An overview of HIV-1 co-receptor function and its inhibitors
Emmanuel G. Cormier and Tatjana Dragic*
Albert Einstein College of Medicine, Department of Microbiology
and Immunology
1300 Morris Park Avenue, Bronx, NY 10461
HIV-1 entry into target cells
To trigger the membrane fusion process that leads to viral entry,
HIV-1 must first interact with CD4 then with a co-receptor [1,
23, 42, 43, 51, 116, 121]. CD4 binding occurs subsequent to less
specific, adhesion factor-mediated interactions with the cell surface
that increase the localized concentration of virions [77].
Binding of the HIV-1 gp120 envelope glycoprotein to CD4 induces
conformational changes in gp120 that create or expose a binding site
for a co-receptor [116, 121]. Once available, the co-receptor
binding site interacts with a complex, discontinuous region of the
co-receptor that involves, but is not limited to the amino-terminal
domain (Nt) [41, 44, 46, 91, 94, 97]. The association of
gp120 with CCR5 or CXCR4 then drives additional conformational
changes within the entire trimeric gp120/gp41 complex that eventually
lead to the insertion of the gp41 fusion peptide into the host cell
membrane, provoking fusion and entry [64].
HIV-1-co-receptor interactions
HIV-1 co-receptors belong to the seven transmembrane G-protein
coupled chemokine receptor family. The evidence accumulated to date
indicates that there are similarities and differences in the way
HIV-1 envelope glycoproteins from R5 and X4 isolates interact with
their respective co-receptors. A cluster of residues in the CCR5 Nt
participates in gp120-binding and is essential for fusion and entry
of both R5 and R5X4 isolates [44, 49, 94]. In contrast,
residues dispersed throughout the extracellular domain of CXCR4 are
involved in gp120 docking, viral fusion and entry [18, 62];
each HIV-1 isolate uses a slightly different subset of CXCR4 residues
in order to gain entry into the target cell. Nevertheless, the gp120
binding sites on CCR5 and CXCR4 comprise negatively charged and
tyrosine residues [27, 44, 49, 50, 94]. Certain mutations in
CXCR4 even enable it to mediate the entry of R5 isolates
[16&endash;18, 120]. Similarities between CCR5 and CXCR4
gp120-binding sites are further underscored by the ability of R5X4
isolates to interact with both co-receptors. These similarities may
account for the ability of a few residue changes in gp120 to induce a
switch in co-receptor usage. It should be noted that the
extracellular loops of CCR5 and CXCR4 also play an indirect role in
viral entry by influencing the overall conformation and/or
oligomerization of the co-receptor proteins.
It is notable, however, that all chemokine receptors described to
date have negatively charged regions in their extracellular domains,
yet most do not mediate HIV-1 entry, and some do so only poorly. It
also seems that the Nts of most if not all chemokine receptors
contain sulfotyrosines. Hence, the unique features that make CCR5 and
CXCR4 efficient HIV-1 co-receptors remain to be identified. Perhaps
it is the way that the different Tyr-Asp-Glu motifs are exhibited on
the surfaces of these receptors, or the ability of CCR5 and CXCR4 to
interact with CD4 or other molecules on the cell surface that
ultimately renders them efficient mediators of viral entry.
Co-receptors and HIV-1 tropism
The selective use of the CCR5 and/or CXCR4 co-receptors is the
molecular explanation of the previous phenotypic categorization of
HIV-1 isolates [9, 37, 52]. CCR5 is the principal co-receptor
for HIV-1 variants that are sexually transmitted and persist within
the majority of infected individuals (R5 isolates). The appearance of
variants that use CXCR4 or both co-receptors (X4 and R5X4 isolates)
signals accelerated CD4+ T-cell loss and disease progression [26,
109]. The phenotypic switch from R5 to X4 viruses in vivo
typically occurs only after several years of infection. This is
surprisingly slow given that changing only a few residues in gp120
can be sufficient to convert an R5 virus into an R5X4 virus in
vitro and that such changes must be occurring continuously given
the error rate of reverse transcription [19&endash;22, 25, 53,
60, 61, 64, 74, 78, 79, 90, 105, 116, 118, 119, 120]. These
observations imply that the transition to CXCR4 usage is specifically
suppressed in vivo [75].
The need for new inhibitors of HIV-1 replication
Death rates due to HIV-1 infection have fallen significantly since
specific inhibitors were developed that antagonize the function of
the viral reverse transcriptase and protease enzymes [56].
Over a dozen different drugs now on the market target these two viral
enzymes. However, the present antiviral cocktails are not well
tolerated by a significant percentage (approximately 25%) of
individuals [69]. There are also increasing concerns about
the long-term metabolic side effects of protease inhibitors, notably
poorly understand problems with fat metabolism [56, 69]. The
increasing emergence and transmission of drug-resistant HIV-1
variants is another problem [129]. Together, the above
factors emphasize the need to identify new classes of antiviral drugs
that can supplement or partially replace existing drug cocktails.
Among the many chemokine receptors that can mediate HIV-1 entry
in vitro [10], only CCR5 and CXCR4 are of
pharmacological importance, since they are the principal co-receptors
used by HIV-1 to enter primary CD4+ T-cells and macrophages. These
are the cells that produce almost the total viral burden in vivo
[19, 76, 132]. In vitro experiments indicate that
a lower level of CCR5 expression can reduce cellular infection by
HIV-1, which may translate into clinical benefit [33, 123].
Blocking the function of CCR5 may not significantly impact human
health since approximately 1% of Caucasians naturally lack CCR5 due
to a protein-disrupting mutation without any detectable consequences
[67, 101]. CCR5 does play a role in the correct functioning
of the mammalian immune system, demonstrated by studies in CCR5
knock-out mice [134]. These animals have a greatly reduced
survival after experimental infection of the brain with
Cyptoccocus neoformans; partial defects in the clearance of
Listeria monocytogenes; reduced IFN-g production after
infection with Leishmania donovani; and an increased
susceptibility to Toxoplasma gondii, due to decreased
production of IL 12 and IFN-g. Whether this matters from the
perspective of inhibitor development is uncertain. The safety of
CXCR4 inhibitors may be more problematic in humans, because knocking
out CXCR4 in mice is lethal [134]. However, a CXCR4-specific
inhibitor was not acutely toxic in adult mice [29].
Co-receptor-targeted inhibitors of HIV-1 entry
We have summarized the properties of the co-receptor inhibitors
described in the literature to date in Tables 1 and 2. The first
inhibitors known to prevent HIV-1 fusion and entry were MIP-1a,
MIP-1b and RANTES, the natural CC-chemokine ligands of CCR5
[24]. The CXC-chemokine SDF-1a has an analogous inhibitory
effect on viral entry via CXCR4 [11, 85]. Variants of
chemokines with increased potency in vitro, usually resulting
from N-terminal modifications to the RANTES or SDF-1a structure, have
since been developed [5, 32, 68, 108, 128, 130]. Chemokines
interfere with HIV-1 replication by several mechanisms: (1) direct
competition between the chemokine and the gp120 glycoprotein for
binding to the co-receptor, (2) a sustained down-regulation of the
co-receptor as a consequence of chemokine binding and signal
transduction, and (3) alteration of the differentiation state of the
target cell that affects HIV-1 replication late in the viral life
cycle [2, 4, 71, 89, 116, 117, 121].
Several CXCR4- and CCR5-specific murine MAbs are known to inhibit
HIV-1 fusion and entry with considerable potency [47, 55, 87,
122]. Co-receptor specific MAbs are not agonists, but most are
antagonists [47, 55, 87, 122]. We and others have shown that
anti-CCR5 MAbs that recognize epitopes in the second extracellular
loop (ECL2) are potent inhibitors of HIV-1 entry even though they
only moderately inhibit gp120 binding to CCR5 [66, 87, 122].
Possibly, these MAbs inhibit important post-gp120 binding steps, such
as conformational changes in CCR5 or its oligomerization
[63]. Few anti-CXCR4 MAbs have been generated and only one
has been extensively characterized. MAb 12G5 recognizes an epitope in
ECL2 and inhibits HIV-1 fusion and entry both in an isolate- and a
cell type-specific manner [73, 111]. Differences in gp120
affinities for CXCR4 and post-translational modifications of CXCR4 in
different cell types could account for these discrepancies. Other
anti-CXCR4 MAbs, whose epitopes remain to be determined, also
variably inhibit the entry of the HIV-1 NL-43 isolate
[57].
Several small molecule inhibitors of CXCR4- and CCR5-mediated
HIV-1 entry are now known. All are receptor antagonists that have no
signaling capacity themselves. The CXCR4 inhibitors T22, ALX40-4C and
AMD3100 (and their derivatives) are highly cationic compounds, having
at least six positively charged atoms at physiological pH [3, 38,
40, 81, 104]. The one small molecule CCR5 inhibitor whose
structure has been described in print, TAK-779, has only one positive
charge [6]. The difference in ligands probably reflects the
surface charges of the two co-receptors; the CXCR4 surface is
strongly anionic, whereas CCR5 has an almost neutral surface. T22,
ALX40-4C and AMD3100 all bind predominantly to the extracellular
domain of CXCR4, especially to the second extracellular loop, with
anionic residues being of particular importance [65, 83]. All
of these compounds antagonize signaling via SDF-1a [38, 40, 81,
104]. Surprisingly, each is completely specific for CXCR4 among
other tested HIV-1 co-receptors, perhaps because of the unique nature
of the CXCR4 extracellular surface. The binding site for TAK-779 on
CCR5 is very different. Our mutagenesis studies have shown that
TAK-779 binds within a pocket formed by transmembrane helices 1, 2, 3
and 7 of CCR5. Some contacts may also be made between TAK-779 and
unidentified residues in the extracellular region, although this
remains to be demonstrated. Once in place, TAK-779 prevents gp120,
but not anti-CCR5 MAbs, from binding to CCR5 [6, 45]. TAK-779
is not an agonist, and does not cause CCR5 down-regulation but it
does inhibit chemokine-induced receptor signaling [6, 45].
TAK-779 also interacts with CCR2b to block signaling and SIVrcm entry
via this co-receptor [6, 45, 131]. The binding pocket for
TAK-779 is probably not unique to CCR5 and similar pockets, likely to
be present in other chemokine receptors, are attractive targets for
drug development.
HIV-1 escape from co-receptor-targeted inhibitors
The following mechanisms of escape from co-receptor inhibitors are
possible: (1) the escape mutant may continue to use the same
co-receptor in an inhibitor-insensitive manner; (2) co-receptor
switching may occur, so that an R5 virus now becomes able to use
CXCR4, or vice versa; and (3) an entirely different
co-receptor may now be used by the escape mutant. In the studies
published to date, the first mechanism is the most common, while the
third has not been found.
The initial study on AMD3100 escape was performed before it was
known that this compound targets CXCR4. It was carried out using
HIV-1NL4-3 in MT-2 cells, which express CD4 and CXCR4, but not CCR5.
The co-receptor-switching pathway was, therefore, unavailable. After
63 passages with increasing AMD3100 doses, the mutant virus had 13
amino acid changes scattered throughout gp120 and still used CXCR4,
but in an AMD3100-insensitive manner [48, 102]. These
observations imply that there may be more than one way for gp120 to
functionally interact with CXCR4. A study with SDF-1a had a similar
outcome: multiple amino acid changes in gp120 occurred during
sequential passages to eventually create a resistant virus that still
used CXCR4 for entry [102]. Interestingly, there was partial
cross-resistance between the AMD3100- and SDF-1a resistant viruses,
and about half of the amino acid substitutions in gp120 were common
to the two escape mutants [102]. When AMD3100 resistance was
selected for by using uncloned X4 or R5X4 primary isolates in PBMC
(which express both CCR5 and CXCR4), R5 viruses become dominant in
the cultures [48].
Escape mutant studies with CCR5-specific inhibitors have been more
limited. An initial report using RANTES derivatives in hu-PBL SCID
mice concluded that co-receptor switching to CXCR4 usage sometimes
occurred [80]. However, escape from a CCR5-specific MAb in
the same animal model did not involve co-receptor-switching (Moore,
J.P. personal communication). Another study using MIP-1a and the R5
virus HIV-1JR-FL in a CCR5+ CXCR4+ cell line found that a 4-6-fold
reduction in sensitivity to CC chemokines occurred after 3 months
without any switch to CXCR4, despite its availability on the target
cells [72].
Concluding remarks
The discovery of the principal HIV-1 co-receptors, CXCR4 and CCR5,
has significantly impacted our understanding of how HIV-1 infects its
target cells and how this relates to viral pathogenesis. New targets
for antiviral drug development have been identified and are now being
exploited. A CCR5 antagonist that blocked HIV-1 entry yet drove
phenotypic evolution to CXCR4 use would be undesirable, because of
the association between X4 and R5X4 viruses with an increased rate of
CD4+ T-cell loss [26, 109]. However, a selection pressure
seems to limit the rate of phenotypic evolution from CCR5 to CXCR4
use in vivo [75]. Furthermore, CCR5-negative
individuals who became HIV-1 infected harbor exclusively CXCR4-using
isolates [76]. The inability of these viruses to use CCR2b,
CCR3, Bonzo, BOB, etc. indicates the irrelevance of these proteins
in vivo. Unless a CCR5-specific inhibitor in some way
interfered with the natural selection pressure that suppresses X4
viruses, blocking HIV-1 entry via CCR5 would not necessarily drive
the rapid emergence of X4 viruses nor viruses that utilize other
co-receptors [75]. CCR5 in particular and the co-receptors in
general therefore represent viable drug targets aimed at slowing
HIV-1 replication and the progression to AIDS.
References