophage-tropic HIV-1 isolates. Here, we have sought to identify
residues in hCCR-5 critical for HIV-1 infection by substitution of
mCCR-5-derived residues into the context of functional chimeric
hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate
that residues 7, 13, and 15 in the first extracellular domain and
residue 180 in the third extracellular domain of CCR-5 are important
for HIV-1 envelope-mediated membrane fusion. Of interest, certain
substitutions, for example, at residues 184 and 185 in the third
extracellular domain, have no phenotype when introduced individually
but strongly inhibit hCCR-5 coreceptor function when present together.
We hypothesize that these changes, which do not preclude chemokine
receptor function, may inhibit a conformational transition in hCCR-5
that contributes to HIV-1 infection. Finally, we report that
substitution of glycine for valine at residue 5 in CCR-5 can
significantly enhance the level of envelope-dependent cell fusion by
expressing cells. The diversity of the mutant phenotypes observed in
this mutational analysis, combined with their wide distribution across
the extracellular regions of CCR-5, emphasizes the complexity of the
interaction between HIV-1 envelope and coreceptor.
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INTRODUCTION |
Infection of cells by human
immunodeficiency virus type 1 (HIV-1) requires interaction of the viral
envelope protein with not only CD4 but also a second cell surface
molecule, termed a coreceptor (reviewed in reference
19). Coreceptor usage varies significantly among
different HIV-1 isolates, although all known coreceptors are members of
the G-protein-coupled chemokine receptor family of
seven-membrane-spanning receptors. The primary coreceptor used by
non-syncytium-inducing, macrophage-tropic (M-tropic) HIV-1 isolates,
which constitute the majority of primary isolates, is CCR-5 (1, 6,
8, 12, 27). In contrast, syncytium-inducing, T-cell-line-adapted
(T-tropic) HIV-1 isolates predominantly use CXCR-4 as a coreceptor
(13). Other chemokine receptors utilized by a small
percentage of generally dualtropic HIV-1 isolates include CCR-2b and
CCR-3 (6, 11). The importance of two orphan chemokine receptors, termed Bonzo/STRL33 and BOB/GPR15, in infection by HIV-1
remains to be established, although these proteins were recently shown
to serve as coreceptors for several simian immunodeficiency virus
and HIV-2 isolates (2, 9). The critical importance of CCR-5
for infection by primary, M-tropic HIV-1 isolates, however, has been
highlighted by the finding that a small percentage of humans lack a
functional CCR-5 gene and as a result appear highly, although not completely, resistant to infection by HIV-1 (17, 22). Importantly, primary T cells derived from such individuals are refractory to infection by M-tropic HIV-1 isolates in vitro (17, 22, 27), thus demonstrating that CCR-5 is the
physiologically relevant coreceptor for the majority of primary
isolates.
At present, relatively little is known about how the viral envelope and
coreceptor interact, although it appears clear that interaction is
dependent upon a prior conformational shift induced by binding of the
envelope gp120 subunit to CD4 (24, 26). This in turn is
believed to lead to the formation of a ternary complex, consisting of
gp120, coreceptor, and CD4, on the surface of the target cell (15,
24, 26). It is unknown how this protein complex then induces the
fusion of the viral and host cell membranes, although the envelope gp41
subunit is believed to play a critical role at this stage.
An important unresolved question is the identity of the amino acid
residues in gp120 and the coreceptor that interact during infection.
However, it is well established that HIV-1 tropism, and hence
coreceptor usage, is largely controlled by a small number of residues
located in the envelope V3 loop (6, 14, 23, 25). Efforts to
identify residues in the CCR-5 coreceptor involved in mediating
infection have thus far largely focused on the functional analysis of
chimeric receptors generated with human CCR-5 (hCCR-5) and a chemokine
receptor lacking coreceptor function, such as the murine CCR-5 homolog
(mCCR-5) (3, 5, 20, 21). These studies have led to three
major conclusions. Firstly, the residues in hCCR-5 involved in
mediating HIV-1 infection are diffuse, being located on at least three
of the four extracellular domains of CCR-5. Secondly, these residues
are functionally redundant, so that several distinct regions of hCCR-5
can suffice independently to confer coreceptor function when
substituted into mCCR-5. Lastly, different HIV-1 envelope proteins
interact differently with CCR-5, such that CCR-5 residues important for
mediating fusion by one envelope protein may be largely irrelevant to
the interaction of CCR-5 with a second envelope protein. Overall, these
data demonstrate that the envelope-CCR-5 interaction is likely to be
highly complex and to involve the interaction of multiple residues in
both proteins.
As noted above, the mCCR-5 chemokine receptor, despite extensive
sequence similarity to hCCR-5, fails to function as an HIV-1 coreceptor
(3, 5, 20). Therefore, it is apparent that one or more of
the 20 extracellular residues that differ between mCCR-5 and hCCR-5
must contribute to the interaction with the HIV-1 envelope protein.
Using mutational analysis in the context of chimeric mCCR-5/hCCR-5
receptors, we have now identified several residues, located in three of
the four extracellular domains of hCCR-5, that play roles in mediating
infection by HIV-1.
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MATERIALS AND METHODS |
Construction of molecular clones and mutants.
Mammalian
expression plasmids encoding hCCR-5, human CXCR-4 (hCXCR-4), and mCCR-5
have been described previously (5). The HMMM chimera
contains hCCR-5 residues 1 through 30, encoding most of the first
extracellular domain, substituted into mCCR-5 (see Fig. 1)
(5). Similarly, the MMHM chimera contains the entire third
extracellular domain of hCCR-5 (residues 167 to 197) in place of the
equivalent sequence in mCCR-5 (5). The MHMM chimera was
generated by recombinant PCR with oligonucleotide primers that
precisely substituted the second extracellular domain of hCCR-5 into
the mCCR-5 context (see Fig. 1). It is important to note that this MHMM
chimera is distinct from the one previously described by Bieniasz et
al. (5) and also termed MHMM. The latter chimera, which
substituted residues 35 to 167 of hCCR-5 into mCCR-5, was not expressed
on the surfaces of transfected cells and was therefore uninformative.
All wild-type and mutant chimeras were expressed by using the pBC12/CMV
mammalian expression vector (7), and all contained
hemagglutinin (HA) epitope tags at their amino termini. Missense
mutations were introduced into the coreceptor chimeras by using
synthetic oligonucleotide primers and the Quick Change mutagenesis kit
(Stratagene) and were verified by using an ABI DNA sequencing kit
(Applied Biosystems). Point mutants are named according to the nature
and location of the mutation; thus, the S7V mutation encodes valine in
place of serine at residue 7 of the HMMM chimera. The expression
plasmids pBC12/CMV/CD4 and pBC12/HIV/SEAP, and the HIV-1 proviral
clones containing the ADA, BaL, 89.6, and IIIB envelope genes, have
been described previously (4, 5, 23). An additional HIV-1
proviral clone, containing the env gene of the M-tropic YU-2
isolate (16), was generated as previously described
(5) by substitution of the appropriate PCR-generated YU-2
SalI-BamHI DNA fragment into the context of the
pIIIB proviral clone.
Cell fusion assay for coreceptor function.
COS and 293T
cells were maintained as previously described (5). Fusion
indicator cells were generated by cotransfection of COS cells (35-mm
cultures) with Lipofectamine and 800 ng of BC12/CMV/CD4, 800 ng of
BC12/HIV/SEAP, and 800 ng of pBC12/CMV-based plasmids expressing one of
the parental, chimeric, or mutant chimeric receptors. Where necessary,
the total amount of plasmid DNA in each transfection was maintained at
2.4 µg by inclusion of the requisite quantity of the parental
pBC12/CMV plasmid. Simultaneously, virus-producing cells were generated
by transfection of COS cells with 2 µg of the relevant HIV-1 proviral
construct. At 48 h posttransfection, producer and indicator cells
were harvested by trypsinization, and equal numbers (5 × 104) were cocultivated in 48-well plates. After 48 h,
culture supernatants were harvested and secreted alkaline phosphatase
(SEAP) activity was determined as described previously (4).
Analysis of CCR-5 expression levels.
293T cells were
transfected with 2 µg of the coreceptor expression plasmids, and
72 h later unfixed cells were stained with a murine monoclonal
antibody (12CA5; Boehringer Mannheim) specific for the HA epitope tag
(10 µg/ml), followed by fluorescein isothiocyanate-conjugated goat
anti-mouse immunoglobulin G (Sigma) (1:200). Surface expression of the
chimeric receptors was then quantified by fluorescence-activated cell
sorting (FACS).
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RESULTS |
Single extracellular domains from hCCR-5 can confer HIV-1
coreceptor function on mCCR-5.
Comparison of the predicted amino
acid sequences of the hCCR-5 and mCCR-5 chemokine receptors shows that
the sequences are ~82% identical, with differences scattered
throughout these related molecules (5, 17, 19). Examination
of the extracellular domains, which are presumably the most relevant to
HIV-1 coreceptor function, reveals that the amino terminal tail of
mCCR-5 has eight substitutions and a 2-amino-acid insertion relative to
the same sequence in hCCR-5 (Fig. 1).
Similarly, the 15-amino-acid second extracellular domain of hCCR-5
differs at 4 residues, while the 31-amino-acid third extracellular
domain differs at 6 residues, from mCCR-5. The fourth extracellular
domain is identical in both molecules. Despite this extensive
similarity, and despite the fact that hCCR-5 and mCCR-5 are both able
to functionally interact with the human chemokines MIP-1
and RANTES,
mCCR-5 has failed to act as a functional coreceptor for any HIV-1
isolate tested thus far (3, 5, 18, 20).
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FIG. 1.
Predicted amino acid sequence of the four extracellular
domains of hCCR-5 (upper lines) and mCCR-5 (lower lines). Identity is
indicated by dots, and deletions are indicated by dashes. The HMMM
chimera substitutes residues 1 to 30 of hCCR-5 into mCCR-5 and
therefore excludes divergent residue 31. The MHMM and MMHM chimeras
represent precise substitutions of the second and third extracellular
domain, respectively, of hCCR-5 into mCCR-5. The location and identity
of the six clustered substitution mutations introduced into the HMMM
(M1 to M3) or MMHM (M4 to M6) chimera are indicated.
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Previously, we have described a quantitative, linear assay for the
measurement of coreceptor-dependent, HIV-1 envelope-induced
cell fusion
(
5). In this assay, indicator cells are generated
by
transfection of, in this case, COS cells with expression vectors
encoding human CD4, a candidate coreceptor, and an indicator construct
consisting of the HIV-1 long terminal repeat linked to the SEAP
indicator gene. In the absence of the HIV-1 Tat
trans-activator,
this construct produces only a minimal
level of SEAP. A second
batch of COS cells are simultaneously
transfected with a full-length
HIV-1 proviral clone. At 48 h after
transfection, these two cultures
are harvested, mixed, and then
cocultivated for a further 48-h
period. Fusion of cells expressing the
HIV-1 envelope protein
with cells expressing CD4 and a functional
coreceptor allows the
HIV-1 Tat protein to activate transcription of
the SEAP gene linked
to the HIV-1 long terminal repeat and results in a
large increase
in SEAP activity in the supernatant media.
In the experiment represented in Fig.
2,
we have used this assay to compare the abilities of three M-tropic
HIV-1 isolates
(ADA, BaL, and YU2), one dualtropic HIV-1 isolate
(89.6), and
one T-tropic HIV-1 isolate (IIIB) to utilize wild-type
forms of
hCCR-5, mCCR-5, and hCXCR-4 as coreceptors. In addition, we
have
also tested the abilities of these viruses to use hCCR-5/mCCR-5
chimeras containing precise substitutions of the first (HMMM),
second
(MHMM), or third (MMHM) extracellular domain of hCCR-5
into an
otherwise entirely mCCR-5 context. As expected, these
data demonstrate
that the three M-tropic HIV-1 isolates and the
dualtropic isolate can
utilize hCCR-5 effectively, while the T-tropic
isolate IIIB is
inactive. In contrast, while both 89.6 and IIIB
can utilize hCXCR-4
efficiently, none of the three M-tropic isolates
gives any detectable
activity. In addition, none of the tested
viruses are able to utilize
mCCR-5 appreciably (Fig.
2).
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FIG. 2.
Analysis of the coreceptor activities of hCCR-5/mCCR-5
chimeras. One set of COS cell cultures was transfected with one of the
indicated HIV-1 proviral expression plasmids or with a control (mock)
vector. A second set was transfected with the pBC12/HIV/SEAP indicator
construct and plasmids expressing CD4 and one of the indicated
amino-terminally HA-tagged chemokine receptors. Cells derived from each
set were then cocultivated starting 48 h posttransfection, and
fusion efficiency was assessed by measurement of supernatant SEAP
activity 48 h later. The data shown represent averages of three
independent experiments, with standard deviations indicated.
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As previously reported by ourselves and others (
5,
20),
viruses able to utilize hCCR-5 can differ widely in the ability
to
utilize specific hCCR-5/mCCR-5 chimeras. Thus, the ADA isolate
utilizes
the MMHM chimera as effectively as wild-type hCCR-5 and
gives readily
detectable activity with both HMMM and MHMM. In
contrast, both the
dualtropic 89.6 and the M-tropic BaL isolates
give little or no
activity with any tested hCCR-5/mCCR-5 chimera
(Fig.
2). Finally, the
YU-2 M-tropic isolate is similar to ADA
in that it can also utilize all
three hCCR-5/mCCR-5 chimeras reasonably
well, although YU-2 does differ
from ADA in that YU-2 utilizes
the MMHM chimera significantly less
effectively than wild-type
hCCR-5. Based on these data, it is apparent
that viruses tropic
for hCCR-5 can nevertheless differ significantly
from one another
in how they interact with at least three of the four
extracellular
domains of CCR-5. In addition, it is also apparent that
for at
least some HIV-1 isolates, the first three extracellular domains
of hCCR-5 can act as functionally redundant envelope binding sites.
Thus, for the M-tropic ADA isolate of HIV-1, each of the first
three
extracellular domains of hCCR-5 is sufficient to mediate
HIV-1
infection when inserted into the otherwise nonpermissive
mCCR-5 context
(Fig.
2). These data, therefore, demonstrate that
although mCCR-5 and
MHMM differ at only four residues, the latter
can nevertheless function
as an HIV-1 coreceptor while the former
does not. Similarly, HMMM and
MMHM differ by only 9 and 6 residues,
respectively, from mCCR-5 yet are
nevertheless able to function
as coreceptors for the ADA strain HIV-1
envelope. Therefore, it
is apparent that several of the amino acid
residues that differ
between hCCR-5 and mCCR-5 must make important
contributions to
the interaction between CCR-5 and the ADA envelope.
Mutational analysis of hCCR-5/mCCR-5 receptor chimeras.
Because the extracellular domains of hCCR-5 are at least in part
functionally redundant in the ability to mediate M-tropic HIV-1
coreceptor function (3, 5, 19), it appears possible that
mutational analysis of wild-type hCCR-5 might be relatively uninformative, as loss of coreceptor function in one extracellular domain would be compensated for by the remaining intact domains. In
contrast, the HMMM, MHMM, and MMHM chimeras represent appropriate contexts in which to map individual residues that mediate M-tropic HIV-1 coreceptor function, in that fusion is then dependent on a
single, nonredundant hCCR-5-derived extracellular sequence.
To map hCCR-5 residues important for HIV-1 ADA fusion, we first
constructed a series of clustered point mutants in the context
of HMMM
(M1, M2, and M3) and MMHM (M4, M5, and M6). As shown in
Fig.
1, these
CCR-5 mutants bear substitutions of the appropriate
mouse sequences in
place of the human sequences in clusters of
from two to four residues
in the first and third extracellular
domains. As shown in Fig.
3, the M1 mutant of HMMM (Y3F, V5G,
and
S7V), as well as the M3 mutant (N13D, Y15G, T16M, and E18A),
were both
only minimally active as coreceptors for HIV-1 when
tested in COS
cells. In contrast, the M2 mutant, bearing a 2-residue
insertion
between hCCR-5 residues 10 and 11, was not significantly
less active
than the parental HMMM chimera. Similarly, both the
M4 (L174F and
S180P) and M5 (Y184H and S185T) mutants of MMHM
showed poor coreceptor
activities, while the M6 mutant (Q188H
and N192S) was as active as the
MMHM parent. Similar data for
each of these wild-type and mutant
chimeric receptors were obtained
for both COS cells (Fig.
3) and
transfected 293T cells (data not
shown).
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FIG. 3.
Analysis of the levels of HIV-1 coreceptor activities
displayed by mutants of HMMM and MMHM. Coreceptor activity was assayed
in COS cells as described in the text by using the ADA viral envelope
protein. All cultures were also transfected with a plasmid expressing
human CD4. These data represent averages of three independent
experiments, with standard deviations indicated. Observed SEAP
activities are given relative to that of the parental hCCR-5
coreceptor, which was arbitrarily set at 100%.
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Point mutants of CCR-5 display negative synergy in their effects on
coreceptor function.
The data presented in Fig. 3 suggest that the
2-amino-acid insertion in the mCCR-5 first extracellular domain (mutant
M2) and the Q188H and N192S substitutions in the third extracellular domain (mutant M6) are unlikely to contribute significantly to the
inability of mCCR-5 to function as an HIV-1 coreceptor. However, these
data also suggest that at least two distinct sequence differences in
both the first and third extracellular domains, defined here by mutants
M1 and M3 and mutants M4 and M5, respectively, must contribute to this
negative phenotype.
To determine if individual residues caused the observed phenotypes, or
whether they instead resulted from a particular combination
of
substitutions, we next tested all of the residues implicated
by the
analysis presented in Fig.
3 as individual missense mutations
of either
HMMM (Fig.
4A) or MMHM (Fig.
4B).
Individual analysis
of the three residues encompassed by the M1
mutation of HMMM revealed
that substitution of phenylalanine for
tyrosine at residue three
(Y3F) had no significant effect on the
activity of HMMM (Fig.
4A). Surprisingly, the V5G mutant of HMMM
displayed significantly
enhanced coreceptor function, giving rise to a
level of cell fusion
that was clearly higher than that seen with the
parental HMMM
chimera and at least as high as that seen with the
wild-type hCCR-5
coreceptor. However, substitution of valine for serine
at residue
seven (S7V) did produce a significant drop in activity,
relative
to the HMMM parent, to a level comparable to that seen with
the
M1 triple mutant. Similarly, for the M3 quadruple mutant, the
individual substitution mutants T16M and E18A had no detectable
effect
on the activity of the HMMM parent (Fig.
4A). In contrast,
both the
conservative N13D and the less conservative Y15G mutations
exerted
marked inhibitory effects. Overall, this analysis identifies
serine 7, asparagine 13, and tyrosine 15 in the first extracellular
domain as
contributing to hCCR-5 coreceptor function. Similar
coreceptor activity
data were obtained for each of these receptor
mutants in both COS cells
(Fig.
4A) and 293T cells (data not shown).
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FIG. 4.
Functional analysis of substitution mutants of
hCCR-5/mCCR-5 chimeras. The indicated missense mutants of HMMM (A),
MMHM (B), and MHMM (C) were analyzed for coreceptor function as
described in the legend to Fig. 3.
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We next wished to address the individual contributions of each of the
two residues substituted in the M4 and M5 mutants of
the MMHM chimera
to the minimal coreceptor function of these molecules
(Fig.
3). As
shown in Fig.
4B, the L174F mutation had only a limited
effect on the
biological activity of the MMHM chimera, while the
S180P mutation
produced dramatic inhibition, apparently accounting
for the entire
phenotype of the M4 double mutant. In contrast,
neither the Y184H nor
the S185T mutation exerted a significant
phenotype when tested alone,
although the M5 double mutant encompassing
both of these changes is
clearly highly compromised in coreceptor
function (Fig.
4B). In the
latter case, it is therefore apparent
that these two substitutions,
while individually innocuous, are
able to exert a synergistic
inhibitory effect when introduced
together. This analysis was repeated
in 293T cells, with very
similar results (data not shown).
Finally, we wished to examine the effects of the four single-residue
differences in the second extracellular domain of hCCR-5/mCCR-5
on
coreceptor function. Surprisingly, as shown in Fig.
4C, none
of these
four residues had any marked effect on the substantial
coreceptor
activity of the MHMM chimera when introduced individually,
although
these four residues represent the only differences between
the active
MHMM chimera and the nonfunctional mCCR-5 molecule.
To examine whether
this result might again represent an example
of negative synergy
between individually innocuous mutations,
we generated three double
missense mutations in the MHMM context,
i.e., A92N/Q102K, Q93E/Q102K,
and D95V/Q102K. As shown in Fig.
4C, each of these double mutants was
in fact highly compromised
in coreceptor function.
Cell surface expression of chimeric CCR-5 receptors.
A
critical concern in measurement of the ability of mutant molecules to
serve as cell surface receptors is that expression levels must be
comparable. We therefore analyzed cell surface expression levels of all
of the described wild-type and mutant chimeric receptors by FACS of
transfected cells with a monoclonal antibody specific for the HA
epitope tag introduced at the amino termini of these proteins. As shown
in Fig. 5, this analysis demonstrated comparable levels of cell surface expression for all tested CCR-5 derivatives (
60% of wild-type hCCR-5). The slight variability that
was observed is unlikely to significantly contribute to the lower level
of coreceptor function observed with some CCR-5 mutants, in that the
V5G mutant of HMMM, which displayed a very high level of coreceptor
activity (Fig. 4A), appeared to display one of the lowest levels of
cell surface expression (Fig. 5).
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FIG. 5.
Relative cell surface expression of wild-type and mutant
hCCR-5/mCCR-5 chimeras. FACS analysis of 293T cells transfected with
HA-tagged forms of the indicated parental and chimeric receptors is
shown. Data are given as mean fluorescence intensities of total cell
populations and are representative of two independent experiments.
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DISCUSSION |
Analysis of the abilities of chimeric receptors derived from
hCCR-5 and mCCR-5 to function as HIV-1 coreceptors has revealed that
HIV-1 isolates can differ greatly in the ability to functionally interact with specific chimeras (Fig. 2). Thus, certain M-tropic HIV-1
isolates, including ADA, YU-2, and SF162, are able to interact effectively with hCCR-5/mCCR-5 chimeras in which only a single hCCR-5
extracellular domain is present (5, 20). In contrast, other
M-tropic isolates, including BaL, M23, and E80, fail to interact
effectively with the same HMMM, MHMM, and MMHM chimeras but will
efficiently fuse to cells expressing hCCR-5/mCCR-5 chimeras in which
any single extracellular domain in hCCR-5 is replaced by the
equivalent mCCR-5 sequence (5, 20). Finally,
certain dualtropic HIV-1 isolates, including 89.6 and GUN-1,
are highly sensitive to any perturbation of hCCR-5 and interact poorly
with almost all hCCR-5/mCCR-5 chimeras (5, 20). From these
data we can conclude that the extracellular sequences in hCCR-5
involved in M-tropic HIV-1 infection are, depending on the particular
isolate tested, either highly (ADA, YU-2, and SF162) or substantially (BaL, M23, and E80) redundant. In contrast, for dualtropic isolates such as 89.6 and GUN-1, this redundancy is quite limited. Therefore, one would predict that mutation of individual residues in hCCR-5 would
be unlikely to significantly affect infection by M-tropic HIV-1
isolates unless (i) the mutant receptor was no longer expressed normally on the cell surface or (ii) the introduced mutation affected the conformation of the CCR-5 receptor such that envelope binding was
sterically hindered. In contrast, such hCCR-5 mutations would be
predicted to be far more likely to exert a significant inhibitory effect on dualtropic HIV-1 infection. In fact, a recently published mutational analysis of hCCR-5 (10) reported no significant
effect by a series of tested mutations on infection by the M-tropic
isolate JR-FL, although substitution of alanine at residues 11, 197, or 276 did inhibit infection by the dualtropic 89.6 isolate
(10).
Because M-tropic isolates of HIV-1 are by far the most common form seen
in vivo (19, 27), it is important to understand how
M-tropic, as opposed to dualtropic, isolates interact with the dominant
coreceptor for M-tropic HIV-1, i.e., the hCCR-5 chemokine receptor.
However, because of the functional redundancy described above,
mutational analysis of hCCR-5 itself might be relatively uninformative.
For this reason, we have instead chosen to perform such a mutational
analysis using a set of three chimeric hCCR-5/mCCR-5 receptor molecules
(HMMM, MHMM, and MMHM) that each contain a single hCCR-5-derived
extracellular domain yet that retain the ability to function as
effective coreceptors for M-tropic HIV-1 isolates such as ADA (Fig. 2).
In this way, we could feel confident that the particular hCCR-5-derived
extracellular domain being subjected to mutational analysis was indeed
playing a critical role in the HIV-1 envelope-mediated fusion process.
In the analysis presented here, we have focused on the small number of
residues which differ between hCCR-5 and mCCR-5, in the expectation
that while at least some of these must contribute to hCCR-5 coreceptor
function, these changes would be very unlikely to affect either cell
surface receptor expression or overall receptor conformation. This
expectation arises from the fact that both hCCR-5 and mCCR-5 are
functional receptors for the human chemokines MIP-1 and
RANTES even though only the former can mediate M-tropic HIV-1
infection (5, 18, 20). This mutational analysis has identified two types of inhibitory effects mediated by substitution of
mCCR-5 residues into hCCR-5. Thus, several individual changes in the
first extracellular domain (S7V, N13D, and Y15G) and one substitution
in the third extracellular domain (S180P) were found to exert marked
inhibitory effects on ADA envelope-mediated cell fusion. Of these, the
most interesting may be N13D, in that this is a relatively conservative
mutation that is perhaps unlikely to significantly affect the global
conformation of the first extracellular domain of hCCR-5. Instead,
these data suggest that asparagine 13 may represent a contact point for
infection by ADA. In contrast, the other three mutations (S7V, Y15G,
and S180P) are less conservative and may therefore act by modifying the
conformation of other, flanking residues. Clearly, distinguishing these
possibilities would require, at minimum, a more intense mutational
analysis or, preferably, knowledge of the molecular structure of
hCCR-5.
An interesting phenomenon uncovered during this analysis is the
existence of marked negative synergy between mutations that individually are phenotypically silent. Thus, the Y184H and S185T mutants of the MMHM chimera have little effect individually, but when
present together in the M5 mutant they exert a marked inhibitory effect
on coreceptor function (Fig. 4B). Similarly, the four residues that
differ between hCCR-5 and mCCR-5 in the second extracellular domain
(Fig. 1) have no effect on HIV-1 coreceptor function by the MHMM
chimera when introduced individually (Fig. 4C) but effectively inhibit
activity when present simultaneously or in certain groups of two
residues. The molecular basis for this effect, of course, is at present
unclear, although it might suggest that multiple substitutions can
influence a segment of hCCR-5 to adopt a different, less favorable
conformation. Alternatively, these mutations may collectively block a
conformational shift in hCCR-5 required for the infection process but
irrelevant to chemokine receptor function. Finally, the possibility
that these residues directly contact the envelope but are highly
functionally redundant, while in our view less probable, cannot be
eliminated at this time. Certainly, it would be of interest to know
whether these mutations block envelope binding by hCCR-5 or, instead,
affect steps subsequent to binding. In either event, these data
demonstrate that certain residues in hCCR-5 play important roles in
HIV-1 infection that would be missed by mutational strategies such as
alanine scanning.
A final, unexpected observation is the finding that the V5G mutant of
the HMMM chimera is a significantly more active coreceptor than HMMM
and is perhaps even more active than hCCR-5 itself (Fig. 4A). The fact
that even the limited mutational analysis reported here can identify
gain-of-function mutants (e.g., V5G), inhibitory point mutants (e.g.,
S180P), and mutants that exert their effects via some form of negative
synergy (e.g., Y184H and S185T) scattered over three of the four
extracellular domains of hCCR-5 emphasizes the remarkable complexity
and plasticity of the functional interaction between hCCR-5 and the
HIV-1 envelope protein.
| |
ACKNOWLEDGMENTS |
We thank Lee Ratner for the ADA envelope clone. The YU-2 envelope
clone was obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH, and was donated by Beatrice
Hahan and George Shaw.
This research was supported by the Howard Hughes Medical Institute and
by the Office of Research and Development, Medical Research Service,
Department of Veterans Affairs, Research Center on AIDS and HIV
Infection, Durham, N.C. T.M.R. is supported by Duke
Interdisciplinary Research Training Program in AIDS grant 2T32AI07392
from the National Institute of Allergy and Infectious Diseases.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Duke University
Medical Center, Box 3025, Room 426 CARL Building, Research Dr., Durham, NC 27710. Phone: (919) 684-3369. Fax: (919) 681-8979.
| |
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Abstract
Introduction
