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Journal of Virology, April 2001, p. 3462-3468, Vol. 75, No. 7
Research Institute for Microbial Diseases,
Osaka University, Osaka,1 Ryukyu
University, Okinawa,2 and Institute
of Medical Science, University of Tokyo,3 and
National Institute of Infectious
Diseases,4 Tokyo, Japan
Received 20 September 2000/Accepted 22 December 2000
CCR5 is an essential coreceptor for the cellular entry of R5
strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893( CCR5 is an essential coreceptor for
the cellular entry of R5 (macrophagetropic, non-syncytium-inducing)
strains of human immunodeficiency virus type 1 (HIV-1) (1, 8, 15,
19-21), which predominate in the early stages of infection
(59). During the course of infection, variants called X4
(T-cell-line tropic, syncytium-inducing) strains emerge (5, 8,
14, 17, 54), which use CXCR4 as a coreceptor (22).
In vitro replication of R5 strains can be blocked by ligands of CCR5,
macrophage inflammatory peptides 1 Mutations in HIV-1 coreceptors and their natural ligand genes have been
shown to modify HIV-1 transmission and disease progression. Caucasians homozygous for a 32-nucleotide deletion in the CCR5 coding
region (CCR5 In addition to the relatively abundant HIV-1 disease-modifying alleles
described above, several less frequent polymorphisms have been found in
the coding region of CCR5 (3, 11, 27, 34, 47). Among them,
CCR5-893(
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3462-3468.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Naturally Occurring Deletional Mutation in the C-Terminal
Cytoplasmic Tail of CCR5 Affects Surface Trafficking of
CCR5
ABSTRACT
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Abstract
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References
) is
a single-nucleotide deletion mutation which is observed exclusively in
Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221-222, 1997).
This mutant gene produces a CCR5 which lacks the entire C-terminal
cytoplasmic tail. To assess the effect of CCR5-893() on HIV-1
infection, we generated a recombinant Sendai virus expressing the
mutant CCR5 and compared its HIV-1 coreceptor activity with that of
wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of
cells expressing the mutant CCR5 revealed that surface CCR5 levels were
greatly reduced in these cells, while cytoplasmic CCR5 levels of the
mutant CCR5 were comparable to that of the wild type. Peripheral blood
CD4+ T cells obtained from individuals heterozygous for
this allele expressed very low levels of CCR5. These data suggest that
the CCR5-893() mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1
transmission and disease progression.
TEXT
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Abstract
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References
and 1
, and RANTES (regulated
on activation normal T-cell expressed and secreted) (4,
16). Recently, monocyte chemoattractant protein 2 was found to
be a natural ligand of CCR5 (9, 23, 49). On the other
hand, replication of X4 strains can be blocked by the CXCR4 ligands
stromal cell derived factor 1 and 1 (SDF-1 and SDF-1)
(10, 42).
32) are resistant to HIV-1 infection (32, 50), while heterozygosity delays disease progression (18, 38). A single valine-to-isoleucine change in the first
transmembrane segment of CCR2 (CCR2-64I), a minor coreceptor for
dualtropic R5X4 strains (15, 20), has a significant impact
on disease progression but not on HIV-1 transmission (30,
53). Homozygosity of a single G-to-A mutation in the 3'
noncoding region of the SDF-1 gene also showed a disease retarding
effect (56), although later studies could not confirm this
effect (39, 55). A CCR5 promoter variant was shown to be
associated with accelerated progression in HIV-1 diseases (35,
37). We recently demonstrated that a single A-to-G mutation in
the promoter region of RANTES was associated with delayed disease
progression in HIV-1-infected individuals in Japan (31).
) is a single-nucleotide deletion which is observed
exclusively in Asians (3). This deletion caused a
frameshift at codon 299 and resulted in premature termination of
translation (Fig. 1). As a result,
the CCR5-893() gene product lacked 51 amino acid residues in
the C-terminal cytoplasmic tail and 3 residues in the last
transmembrane segment, and it gained 10 amino acid residues encoded in
the 1 open reading frame in its C terminus. The CCR5-893() gene
product is thus composed of 308 amino acids.
View larger version (43K):
[in a new window]
FIG. 1.
Structure of the CCR5-893( ) gene product. Outlined
letters in gray circles denote amino acid residues which are not
present in the CCR5-893() gene product; those in black circles denote
amino acid residues generated by the frameshift.
To assess the effect of CCR5-893() on HIV-1 infection, we used a
recombinant Sendai virus (SeV) vector to express the wild-type and mutant CCR5 and examined their ability to support CD4-dependent cell fusion mediated by an HIV-1 envelope protein of the R5
strain SF162. Recombinant SeVs were generated as described previously (26, 28, 29), and HIV-1 coreceptor activity was examined by a recombinant vaccinia virus-based gene activation assay using a
-galactosidase gene as a reporter (40, 41). Mouse L
cells lacking endogenous CCR5 expression were used for this experiment. Since none of the extracellular domains of CCR5 were affected by this
deletion, we initially anticipated that this deletion had no effect on
HIV-1 coreceptor activity. On the contrary, the CCR5-893() product
showed a greatly reduced ability to support R5 envelope-mediated cell
fusion compared with wild-type CCR5 (Fig.
2A). A similar result was obtained when
we used a vaccinia virus vector to express CCR5 (Fig. 2B) (33,
52). Northern blot analyses confirmed the presence of comparable
amounts of CCR5 mRNA in cells infected with SeV expressing wild-type
CCR5 and in those infected with SeV expressing CCR5-893() (data not shown). Furthermore, CD4+ MT4 cells infected with SeV
expressing wild-type CCR5 supported replication of SF162 five times
better than those infected with SeV expressing CCR5-893() (Fig. 2C).
These data clearly indicated that the CCR5-893() product had a
reduced coreceptor activity for HIV-1 entry.
|
We then analyzed levels of cell surface CCR5 by flow cytometry. Jurkat
and L cells were used in this experiment. Nine hours after
infection of SeVs expressing the wild-type and mutant CCR5, cells were
stained with T227, a rat monoclonal immunoglobulin G1 (IgG1) antibody
directed against the N-terminal extracellular domain of CCR5 (Y. Tanaka
et al., unpublished data), followed by
fluorescein-5-isothiocyanate (FITC)-labeled anti-rat IgG. Stained cells
were fixed in 1% formaldehyde in phosphate-buffered saline (PBS) and
analyzed by FACScan (Becton Dickinson, San Jose, Calif.). As shown in
Fig. 3, the CCR5-893() product was
poorly detected on the surface of all the cell lines examined, whereas
wild-type CCR5 was readily detected on the cell surface (Fig. 3A and
B). When cells were permeabilized with 0.05% saponin and 0.2% bovine serum albumin in PBS before staining, strong staining was
observed in cells expressing the CCR5-893() product (Fig. 3D and E).
Intracellular fluorescence intensity of the CCR5-893()
product was comparable to that of the wild type. These data suggested
that the mutant CCR5 was synthesized as efficiently as the wild type,
but its surface trafficking was greatly impaired by the mutation. A
similar result was obtained when we used monocytic U937 cells (data not shown).
|
To determine the subcellular localization of the
CCR5-893() product, we used immunofluorescence confocal
microscopy. Jurkat and HeLa cells infected with SeV expressing
wild-type CCR5 or CCR5-893() were fixed in 3% paraformaldehyde in
PBS, permeabilized with 0.05% saponin and 0.2% bovine serum albumin
in PBS, and incubated with rat monoclonal antibody T227 directed
against human CCR5 and rabbit polyclonal antibodies directed against
calnexin (Stressgen). Bound antibodies were then detected with
FITC-conjugated goat antibody directed against rat IgG (American
Qualex Antibodies, San Clemente, Calif.) or Cy5-conjugated
goat antibody directed against rabbit IgG (Amersham-Pharmacia
Biotech). Indirect immunofluorescence was visualized with a Fluoview
FV300 laser confocal microscope system (Olympus, Tokyo, Japan). As
shown in Fig. 4, fluorescent signals of
CCR5 were mainly observed on the cell surface in cells expressing
wild-type CCR5. Calnexin, an endoplasmic reticulum (ER) chaperone that
assists in the folding of proteins in the ER, was observed in the
perinuclear regions of the cells and showed only a partial
colocalization with CCR5 (Fig. 4). In contrast, distribution of the
CCR5-893() product was limited to the perinuclear regions, and almost
all CCR5-specific signals were surrounded by calnexin-specific signals
(Fig. 4). These data indicated a clear colocalization of the
CCR5-893() product and calnexin in both cell types and suggested that
the CCR5-893() product was retained in ER. Similar results were
obtained when we used U937 cells or 2D7, a mouse monoclonal antibody
against the second extracellular loop of CCR5 (data not shown).
|
To investigate whether the additional 10 amino acid residues generated
by the frameshift are responsible for the inefficient surface
trafficking of the CCR5-893() product, we introduced a stop codon at
position 299 of CCR5 to remove these 10 amino acid residues from the
mutant CCR5. A recombinant vaccinia virus was used to express this
artificial mutant CCR5, designated CCR5-stop. L cells infected with the
recombinant vaccinia virus expressing CCR5-stop did not support
CD4-dependent R5 envelope-mediated membrane fusion (Fig. 2B). Surface
expression of CCR5-stop in CV-1 cells was more severely impaired than
that of the CCR5-893() product (Fig. 3C), although intracellular
staining of CCR5-stop was comparable to that of the CCR5-893()
product (Fig. 3F). These data indicated that lack of the entire
cytoplasmic tail rather than the addition of 10 aberrant amino acid
residues was responsible for inefficient surface trafficking of
the mutant CCR5. Previously, Gosling et al. reported that a
deletion after amino acid 308 of CCR5 did not abolish its HIV-1
coreceptor activity (24). Similarly, Alkhatib et al.
demonstrated that truncation after amino acid 307 did not alter surface
expression, chemokine binding, and HIV-1 coreceptor activity of CCR5
(2). Therefore, the eight amino acid residues from
positions 299 to 306 seem to be important for efficient surface trafficking of CCR5.
CCR532 was reported to affect expression of wild-type CCR5
(7). To determine whether CCR5-893() also has a
dominant negative effect on wild-type CCR5 expression, we
simultaneously inoculated Jurkat cells with SeV expressing wild-type
CCR5 and SeV expressing CCR5-893(). Six hours after infection,
surface expression of CCR5 was examined by flow cytometry. As
shown in Fig. 3G, coinfection with SeV expressing CCR5-893() severely
reduced the levels of CCR5 expression on the cell surface compared to
coinfection with the parental Z strain of SeV. This finding indicated
that CCR5-893() dominantly affects wild-type CCR5 expression as
the CCR532 does.
To determine whether individuals carrying CCR5-893() exhibit reduced
cell surface expression of CCR5, we analyzed levels of CCR5 on the
surface of primary peripheral blood CD4+ cells.
CD4+ cells were positively selected from peripheral blood
mononuclear cells by using MACS CD4 MicroBeads (Miltenyi Biotec,
Nordrhein-Westfalen, Germany) and stained with T227 rat monoclonal
antibody directed against CCR5 followed by FITC-conjugated goat
antibody directed against rat IgG (American Qualex Antibodies) and a
peridinin chlorophyll protein (PerCP)-conjugated mouse monoclonal
antibody directed against HLA-DR (Becton Dickinson).
Stained cells were fixed in 1% formaldehyde in PBS and analyzed by
FACScan (Becton Dickinson) (Fig. 5A). In
the first set of experiments (Ex. 1 in Fig. 5B and C), we analyzed one
heterozygote for CCR5-893() and 30 nonmutant individuals. We then
analyzed three heterozygotes for CCR5-893() and nine nonmutant
individuals in experiment 2 in Fig. 5B and C. CD4+ cells
were further divided into HLA-DR+ cells and
HLA-DR
cells to normalize the activation status of
each individual, which is known to affect the levels of CCR5 expression
(58). The proportion of HLA-DR+
CD4+ cells varied among individuals, ranging from 3 to 19%
of total CD4+ cells. As reported previously
(48), more HLA-DR+ CD4+ cells than
HLA-DR CD4+ cells express CCR5.
Nevertheless, both HLA-DR+ and HLA-DR
CD4+ cells from individuals heterozygous for CCR5-893()
expressed significantly lower levels of CCR5 than those from nonmutant
individuals (Fig. 5B and C). These data suggested that CCR5-893()
also reduced CCR5 expression in cells that can be actual targets for
HIV-1 infection. Furthermore, CD8+ cells from the
heterozygote for CCR5-893() also showed reduced levels of CCR5
expression (data not shown).
|
Above finding prompted us to investigate whether CD4+ cells
from heterozygotes for CCR5-893() show reduced sensitivity to infection with HIV-1 R5 strains. For this, 106
CD4+ cells obtained from two heterozygotes and two
nonmutant individuals were stimulated with phytohemagglutinin for 2 days and then inoculated with 25 ng of p24 of HIV-1 strain SF162.
Culture supernatants of infected cells were periodically assayed for
levels of p24 core antigen (RETRO-TEK, Buffalo, N.Y.). As shown in Fig.
5D, HIV-1 strain SF162 grew to significantly lower titers in cells from
two heterozygotes than in those from two nonmutant individuals. This
result suggested that CD4+ cells from individuals
heterozygous for CCR5-893() had reduced sensitivity to infection with
HIV-1 R5 strains.
To determine the frequency of CCR5-893() in non-HIV-1-infected and
HIV-1-infected individuals in Japan, we analyzed CCR5 genes of 156 non-HIV-1-infected and 207 HIV-1-infected individuals. DNA was
extracted from peripheral blood mononuclear cells by a method
previously described (31). Informed consent was obtained from all subjects. DNA fragments corresponding to a 1,194-bp region spanning the entire open reading frame of CCR5 were PCR amplified using
primers CKR5a+ (5'-CAGTTTGCATTCATGGAGGG-3') and CKR5a
(5'-CTAAGCCATGTGCACAACTC-3'). PCR was performed in a 50-µl
reaction mixture containing 1 µg of DNA by 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. Amplified DNA fragments
were purified and sequenced using primer CKR5c+
(5'-CTGTGTTTGCGTCTCTCC-3'). Sequencing reactions were
performed by the dideoxy-chain termination method using an ABI Prism
377 (Applied Biosystems) automated DNA sequencer. We found three
heterozygotes among non-HIV-1-infected and two among HIV-1-infected
individuals. The calculated allele frequencies were approximately 1%
in non-HIV-1-infected and 0.5% in HIV-1-infected individuals, showing
a tendency toward reduced frequency in HIV-1-infected individuals,
although this difference did not reach statistical significance. Since
both non-HIV-1-infected and HIV-1-infected individuals carry
CCR5-893(), heterozygosity for this allele does not confer complete
resistance to initial HIV-1 transmission. Although homozygosity of
CCR5-893() is highly likely to confer at least partial resistance to
initial HIV-1 transmission, the frequency of this allele appeared to be
too low for evaluation of the epidemiological consequence of this
allele in Japan. The clinical effect of this allele may be similar to
that of CCR532, since heterozygosity for CCR532 also failed to
protect from HIV-1 transmission, while this genotype was associated
with delayed disease progression (18, 38). We are
currently analyzing samples from other Asian ethnic groups including
Chinese and Thai to determine whether CCR5-893() is more prevalent in
these populations.
With respect to cytoplasmic retention of chemokine receptors, two
precedents have been reported. The chemokine receptor CCR2, which shows
more than 75% amino acid sequence homology with CCR5, arises in two
isoforms, CCR2A and CCR2B, by differential splicing from a single gene.
They differ only in their carboxyl-terminal cytoplasmic tails
(12). The amino acid sequence of the CCR2B cytoplasmic
tail showed 76% identity with that of CCR5, whereas the corresponding
region of CCR2A showed little homology with CCR5. A recent study by
Wong et al. showed that CCR2A was predominantly detected in the
cytoplasm of transfected cells, whereas CCR2B was efficiently
trafficked to the cell surface (57). Truncation analyses
of CCR2A further suggested the presence of a cytoplasmic retention
signal in the carboxyl tail. In the case of CCR5, however, our
truncation analysis suggested that the absence of the entire cytoplasmic tail rather than the addition of aberrant amino acid residues generated by the frameshift was responsible for the poor trafficking of the mutant CCR5. Therefore, the molecular mechanism of
cytoplasmic retention of CCR5-893() may be different from that of
CCR2A. Further studies, including the identification of factors
interacting with the cytoplasmic tails of CCR2A, CCR2B, and CCR5, would
be necessary to define the precise mechanisms responsible for
cytoplasmic retention of these chemokine receptors. Also, it would be
interesting to analyze the interaction between the CCR5-893() product
and ER chaperones, since our confocal microscopic observation of cells
expressing the mutant CCR5 suggested that the mutant protein was
retained in ER.
Another example of cytoplasmic retention of the chemokine receptor is
US28, encoded by a cytomegalovirus. This chemokine receptor also showed
HIV-1 coreceptor activity (45) and was reported to be
retained in the cytoplasm of Cf2Th cells but not COS-1, U87, or HeLa
cells (43). Similarly, our data showed that surface expression of CCR5-893() was more severely impaired in Jurkat (Fig.
3A) and U937 (data not shown) cells than in epithelial CV1 cells (Fig.
3C). It is possible that factors affecting surface trafficking of these
chemokine receptors distribute differently in different cell types and
that the molecular mechanism controlling cytoplasmic retention of US28
may be identical or similar to that of CCR5-893().
CCR532 was mainly observed in Caucasians (18, 32, 38,
53), whereas CCR5-893() was exclusively observed in Asians (3). Furthermore, CCR524, another nonfunctional allele
of CCR5, was also observed in sooty mangabey and red-capped mangabey monkeys (13, 44). Distribution of multiple nonfunctional
alleles of CCR5 in various human and primate populations suggested the presence of a certain selective pressure favoring those nonfunctional alleles during evolution of humans and lower primates. It is unlikely that HIV-1 infection itself could be the selective pressure, since the
HIV-1 epidemic is a recent event. CCR5 has been shown to be involved in
several inflammatory diseases (6, 46), and CCR532 was
associated with a reduced risk of severe symptoms in patients with
multiple sclerosis and asthma (25, 51). Therefore, it is
possible that impaired function of CCR5 might be advantageous for
survival of individuals with autoimmune or inflammatory diseases. Alternatively, nonfunctional alleles of CCR5 might have been selected for by variola virus epidemics, since it was recently reported that
poxvirus infection was facilitated by CCR5 (36).
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ACKNOWLEDGMENTS |
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The first two authors contributed equally to this work.
We thank all of the blood donors described in this study, David Chao
and Jun-ichi Sakuragi for critical discussions, and Akio Nomoto and
Shusuke Kuge for guiding the confocal microscopic observations. pG1T7-gal was kindly supplied by E. Berger.
This work was supported by grants from the Ministry of Education, Science, Sports and Culture, the Ministry of Health and Welfare, and the Organization for Pharmaceutical Safety and Research (OPSR), Japan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8346. Fax: 81-6-6879-8347. E-mail: shioda{at}biken.osaka-u.ac.jp.
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Journal of Virology, April 2001, p. 3462-3468, Vol. 75, No. 7
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3462-3468.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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