JOURNAL OF VIROLOGY, Oct. 2009, p. 9694–9708
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 19
Human Immunodeficiency Virus Type 1 V1-to-V5 Envelope Variants
from the Chronic Phase of Infection Use CCR5 and Fuse More
Efficiently than Those from Early after Infection?
Behzad Etemad,1Angela Fellows,1Brenda Kwambana,1Anupa Kamat,1Yang Feng,2
Sandra Lee,2and Manish Sagar1*
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139,1and
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of
Public Health, Boston, Massachusetts 021152
Received 8 May 2009/Accepted 8 July 2009
Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein modifications over the course of
infection have been associated with coreceptor switching and antibody neutralization resistance, but the effect
of the changes on replication and host cell receptor usage remains unclear. To examine this question, unique
early- and chronic-stage infection envelope V1-toV5 (V1-V5) segments from eight HIV-1 subtype A-infected
subjects were incorporated into an isogenic background to construct replication-competent recombinant
viruses. In all subjects, viruses with chronic-infection V1-V5 segments showed greater replication capacity than
those with early-infection V1-V5 domains in cell lines with high levels of both the CD4 and the CCR5 receptors.
Viruses with chronic-infection V1-V5s demonstrated a significantly increased ability to replicate in cells with
low CCR5 receptor levels and greater resistance to CCR5 receptor and fusion inhibitors compared to those
with early-infection V1-V5 segments. These properties were associated with sequence changes in the envelope
V1-V3 segments. Viruses with the envelope segments from the two infection time points showed no significant
difference in their ability to infect cells with low CD4 receptor densities, in their sensitivity to soluble CD4, or
in their replication capacity in monocyte-derived macrophages. Our results suggest that envelope changes,
primarily in the V1-V3 domains, increase both the ability to use the CCR5 receptor and fusion kinetics. Thus,
envelope modifications over time within a host potentially enhance replication capacity.
The human immunodeficiency virus type 1 (HIV-1) viral
envelope glycoprotein evolves over the course of infection (24,
78), with the portion from constant region 2 to variable loop 5
(C2-V5) diversifying at an approximate rate of 1% per year in
the absence of antiretroviral medications (77). The envelope
glycoprotein variable loops 1 and 2 (V1-V2) expand and add
more glycosylation sites over the course of infection (21, 76).
These envelope changes arise primarily due to errors during
reverse transcription, the high rate of viral replication, and
recombination (25, 42, 58, 86). The rate of mutation fixation in
a virus population, however, depends on both the level of viral
replication and, more importantly, the selective advantage or
disadvantage conferred by the mutation. The host immune
response and the replication capacity in the available target
cells primarily drive this selection (12, 51). Envelope modifi-
cations that confer an advantage in evading the host humoral
immune response and/or increase the efficiency of target cell
infection and replication are likely favored over the course of
an infection within a subject.
Studies with the simian immunodeficiency virus/macaque
model, simian human immunodeficiency virus, and HIV-1
have shown that envelope modifications that occur over the
course of an infection confer antibody neutralization resistance
(8, 9, 72, 85). The host neutralizing antibodies target specific
epitopes on the circulating viral envelope glycoproteins, but
viruses evolve to escape these responses (70, 85). We have
previously shown that in HIV-1 subtype A-infected individuals,
changes in the envelope glycoprotein V1-V2 loops account for
some of the observed neutralization resistance to autologous
plasma (76). Besides influencing the sensitivity to the host
neutralizing-antibody response, envelope modifications that
occur over the course of infection also potentially affect host
cell receptor interactions and replicative capacity in different
HIV uses the CD4 receptor along with a coreceptor, such as
CCR5 and/or CXCR4, for host cell attachment and fusion
(11). Early in infection, most HIVs use the CCR5 coreceptor,
and over time, HIVs often acquire an ability to use a wider
variety of coreceptors, such as CXCR4 (7). In subtype B
HIV-1, coreceptor usage has been mapped primarily to the
envelope V3 loop (15, 19, 20, 28), while in non-subtype B
viruses, other portions of the envelope sequence such as the
V1-V2 loops may influence coreceptor usage (26). Because
coreceptor switching occurs less frequently in subtype A vi-
ruses (26, 29), envelope glycoprotein modifications that occur
over the course of infection may affect cell entry efficiency
primarily by altering CD4 or the CCR5 receptor utilization.
We have previously shown that evolution in the envelope gly-
coprotein V1-V2 loops can have modest effects on cell entry
efficiency in cells with limiting levels of receptors (76). Modi-
fications in the V1-V2 loops and other envelope segments are
constrained because antibody neutralization resistance needs
* Corresponding author. Mailing address: Brigham and Women’s
Hospital, Harvard Medical School, 65 Landsdowne Street, Room 447,
Cambridge, MA 02139. Phone: (617) 768-8372. Fax: (617) 768-8738.
?Published ahead of print on 22 July 2009.
to be attained while preserving the ability to bind host recep-
tors and enter cells. The effects of envelope sequence changes
that occur over the course of infection on host cell receptor
interactions and replicative capacity remain largely undefined,
especially for non-subtype B HIV-1.
In the present study, we incorporated previously isolated
unique early- and chronic-infection V1-V5 envelope segments
into a parental virus. As opposed to the majority of studies
which examine HIV-1 envelope glycoprotein phenotypes using
viral pseudotypes, we constructed replication-competent re-
combinant viruses. We found that engineered viruses with
V1-V5 segments from the chronic phase of infection had sig-
nificantly increased replication capacity compared to HIVs
with V1-V5 portions from early infection. In addition, variants
with chronic-infection V1-V5s also had significantly greater
replication capacity in cells with low CCR5 densities and lower
sensitivity both to the CCR5 antagonists TAK779 and PSC-
RANTES and to the fusion inhibitor T-20, compared to HIVs
with V1-V5 portions from early in infection. These properties
were associated primarily with sequence changes within the
V1-V3 envelope domains. These results in conjunction with
our previous studies suggest that envelope sequence changes
within the envelope V1-V3 domain may lead to antibody neu-
tralization resistance and a concomitant increase in the ability
to use the CCR5 receptor and faster fusion capacity.
MATERIALS AND METHODS
Subjects and envelope sequences. A first-round PCR product encompassing
V1-V5 sequences had been obtained previously from peripheral blood mononu-
clear cell samples from nine antiretroviral-naïve subtype A-infected women who
acquired HIV-1 through heterosexual contact (Table 1) (76). Each sequence was
derived from an independent PCR starting with one template copy to minimize
resampling bias (74). Previously, we isolated V1-V3 sequences from this first-
round product using a second nested PCR. In this study, we used the same
first-round amplified product to amplify V1-V5 sequences using primers Env10S
(5?-CACTTCTCCAATTGTCCCTCAT-3?; corresponding to nucleotide posi-
tions 7647 to 7668 in the HXB2 genome) and Env15 (5?-CCATGTGTAAAGT
TAACCCC-3?; HXB2 nucleotide positions 6576 to 6595). Amplified products
were cleaned using Qiaquick (Qiagen) and directly sequenced.
Construction of recombinant viral plasmids. We modified a previously de-
scribed yeast gap repair homologous recombination method (43) to incorporate
unique V1-V5 PCR-amplified envelope fragments into a full-length subtype A
HIV-1 clone (Q23-17) (63) (Fig. 1). To incorporate the Q23 HIV-1 sequences
into a Saccharomyces cerevisiae and Escherichia coli shuttle vector, NotI and
XhoI digestion was used to isolate the full-length Q23 sequence, and this frag-
ment was cloned into the multiple-cloning site of pRS315 (New England Bio-
labs). Within Q23, nef sequences were replaced by the HIS3 gene, which allows
yeast to synthesize histidine. The selection marker, HIS3, was amplified from
pRS313 (New England Biolabs) using primers 5?-AAGTGGTCAAAAAGTAGCA
TAGTTCTGGTGGCAATGATTGAAATAAATTCCCTTTAAGAGC-3? and 5?-
underlined portion of each primer denotes the segments homologous to Q23 nef
sequences. Yeast (strain S288C) was transformed with the HIS3 gene PCR
fragment and BmgBI-linearized pRS315-Q23 plasmid using the lithium acetate
(LiAc) technique (13). (BmgBI cuts within the Q23 nef sequence.) Transformed
yeast cells were selected and expanded on complete minimal medium (CMM)
without leucine or histidine. Yeast plasmids were extracted using glass beads, and
a portion of the crude extract was electroporated into E. coli. Transformed E. coli
were confirmed to have the plasmid pRS315-Q23?nef-HIS3 by restriction en-
zyme digestions. Within pRS315-Q23?nef-HIS3, the V1-V5 portion of the Q23
envelope gene was replaced by the selection marker URA3 to enable different
V1-V5 envelope segments to be shuttled into the plasmid. URA3 encodes the
orotidine 5?-phosphate decarboxylase protein involved in uracil biosynthesis. The
URA3 gene was amplified from pRS316 (New England Biolabs) using primers
C-3? and 5?-CACTTCTCCAATTGTCCCTCATATCTCCTGGTATTTCACAC
CGCAGGG-3?. The underlined segment of each primer highlights portions
homologous to Q23 envelope sequences (analogous to primers Env15 and
Env10S, respectively). Yeast were transformed with the URA3-amplified PCR
fragment and BglII-linearized pRS315-Q23?nef-HIS3 (BglII cuts within the
envelope V1-V5 region). Yeast cells were grown on CMM plates lacking leucine,
histidine, and uracil. Crude extract from a transformed yeast colony was elec-
troporated into E. coli to isolate and expand the plasmid pRS315-Q23?nef-
HIS3?V1-V5-URA3. Different V1-V5 envelope fragments were shuttled into
pRS315-Q23?nef-HIS3?V1-V5-URA3 using LiAc transformation. Briefly,
pRS315-Q23?nef-HIS3?V1-V5-URA3 was linearized with StuI, which cuts
within the URA3 gene. Linearized pRS315-Q23?nef-HIS3?V1-V5-URA3 and
amplified V1-V5 envelopes were used to transform yeast, and yeast was grown on
CMM plates lacking leucine and histidine and with 5-fluoro-1,2,3,6-tetrahydro-
2,6-dioxo-4-pyrimidine carboxylic acid (FOA). FOA inhibits the growth of yeast
that expresses the URA3 gene. Yeast colonies on CMM plates lacking leucine
and histidine and with FOA were screened for the HIV-1 envelope gene by PCR.
Plasmids were expanded in E. coli, and incorporation of the different V1-V5
envelope segments within Q23 was confirmed by sequence analysis.
Virus stocks and titers. Viral stocks were prepared by transiently transfecting
293T human embryonic kidney fibroblast cells using the Fugene protocol (Roche
Molecular Biochemicals). The 293T cells were maintained in Dulbecco’s modi-
fied Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 U of penicillin per ml, and 100 ?g of streptomycin per ml
(DMEM complete). After 48 h in culture, the supernatant was filtered through
a 0.45-?m filter, aliquoted, and stored at ?80°C until use. The infectious dose of
each virus stock was determined by infecting TZM-bl cells with a range of viral
dilutions by directly counting ?-galactosidase (?-gal)-positive “blue” foci at 48 h
postinfection as previously described (32, 76). TZM-bl cells were obtained
through the NIH AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH (NIH-ARRRP) (84).
TABLE 1. Subjects, sequences, and clones
Early infectionChronic infection
aDuration from the estimated date of infection to the day of sample collection.
bNumber of independent sequences analyzed.
cNumber of replication-competent recombinant viruses with unique V1-V5 segments.
VOL. 83, 2009USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME9695
Site-directed mutagenesis. The E370D mutation was introduced into pHXB-
2-env using the QuikChange site-directed mutagenesis kit (Stratagene) with
primers 5?-GGAGGGGACCCAGACATTGTAACGCACAG-3? and 5?-CTGT
GCGTTACAATGTCTGGGTCCCCTCC-3?. pHXB-2-env was obtained from
the NIH-ARRRP (52). The wild-type HXB-2 envelope plasmid and HXB-2
E370D mutated envelope plasmid were used to make virus pseudotypes with a
Q23?Env plasmid as previously described (41, 76). The YU2 K421D mutated
virus was constructed by site-directed mutagenesis of pYU2, using primers 5?-C
ACACTCCCATGTAGAATAGATCAAATTATAAATATGTGGC-3? and 5?-
was obtained from the NIH-ARRRP (39). Site-directed mutagenesis was per-
formed on an EcoRI/SalI segment of pYU2 subcloned into the multiple-cloning
site of pRS316 (NE Biolabs). The EcoRI/SalI portion with the site-directed
mutation was reinserted into the original pYU2 to obtain pYU2 K421D. The
HXB-2 E370D and YU2 K421D mutations were confirmed with sequence anal-
Sensitivity to inhibitors. Sensitivity to soluble CD4, PRO1008 (Progenics),
CCR5 antagonists TAK779 and PSC-RANTES, and fusion inhibitor T-20 was
assessed on TZM-bl cells. TAK779 and T-20 were obtained through the NIH-
ARRRP (2). Sensitivity was examined in 96-well plates in the presence of twofold
serial dilutions of the inhibitor. Soluble CD4 was incubated with 500 infectious
particles (IP) of each virus for 1 h at 37°C prior to adding 1 ? 104TZM-bl cells
in each well. TAK779, PSC-RANTES, or T-20 was incubated with 1 ? 104
TZM-bl cells for 1 h at 37°C prior to adding 500 IP of each virus. After 48 h, all
infections were assessed for ?-gal expression using Galacton-Light Plus (Applied
Biosystems). Relative light units in wells without any infectious virus were used
as the background level, and this was subtracted from the relative light units of
each well. The level of ?-gal expression in the presence of serially diluted
inhibitor versus medium alone was used to determine the percentage of inhibi-
tion. Data were fitted to estimate the 50% inhibitory concentration (IC50). All
IC50s were calculated from a minimum of two independent experiments, and
within each experiment all infections were performed in triplicate.
Replication kinetics on cells with various levels of cell surface receptors. Cell
lines with various CD4 and CCR5 surface receptor densities were kindly pro-
vided by Emily Platt and David Kabat (62). All cells were plated 1 day prior to
infection at a density of 4 ? 104cells per well in a 24-well dish. The next day, the
medium was removed and cells were incubated in the presence of 20 ?g/ml
DEAE-dextran with 500 IP in a total of 100 ?l of DMEM complete. After 2 h,
cells were washed with phosphate-buffered saline, and 1 ml of fresh DMEM
complete was added to each well. Cells were incubated at 37°C, and all of the
medium was collected and replaced with fresh DMEM complete on the first and
fourth days of infection. Cells were expanded to a T25 flask after the fourth day.
Medium was also collected on day 7 after infection. p24 levels were assessed on
medium collected from days 1, 4, and 7 after infection using a p24 enzyme-linked
immunosorbent assay (Perkin-Elmer). The day 7 p24 levels in the cells with
limiting levels of receptors were normalized relative to the day 7 p24 level in the
JC53 cells with high levels of CD4 and CCR5.
Determination of coreceptor use. Coreceptor usage was determined using the
GHOST cell assay as previously described (41, 48). Briefly, 4 ? 104
GHOST(3)CXCR4 or GHOST(3)X4/R5 cells were plated in a 24-well dish 1 day
prior to infection. GHOST(3)X4/R5 and GHOST(3)CXCR4 cells express the
CD4 receptor with both the CCR5 and the CXCR4 coreceptors or the CXCR4
coreceptor alone, respectively. In addition, green fluorescent protein (GFP) is
under the transcriptional control of the HIV-2 long terminal repeat in these cells.
Thus, infection can be assessed by flow cytometric measurement of GFP staining.
Cells were exposed to 10,000 infectious virions, and wells were washed 2 h after
exposure. Cultures were incubated for 2 days prior to fluorescence-activated cell
sorter analysis for GFP staining of 10,000 events.
Replication kinetics on MDMs. Peripheral blood mononuclear cells (PBMCs)
were isolated using the Ficoll-Hypaque density centrifugation method from buffy
FIG. 1. Construction of viruses using yeast gap repair homologous recombination. (A) A full-length HIV-1 clone was isolated and ligated into
the multiple-cloning site of pRS315 (New England Biolabs). pRS315 sequences permit plasmid replication in both bacteria and yeast, and pRS315
also contains the beta-isopropylmalate dehydrogenase gene for the leucine synthetic pathway (LEU2). Thus, yeast with the recombined plasmid
can be selected in leucine dropout media. (B) The yeast selection gene, for histidine (HIS3), was amplified with primers that contained the HIS3
gene sequences at the 3? end flanked by nef homologous sequences at the 5? end. This PCR product and the HIV-pRS315 plasmid linearized by
endonuclease digestion within the nef gene were used to transform yeast and recover the HIV-pRS315-?nef-HIS3 plasmid. (C) The URA3 gene
was amplified from pRS316 (New England Biolabs) using primers that contained the URA3 gene sequences at the 3? end flanked by sequences
homologous to the HIV-1 envelope. URA3 encodes the orotidine-5?-phosphatase decarboxylase protein involved in the biosynthesis of uracil. Yeast
was transformed with HIV-pRS315-?nef-HIS3 plasmid linearized by endonuclease digestion within the env gene and the URA3 PCR product.
Yeast with the recombined plasmid (HIV-pRS315-?nef-HIS3-?Env) was selected in leucine, histidine, and uracil dropout media. (D) Yeast was
transformed with the HIV-pRS315-?nef-HIS3-?Env plasmid linearized with endonuclease digestion within URA and an envelope PCR product
of interest. Recombined plasmid was isolated from yeast selected in leucine and histidine dropout media enriched with FOA. URA3 converts FOA
to a toxic product which inhibits yeast with URA3 expression.
9696ETEMAD ET AL.J. VIROL.
coats obtained from local blood banks. Monocytes were isolated using the Percoll
gradient method (14). Monocytes were incubated in macrophage SFM medium
(Gibco BRL) supplemented with 10% human serum, 5% fetal bovine serum, 1
mM L-glutamine, 100 U of penicillin per ml, and 100 ?g of streptomycin per ml
(complete macrophage medium) for 5 to 7 days prior to infection. Between 0.5 ?
106and 1.0 ? 106monocyte-derived macrophages (MDMs) were plated per well
in a 24-well plate. Cells were infected with 5,000 IP of virus in the presence of
20 ?g/ml DEAE-dextran. Cells were washed after 2 h of viral exposure. All of the
medium was collected and replaced with fresh complete macrophage medium on
days 1, 4, 7, 10, and 14. All viruses were evaluated for their ability to replicate in
MDMs from a minimum of two different blood donors. Replicative capacities of
all recombinant viruses with V1-V5 segments from the same subject were always
compared on MDMs obtained from the same donor.
Construction of chimeric V1-V5 envelope segments. Overlap PCR was used to
generate chimeric V1-V5 envelope fragments. The V1-V2 and C2-V5 envelope
portions were amplified from the plasmids with the V1-V5 envelope segment of
interest using primers Env 15 and 5?-TGAGGTATTACAATTTATTAATCTA
TA-3? and Env 10S and 5?-TATAGATTAATAAATTGTAATACCTCA-3?, re-
spectively. The V1-V3 and C3-V5 envelope domains were amplified from the
V1-V5 envelope segment of interest using primers Env15 and 5?-GTGTTGTA
ATTTCTAGATCCCCTCCTG-3? and Env 10S and 5?-CAGGAGGGGATCTA
GAAATTACAACAC-3?, respectively. Amplified segments were gel purified
and combined in a PCR with primers Env10S and Env15 to construct the
chimeric V1-V5 domains. Construction of all chimeric V1-V5 envelope segments
was verified by sequence analysis.
Statistical analysis. The average from the multiple independent experiments
was calculated for each recombinant virus for the replicative-capacity and inhib-
itor sensitivity assays. Differences among early- and chronic-infection viruses
were assessed by two independent statistical tests. First, the value from the
early-infection V1-V5 recombinant virus was compared to the median value for
the multiple viruses with chronic-infection V1-V5 segments using the Wilcoxon
matched-pairs signed-rank test. Second, the Exact Wilcoxon-Mann-Whitney test,
stratified by subject, was used for an aggregate comparison between early- and
chronic-infection viruses. The two tests showed that the same properties were
significantly different (P ? 0.05) between the early- and chronic-infection vari-
ants except for replication in the cells with low CCR5 levels; in this case the Exact
Wilcoxon-Mann-Whitney test demonstrated a significant difference, while with
the Wilcoxon matched-pairs signed-rank test a trend was observed (P ? 0.1).
Only the P values from the Exact Wilcoxon-Mann-Whitney test are presented
because it accounts for the measurements from all the multiple chronic-infection
variants as opposed to reducing them into a single median value. All P values are
based on a two-sided test. All statistical analyses were done with Intercooled
Stata version 8.0 (Stata Corporation, College Station, TX) and SAS version 8.2
(SAS Institute, Cary, NC).
V1-V5 sequences. Sequences were generated from samples
taken at 1 to 6 months postinfection and from approximately
24 to 47 months after estimated infection as described previ-
ously (Table 1) (76). The median interval of time for the
analyzed sequences was about 35 months (range, 18 to 46
months). In previous studies, we have shown that five (QA284,
QA779, QB670, QC168, and QC449) of the nine subjects ex-
amined in this study had strictly homogenous envelope se-
quences early in infection (74, 75). Thus, for these subjects,
only one early-infection V1-V5 sequence was isolated, and for
the remaining four subjects, multiple sequences (median, 3;
range, 3 to 4) were isolated from early in infection (Table 1).
For all subjects, multiple sequences (median, 4; range, 2 to 9)
were isolated and examined from the chronic-infection sample.
Similar to our previous results, early-infection sequences
showed a significantly lower median number of predicted N-
linked glycosylation sites (PNGS) within the V1-V2 domain
(median, 5; range, 3 to 8) than did sequences from the chronic
phase of infection (median, 6; range, 4 to 9; P ? 0.03) (76).
There was no significant difference in V1-V2 and V1-V4 length
or in the number of V1-V4 PNGS between the early- and
chronic-infection sequences. As before, neighbor-joining phy-
logenetic analysis showed that sequences at both time points
within each subject clustered together, suggesting that there
was no contamination or reinfection by two different partners
(data not shown) (76).
Generation of replication-competent recombinant viruses.
To assess the effects on envelope function from the changes
that occur in the V1-V5 segments over time, we constructed
replication-competent recombinant viruses incorporating dif-
ferent V1-V5 sequences within a HIV-1 subtype A Q23-17
background. Q23-17 is a replication-competent clone derived
from a subject’s sample approximately 1 year after estimated
infection (63). Because sequences early in infection are rela-
tively homogeneous, only the predominant V1-V5 sequence
from early in infection was incorporated into the full-length
HIV-1 clone. All unique chronic-infection V1-V5 segments,
however, were inserted into the Q23-17 HIV-1 clone. The
median titer of the replication-competent recombinant viruses
was 5.4 ? 105IP per ml (range, 1.0 ? 105to 2.1 ? 106). Some
chronic-infection V1-V5 variants in QA203 (one of four),
QA779 (one of seven), QB424 (three of four), QB596 (one of
eight), QB670 (nine of nine), QC168 (one of five), and QC890
(one of five) did not yield an infectious titer greater than
20 IP/ml, the limit of detection for the titer assay (Table 1). No
further studies were pursued with the QB670 envelopes from
either time point because no replication-competent recombi-
nant virus with the QB670 V1-V5 chronic-infection sequence
could be generated. It should be noted that we introduced
V1-V5 segments within a heterologous backbone, and thus
these constructs may not accurately reflect the properties of
the original full-length parent envelope, which is potentially
evident by the nonfunctional envelopes.
Replication capacity. We examined replication efficiency dif-
ferences among viruses with V1-V5 envelope segments from
different times during infection in a cell line with high levels of
the CD4 and CCR5 receptors, JC53 (62). Thus, host cell re-
ceptors were at a consistently high level in all infections, as
opposed to the case for PBMCs, where there is extensive donor
variability, especially in CCR5 density (47, 69). All recombi-
nant viruses showed more than a 100-fold increase in p24
antigen levels from day 1 (median, 4.2 pg/ml; range, 0 to
20.8 pg/ml) to day 7 (median, 1,105 pg/ml; range, 222.9 to
5,534.0 pg/ml), suggesting that all viruses replicated in cells
with high CD4 and high CCR5 cell surface receptor concen-
trations. In all subjects, early-infection variants replicated to a
lower level than the median for the chronic-infection viruses,
although these differences in some individuals (QB596 and
QC449) were relatively small (Fig. 2). Early-infection viruses
had significantly lower p24 levels (median, 713.0 pg/ml; range,
222.9 to 2,681.4 pg/ml) at day 7 after infection in the JC53 cells
than the chronic-infection variants (median, 1,527.8 pg/ml;
range, 727.1 to 5,534.0 pg/ml) (P ? 0.01).
Receptor utilization. Because host cell entry is the rate-
limiting step in HIV-1 replication (3, 44, 65) and entry is
potentially limited by receptor binding and fusion (46, 61), we
hypothesized that early- and chronic-infection viruses had dif-
ferences in receptor attachment and fusion kinetics. Previous
studies suggest that sensitivity to receptor inhibitors correlates
with the affinity of the viral envelope for the host cell receptor
(59, 66). Sensitivities to a small-molecule CCR5 inhibitor,
VOL. 83, 2009 USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME 9697
TAK779 (2), and to a soluble CD4 molecule, PRO1008-1 (Pro-
genics), were used as surrogate markers to measure CCR5 and
CD4 utilization, respectively. To demonstrate that inhibitor
sensitivity correlates with binding capacity, we tested viruses
with previously documented receptor affinity differences. A
YU2 envelope with a lysine (K)-to-aspartic acid (D) mutation
at position 421 (K421D) within the envelope bridging sheet
region has markedly lower CCR5 binding than the wild-type
YU2 (71). The YU2 K421D virus (IC50, 2.1 nM ? 0.6 nM) had
an approximately 18-fold greater sensitivity to TAK779 than
the wild-type YU2 virus (IC50, 37.7 nM ? 12.6 nM). The
HXB2 envelope with a glutamic acid (E)-to-aspartic acid (D)
mutation at envelope position 370 (E370D) results in de-
creased affinity for the CD4 receptor compared to the wild-
type HXB2 envelope (49). HXB-2 E370D pseudotypes (IC50,
1.3 ?g/ml ? 1.1 ?g/ml) had approximately 13-fold greater
sensitivity to soluble CD4 than wild-type HXB-2 pseudotypes
(IC50, 27.3 ?g/ml ? 3.4 ?g/ml).
To document differences in receptor utilization between chi-
meric envelopes with V1-V5 segments from the early and
chronic phases of infection, we examined sensitivity to TAK779
and soluble CD4. The various recombinant viruses with differ-
ent V1-V5 envelope segments demonstrated large variations in
TAK779 IC50s, from 0.4 to 156.8 nM (Fig. 3A). In all subjects
(except QB424), early-infection HIVs had lower TAK779
IC50s than the median TAK779 IC50s from all the chronic-
infection viruses, although in QC449, the difference was less
than twofold. In aggregate, early-infection variants (median,
10.0 nM; range, 7.1 to 105.2 nM) showed approximately four-
to fivefold greater sensitivity to TAK779 than viruses with
chronic-infection V1-V5 domains (median, 46.4 nM; range, 0.4
to 156.8 nM) (P ? 0.01).
TAK779 is an allosteric inhibitor (2), and some envelopes
with acquired resistance to small-molecule CCR5 inhibitors
display continued susceptibility to CCR5 chemokines (64, 81),
which suggests that sensitivity to TAK779 may not necessarily
correlate with an ability to utilize the native CCR5 receptor.
Thus, we examined the sensitivities of the acute- and chronic-
infection envelopes to a CCR5 chemokine, PSC-RANTES, a
competitive inhibitor. Sensitivity to PSC-RANTES varied from
0.01 to 0.81 nM (Fig. 3B). Similar to TAK779, early-infection
variants (median, 0.18 nM; range, 0.04 to 0.45 nM) were sig-
nificantly more sensitive to PSC-RANTES than the chronic-
infection viruses (median, 0.37 nM; range, 0.01 to 0.81 nM)
(P ? 0.002).
Chronic-infection viruses may potentially have decreased
CCR5 inhibitor sensitivity compared to early-infection variants
because they may use CXCR4 receptor for cell entry. To ex-
clude this possibility, we investigated coreceptor usage among
our recombinant viruses using the GHOST cell assay. GFP
staining was observed in more than 5% of the GHOST(3)
CXCR4 cells after infection with a known CXCR4-using virus
(LAI) (56) and in fewer than 0.2% of the cells with a CCR5-
exclusive virus (JRCSF) (6). Recombinant viruses with early-
or chronic-infection V1-V5 envelope segments showed a me-
dian of 0.2% GFP-positive cells (range, 0.1% to 0.3%). On the
other hand, a median of 4% (range, 2% to 16%) of
GHOST(3)X4/R5 cells were GFP stained after infection with
the same recombinant viruses. This suggests that none of the
recombinant viruses utilized the CXCR4 coreceptor for cell
entry. Thus, chronic-infection viruses do not have decreased
sensitivity to CCR5 antagonists because of the ability to use
CXCR4 as a coreceptor.
Among the diverse viruses, soluble CD4 IC50s varied
from 6.2 to 22.4 ?g/ml (Fig. 3C). Unlike TAK779 and PSC-
RANTES sensitivity, in aggregate, early-infection variants
showed no significant difference in soluble CD4 sensitivity
(median, 13.6 ?g/ml; range, 10.5 to 19.7 ?g/ml) compared to
chronic-infection viruses (median, 13.3 ?g/ml; range, 6.2 to
22.4 ?g/ml) (P ? 0.9).
To further confirm that chronic- versus early-infection vi-
ruses had an increased ability to use CCR5 and no significant
FIG. 2. Replication in cells with high levels of CD4 and CCR5 receptors (JC53). Each graph shows the p24 production 7 days after infection
for virus with early-infection V1-V5 segments (black bars) and viruses with chronic-infection V1-V5 sequences (white bars). Note that the y axis
scale, which depicts p24 levels, is different in each graph. The subject identification is denoted above each graph, and the V1-V5 segment
identification is below each column. All infection levels represent mean values from two or more independent experiments. The error bars show
the standard deviations.
9698ETEMAD ET AL. J. VIROL.
FIG. 3. Sensitivity to TAK779 (A), PSC-RANTES (B), and soluble CD4 (C) of viruses with early-infection V1-V5 portions (black bars) or
chronic-infection V1-V5 segments (white bars). The y axis shows the IC50s for each inhibitor. Note that the y axis scale is different in each graph.
The subject identification is denoted above each graph, and the V1-V5 segment identification is below each column. All IC50s represent mean
values from two or more independent experiments. The error bars show the standard deviations.
difference in CD4 utilization, we examined replication capacity
in cells with limiting levels of receptors. We reasoned that
viruses with an envelope glycoprotein that has a greater ability
to utilize the CCR5 receptor should be able to replicate effi-
ciently in JC10 cells, which have a low CCR5 density and high
levels of CD4. Furthermore, viruses with envelopes that have
higher CD4 binding should have better replication in RC49
cells, which express low levels of CD4 and medium density of
CCR5. As controls, we observed that the YU2 K421D virus
was unable to replicate in the JC10 cells with low CCR5 den-
sity. On the other hand, the wild-type YU2 produced 39.4% ?
7.9% p24 in JC10 cells relative to the JC53 cells with high
levels of CCR5 at day 7 after infection. Entry in the RC49 cells
with low CD4 densities relative to the JC53 cells with high CD4
and high CCR5 concentrations was around fourfold lower for
the HXB2 E370D (6.1% ? 1.1%) compared to the wild-type
HXB-2 pseudotypes (25.3% ? 3.5%) at day 2 after infection.
In the JC10 cells with low CCR5 receptor concentrations,
relative infection levels at day 7 after infection varied from 0.3
to 29.9% for the early- and chronic-infection viruses (Fig. 4A).
In all subjects except QB424, early-infection variants had lower
relative infection levels in JC10 cells than the median of the
relative infection levels of the chronic-infection viruses. These
differences were less than twofold in two subjects (QB596 and
QC449). Recombinant viruses with early-infection V1-V5 seg-
ments (median, 6.6; range, 0.4 to 21.7) showed significantly
lower relative infection levels in the JC10 cells than engineered
HIVs with V1-V5 portions from the chronic phase of infection
FIG. 4. Relative replication in cells with high CD4 and low CCR5 levels (JC10) (A) and in cells with low CD4 and medium CCR5 levels (RC49)
(B) among viruses with early-infection V1-V5 portions (black bars) or chronic-infection V1-V5 segments (white bars). In each graph, the y axis
shows the infection levels in the respective cell lines. Note that the y axis scale is different in each graph. These values are normalized relative to
the infection levels in the high-CD4, high-CCR5 (JC-53) cell line. The subject identification is denoted above each graph, and the V1-V5 segment
identification is below each column. All infection levels represent mean values from two or more independent experiments. The error bars show
the standard deviations.
9700ETEMAD ET AL.J. VIROL.
(median, 18.3%; range, 0.3 to 29.9%) (P ? 0.01). In the RC49
cells with low levels of CD4, relative infection levels varied
from 0 to 41.2% (Fig. 4B). There was no significant difference
in relative infection levels in the RC49 cells between the early-
infection variants (median, 5.5; range, 1.0 to 15.1) and the
chronic-infection variants (median, 4.8; range, 1.7 to 10.9)
(P ? 0.9).
Because early- and chronic-infection envelopes from the
eight different subjects showed similar relationships between
CCR5 inhibitor IC50s and replicative capacity in the low-CCR5
cells, we examined the correlation between these assays. The
TAK779 IC50was significantly correlated with relative infec-
tion levels in the low-CCR5 cells (? ? 0.6, P ? 0.001; Spear-
man rank correlation) (Fig. 5A) and with sensitivity to PSC-
RANTES (? ? 0.8, P ? 0.001; Spearman rank correlation)
(Fig. 5B). Although none of these assays directly measures
affinity for the CCR5 receptor, collectively our results suggest
that they may be highly correlated surrogate markers for an
envelope’s ability to bind the CCR5 receptor. In contrast, the
soluble CD4 IC50showed no significant correlation with rela-
tive infection levels in the low-CD4, medium-CCR5 cell line
(? ? 0.03, P ? 0.9; Spearman rank correlation), suggesting that
these surrogate markers for CD4 use may be influenced by
other factors, such as gp120 shedding or the ability to bind the
coreceptor after CD4 engagement.
Fusion capacity. Differences in cell entry and replication
between early- and chronic-infection viruses may also be influ-
enced by fusion kinetics. Previous studies have suggested that
sensitivity to the fusion inhibitor T-20 correlates with fusion
kinetics (66). Although all envelopes harbored an isogenic
transmembrane domain, gp41, the diverse recombinant viruses
showed great variation in T-20 IC50s from 0.02 to 2.8 ?g/ml
(Fig. 6). In all subjects except QC890, early-infection HIVs had
T-20 IC50s lower than the median T-20 IC50s from all the
chronic-infection viruses, although in three subjects (QB424,
QB596, and QC449) the difference was less than twofold.
Early-infection variants (median, 0.3 ?g/ml; range, 0.2 to 1.5 ?g/
ml) were approximately two- to threefold more sensitive to
T-20 than viruses with chronic-infection V1-V5 domains (me-
dian, 0.8 ?g/ml; range, 0.3 to 1.4 ?g/ml) (P ? 0.01; Wilcoxon
matched-pairs signed-rank test).
FIG. 5. Association between sensitivity to TAK779 and both rela-
tive replication in cells with high CD4 and low CCR5 levels (JC10)
(A) and sensitivity to PSC-RANTES (B). The y axis shows the relative
infection in JC10 cells (A) and sensitivity to PSC-RANTES (B). The x
axis shows the TAK779 sensitivity for each chimeric envelope, repre-
sented by individual dots. The line shows the best-fit linear regression
curve, with the correlation coefficient listed on the top of the graph.
FIG. 6. Sensitivity to fusion inhibitor T-20 among viruses with chronic-infection V1-V5 segments (white bars) versus those with early-infection
V1-V5 portions (black bars). The y axis shows the IC50s against the fusion inhibitor. Note that the y axis scale is different in each graph. The subject
identification is denoted above each graph, and the V1-V5 segment identification is below each column. All IC50s represent mean values from two
or more independent experiments. The error bars show the standard deviations.
VOL. 83, 2009USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME9701
Genotypic mapping of the differences to CCR5 and fusion
inhibitors. To identify the envelope determinants for the
difference in CCR5 utilization and fusion capacity among ear-
ly- and chronic-infection V1-V5 segments, we constructed chi-
meric V1-V5 envelope domains. Chimeric V1-V5s were cre-
ated with the subject’s early-infection V1-V5 envelope portion
and the chronic-infection V1-V5 envelope segment that dem-
onstrated the highest IC50against CCR5 inhibitors in seven
subjects. No chimeric envelopes were generated from QC449
V1-V5 segments because viruses with early- and chronic-infec-
tion envelope regions from this subject showed minimal dif-
ferences in CCR5 utilization (Fig. 3A and B and 4A). Overlap
PCR was used to create four sets of chimeric V1-V5 segments
for each subject. We constructed V1-V5s with (i) early-infec-
tion V1-V2 and chronic-infection C2-V5 (V1V2E-C2V5L), (ii)
chronic-infection V1-V2 and early-infection C2-V5 (V1V2L-
C2V5E), (iii) early-infection V1-V3 and chronic-infection
C3-V5 (V1V3E-C3V5L), and (iv) chronic-infection V1-V3 and
early-infection C3-V5 (V1V3L-C3V5E) segments. These chi-
meric V1-V5 envelope segments were incorporated in the full-
length Q23 clone using the yeast gap repair homologous re-
combination system. In all cases except QA203, the TAK779
IC50s of the V1-V3/C3-V5 chimeras were within twofold of
that of the parent sequence from which the V1-V3 portion was
obtained (Fig. 7A). Thus, the V1V3L-C3V5E chimeras com-
posed of chronic-infection V1-V3 and early-infection C3-V5
segments showed TAK779 IC50s similar to that of the parent
virus with the chronic-infection V1-V5 segment, and the V1-
V3E-C3V5L showed TAK779 sensitivity similar to that of the
virus with early-infection V1-V5 domains. The V1-V3/C3-V5
chimeras’ sensitivity to PSC-RANTES was also similar to that
of the parent sequence from which the V1-V3 portion was
obtained (Fig. 7B). In the cases where the chronic-infection
variant replicated more efficiently than the early-infection vari-
ant in the cells with low CCR5 densities (JC10), relative rep-
lication in the JC10 cells was higher among the V1V3L-C3V5E
than among the V1V3E-C3V5L chimeras, except in QA203
(Fig. 7C). In QB424, where QB424-17E replicated more effi-
ciently than QB424-5C in JC10 cells, the V1V3E-C3V5L pro-
duced greater relative amounts of p24 than the V1V3L-
C3V5E. Similarly, except for QB424, the V1V3L-C3V5E
versus the V1V3E-C3V5L chimeras were less sensitive to fu-
sion inhibitor T-20, if the chronic- compared to the early-
infection V1-V5 showed a higher T-20 IC50(Fig. 7D). In
QC890, where QC890-15E was mildly less sensitive to T-20
than QC890-16C, the V1V3E-C3V5L chimera also had a min-
imally higher T-20 IC50than the V1V3L-C3V5E chimera. Col-
lectively, this suggests that V1-V3 sequences mainly influence
the difference in CCR5 utilization and fusion capacity between
early- and chronic-infection viruses. Interestingly, in all cases
except QC890, viruses with early-infection V1-V2 and chronic-
infection C2-V5 portions consistently demonstrated greater
resistance to the CCR5 antagonists and higher replication ca-
pacity in cells with low CCR5 receptor densities than viruses
with chronic-infection V1-V2 and early-infection C2-V5 seg-
ments (Fig. 7A, B, and C). In most cases, these differences
among the V1-V2/C2-V5 chimeras, however, were relatively
small. A similar pattern was not observed among the V1-V2/
C2-V5 chimeras for T-20 sensitivity (Fig. 7D). Thus, the spe-
cific envelope segment (V1-V2 or V3) that influence CCR5
usage and fusion capacity differences among early- and chronic-
infection variants cannot be identified among these different
Replication capacity in MDMs. To examine the biological
relevance of CCR5 utilization differences among early- and
chronic-infection viruses, we examined replication capacity in
primary cells with known limiting levels of CD4 and CCR5
receptors. Macrophages express lower surface levels of both
CD4 and CCR5 compared to CD4?T cells (17, 37, 50, 55, 88).
In addition, viruses that use the CCR5 coreceptor often have
large differences in macrophage tropism (60), and this could
potentially relate to enhanced CCR5 usage (40). MDMs were
isolated from PBMCs using standard Percoll gradient methods
and infected with different viruses. Infections were monitored
by p24 antigen levels at days 1, 4, 7, 10, and 14.
Recombinant viruses with a subject’s V1-V5 segments from
early and chronic infection displayed various replication capac-
ities in MDMs (Fig. 8). There was no consistent pattern in the
replication difference between recombinant viruses with early-
versus chronic-infection V1-V5 segments. In some subjects
(QA203, QA284, and QA779), all viruses with chronic- versus
early-infection V1-V5s showed higher replication in MDMs. In
one subject (QB596), the virus with early-infection V1-V5
demonstrated greater replication in MDMs than the recombi-
nant viruses with chronic-infection V1-V5s. In the remaining
subjects, there was no significant difference in replication ca-
pacity between viruses with early- and chronic-infection V1-V5
segments. In addition, there was no significant correlation be-
tween the highest p24 level in MDMs over the course of in-
fection and CCR5 inhibitor IC50s, soluble CD4 IC50s, and
replicative capacity in cells with low CD4 or low CCR5 densi-
ties. These data suggest that CCR5 utilization differences
among early- and chronic-infection viruses do not confer rep-
lication capacity differences in MDMs.
In this study of sequences from eight individuals with HIV-1
subtype A infection, we showed that modifications within the
V1-V5 envelope segments confer increased replication capac-
ity over the course of infection (Fig. 2). Previous studies with
subtype B HIV-1 have also demonstrated that ex vivo replica-
FIG. 7. Early and chronic chimeric envelope TAK779 IC50s (A), PSC-RANTES IC50s (B), relative replication in high-CD4, low-CCR5 cells
(JC10) (C), and T-20 IC50s (D). V1V2E-C2V5L (blue), V1V2L-C2V5E (green), V1V3E-C3V5L (red), and V1V3L-C3V5E (yellow) chimeras are
shown. For reference, viruses with early-infection V1-V5 (black bars) and chronic-infection V1-V5 (white bars) segments from which the envelope
portions for the chimeras were derived are also shown. The y axis shows the IC50s for each inhibitor (A and C) and relative replication levels (B).
The y axis scale is different among the graphs. Subject identifications are denoted above each graph. All values represent mean values from two
or more independent experiments with viral stocks from two separate preparations. The error bars show the standard deviations.
VOL. 83, 2009USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME9703
tion capacity increases over the course of infection (4, 34, 80).
Similarly, in the simian immunodeficiency virus/macaque
model, longitudinally collected viruses demonstrate greater in
vivo replication than variants isolated from early in infection
(31). We also showed that longitudinally isolated V1-V5 enve-
lope segments conferred significantly increased resistance to
CCR5 inhibitors, TAK779 and PSC-RANTES (Fig. 3A and B).
In addition, we demonstrated that in the majority of subjects,
V1-V5 domains from the chronic phase of infection, compared
to those from early after HIV-1 acquisition, led to increased
replication capacity in cells with low CCR5 densities (Fig. 4A).
From these significantly correlated measures of CCR5 use
(Fig. 5), we concluded that viruses from the chronic phase of
infection are more efficient at utilizing CCR5 than variants
isolated early after infection. Among the longitudinally col-
lected variants from the different subjects, we also found that
resistance to fusion inhibitors increases over time, suggesting
that chronic-infection viruses are more fusogenic than isolates
from early in infection (Fig. 6). Previous studies and our con-
trols suggest that these measurements could be surrogate
markers for coreceptor affinity and fusion kinetics (59, 66).
Because cell entry is potentially the rate-limiting step in HIV-1
replication (3, 44, 65), in the aggregate, our data suggest that
increases in replication capacity over the course of infection
are potentially related to greater affinity for the CCR5 receptor
and/or faster fusion kinetics.
A previous study has suggested that acute- and chronic-
infection variants are not significantly different in their sensi-
tivities to CCR5 and fusion inhibitors (73). In that study, how-
ever, the virus isolates from the acute and chronic phases of
infection were obtained from different subjects. In contrast to
that publication and similar to our results, other studies which
have examined longitudinally collected variants have suggested
that viruses become more resistant to CCR5 and fusion inhib-
itors over time (27, 33, 68). In these studies, PBMC cocultures
were used to demonstrate that viruses from the chronic phase
of infection have differential susceptibility to receptor and fu-
sion inhibitors compared to late-stage variants. In contrast to
these studies, we examined subtype A viruses as opposed to the
presumably subtype B HIVs examined in the previous publi-
cations. In addition, we avoided in vitro adaptation that may
occur among viruses in PBMC cocultures by amplifying V1-V5
envelope segments and incorporating them into a full-length
HIV-1 clone using yeast gap repair methodology. One of the
major differences between our study and previous publications,
however, is that we examined differences among variants from
the early and chronic phases of infection as opposed to com-
paring viruses from the chronic and late stages of disease.
Collectively, these studies suggest that both subtype A and B
HIVs found early in infection are extremely sensitive to CCR5
antagonists and fusion inhibitors, and sensitivity to these com-
pounds progressively decreases over later times in infection.
Studies from our group and others demonstrate that there is a
strong correlation between sensitivity to TAK779 and clinically
relevant CCR5 antagonists, such as maraviroc and vicriviroc
(81, 87; T. Henrich, M. Sagar, and D. Kuritzkes, unpublished
data). Potential implications from these studies are that che-
mokine antagonists and fusion inhibitors may be ideal thera-
peutic drugs early in infection. In addition, although CCR5
utilization increases over time, HIV-1 infection starts with vari-
ants highly sensitive to CCR5 inhibitors, which further justifies
the exploration of CCR5 inhibition as a potential means to
interrupt transmission (36, 83).
Another major difference between our work and the major-
ity of previously published studies is that we examined enve-
lope phenotypic differences using replication competent vi-
ruses as opposed to viral pseudotypes. We employed a
modified yeast gap repair homologous recombination system
to generate a large number of recombinant replication-com-
petent viruses (Fig. 1). Within the HIV field, the majority of
studies examining envelope glycoprotein phenotypic differ-
ences employ virus pseudotypes. Although, pseudoviruses are
highly conducive to high-throughput analysis of a large number
of envelopes, there are a number of inherent limitations. First,
FIG. 8. Replication in MDMs. Each graph shows the p24 production of virus with early-infection V1-V5 segment (filled symbols) and viruses
with chronic-infection V1-V5 sequences (open symbols) over time in MDMs. Note that the y axis scale, which depicts p24 levels, is different in each
graph. The subject identification is denoted above each graph, and the V1-V5 segment identification is documented in the insets.
9704ETEMAD ET AL.J. VIROL.
the virus pseudotypes are restricted to a single round of rep-
lication, and thus, phenotypic differences conferred by the viral
envelope glycoproteins, such as replication capacity, cannot be
examined over multiple replication cycles. Second, pseudovi-
ruses often display different phenotypes compared to replica-
tion-competent viruses. For instance, single-cycle pseudotypes
compared to replication-competent chimeric viruses have
demonstrated different sensitivities to CCR5 inhibitors on the
same target cells (64). Third, the number of envelope glyco-
proteins expressed on a viral particle may be different among
pseudoviruses versus replication-competent viruses (60). This
may relate to differences in the number of defective envelopes
and glycoprotein processing among the two types of recombi-
nant viruses. Envelope glycoprotein properties need to be ex-
amined in more detail among replication-competent recombi-
nant viruses and viral pseudotypes to definitely document the
differences among these two virus constructs.
The exact biological mechanism for increased CCR5 utili-
zation among chronic-stage viruses compared to variants early
after infection remains unclear. Although, TAK779 is an allo-
steric (2) and not a competitive inhibitor, TAK779 sensitivity
correlates with affinity for the CCR5 receptor (66). Further-
more, TAK779 IC50s correlate with sensitivity to the CCR5
competitive inhibitor RANTES (Fig. 5B) (40, 68). Thus, we
suggest that increased CCR5 usage is because of a greater
affinity for the CCR5 receptor. Another potential mechanism
for higher CCR5 utilization is that chronic-stage envelopes
could bind a broader array of CCR5 conformations. Indeed,
natural CCR5 ligands, such as RANTES, can trigger internal-
ization, as well occupy and presumably distort the receptor
(54). Thus, in vivo, the CCR5 receptor may have different
conformations in the presence of chemokines. It has been
shown that envelopes from the late phase of infection com-
pared to the variants from the chronic stage of disease display
an increased ability to bind a broad range of chimeric CCR5
receptors (30). Therefore, an ability to bind different structural
forms of the CCR5 receptor may explain the increased CCR5
usage among chronic-stage variants compared to the early-
infection isolates. Finally, it also possible that chronic- and
early-stage viruses may have similar binding to the CCR5 re-
ceptor, but after CCR5 attachment, viruses with chronic- ver-
sus early-stage V1-V5s may have a higher propensity for pro-
ceeding to fusion; this may account for the differences in CCR5
utilization. Previous studies have suggested that CCR5 affinity
is directly correlated with fusion capacity (66), and thus viruses
with chronic- versus early-infection V1-V5s may possess both
greater affinity for the CCR5 receptor and increased fusion
capacity. Thus, our studies cannot distinguish whether in-
creased replication capacity among the chronic-stage variants
is due to increased CCR5 utilization and/or faster fusion ki-
Interestingly, both sensitivity to CCR5 inhibitors and differ-
ential fusion capacity have been previously mapped to se-
quence changes within the envelope V3 loop and the bridging
sheet, which are important for coreceptor binding (16, 66, 67).
Among our eight subjects, differences among the early- and
chronic-infection envelope sequences were evident primarily in
the V3 loop and not in the bridging sheet, which consists of ?
strands 2, 3, 20, and 21 (35, 53). V3 loop modifications, how-
ever, are not solely responsible for the phenotypic differences
observed among early- and chronic-infection envelopes. Our
chimeric envelope data suggest that sequence changes within
the V3 loop in conjunction with differences in the V1-V2 loops
influence CCR5 usage (Fig. 7A, B, and C). Furthermore, al-
though each envelope harbored an isogenic transmembrane
domain, notable differences were observed in the sensitivity to
fusion inhibitor T-20 among early- and chronic-infection
V1-V5 segments, and our envelope chimera data suggest that
changes within the V1-V3 domains also affected this pheno-
type (Fig. 7D). Because of numerous and diverse changes in
the V1-V3 domains between the early- and chronic-infection
envelope isolates, we were unable to identify canonical enve-
lope modifications as being responsible for the enhanced
CCR5 utilization and fusion capacity. Interestingly, previously
identified polymorphisms, such as changes at positions 318 and
319 of the V3 loop (HXB2 numbering), which have been as-
sociated with differential sensitivity to CCR5 inhibitors (40),
were highly conserved among early- and chronic-infection en-
velope sequences. The 318/319 consensus tyrosine (Y)/alanine
(A) motif was modified to serine (S)/A in QA284-8C, Y/threo-
nine (T) in QA779-5C and QA77913C, and Y/glycine (G)
among all QC449 variants. Thus, in our study, these exclusive
318/319 modifications within the V3 loop did not influence the
majority of observed changes in CCR5 usage. Our studies
contrast with other publications potentially because of the dif-
ferences in the subtype of the virus and because we examined
longitudinally isolated viral variants from natural infection as
opposed to laboratory-derived viral strains.
Progressively decreasing sensitivity to CCR5 entry inhibitors
provides insight into the selection mechanisms acting on the
virus during the course of infection within a host. The host
antibody neutralizing response is a well-described selection
force that drives evolution in the HIV-1 envelope gene (10, 21,
85). Our results imply that receptor use may be another po-
tential selection mechanism driving changes in the viral enve-
lope glycoprotein. After HIV-1 acquisition, a large percentage
of memory CD4?CCR5?T cells are eliminated from the
mucosal tissues (5, 45). In addition, CCR5 receptor levels on
the remaining target cells may be downregulated through high-
level expression of chemokines such as RANTES, MIP-1?, and
MIP-1? (1, 79). Both processes likely decrease the availability
of CD4?T cells with high levels of the CCR5 receptor. The
dearth of these cell types potentially forces HIV-1 to evolve
envelopes with an increased ability to use low levels of the
CCR5 receptor and/or switch to CXCR4 use late in infection.
The need to evolve a greater ability to use low levels of CCR5
is likely especially true for subtype A HIV-1, where use of
other coreceptors, such as CXCR4, has been infrequently doc-
umented (26, 29). It should be noted, however, that not all
individuals, such as QB424, displayed an enhanced ability to
use CCR5 over time, suggesting that the potential selection
forces or virus responses may be different in some hosts.
Chronic-stage viruses showed increased CCR5 utilization
compared to early-phase viruses even though envelope glyco-
proteins expand variable loops and increase the number of
glycosylated amino acids. Similar to our previous investigations
with the same subjects (76), we found that chronic-stage en-
velopes had a significantly higher number of glycosylated res-
idues than viruses early after HIV-1 acquisition. Envelope vari-
able loop expansion and increased glycosylation likely evolve
VOL. 83, 2009 USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME9705
to conceal conserved antigenic portions on the viral envelope
glycoprotein, such as the CD4 and/or the coreceptor binding
site (35, 53), from the host neutralizing antibody response.
Shielding these domains, however, may hinder access of the
viral envelope glycoprotein for the host cell receptors. Our
results imply that increased glycosylation did not adversely
affect CCR5 binding. Interestingly, we also found no difference
either in sensitivity to a CD4 inhibitor, soluble CD4, or in
replication capacity in cells with limiting levels of the CD4
receptor among chronic- and early-stage envelope V1-V5 seg-
ments (Fig. 3B and 4B). It has been hypothesized that HIV-1
envelopes with an increased ability to use low levels of CD4
evolve in the absence of humoral immune pressure, such as in
the central nervous system, an immunologically privileged site
(18, 57). Our data suggest that the converse does not neces-
sarily hold, because neutralizing antibodies, which we have
previously documented in these subjects (76), do not lead to
envelope modifications that decrease the efficiency of CD4
After observing increased CCR5 utilization among chronic-
stage envelopes, we hypothesized that envelopes from late in
infection were more likely to replicate in primary cells with
limiting levels of CCR5, such as macrophages. Indeed, some
previous studies suggest that isolates from late in disease are
more macrophage-tropic than those from early in infection
(23, 38, 82). Furthermore, it has been suggested that higher
CCR5 binding confers macrophage tropism (22). We, however,
observed no significant differences among early envelopes ver-
sus chronic-stage variants in their ability to replicate in MDMs
(Fig. 8). Therefore, our observations support previous conclu-
sions that replication capacity in macrophages does not corre-
late with an ability to use CCR5 (59). Because we did not
observe significant differences in CD4 receptor use, however,
we cannot directly corroborate that CD4 affinity predominately
influences macrophage tropism as has been previously sug-
In the same subjects analyzed in this study, we have previ-
ously shown that changes within the V1-V2 envelope loops
confer significantly increased neutralization resistance to au-
tologous plasma (76). Although we did not directly assess neu-
tralization sensitivity of the chimeric viruses in this study to
autologous plasma, collectively our results imply that evolution
within the envelope glycoprotein over the course of infection
that occurs in response to the host antibody response does not
necessarily confer a fitness cost in terms of receptor usage and
replication capacity. It should be noted, however, that effects
on entry and replication of the specific modifications that con-
fer neutralization escape will need to be examined in detail to
validate this hypothesis. In summary, our data suggest that
modifications within the envelope V1-V3 lead to antibody neu-
tralization escape, an increased ability to utilize the CCR5
receptor, and faster fusion kinetics.
We thank Jawad Kiani, Sonam Sheth, and Laura Cohen for assis-
tance with sequence analysis and PCR amplifications; Nikolaos Chat-
ziandreou and Ines Freitas for help with the PSC-RANTES sensitivity
assay; Cammie Lesser for yeast materials and protocols; Emily Platt
and David Kabat for cell lines with various levels of CD4 and CCR5;
Oliver Hartley for PSC-RANTES; and Julie Overbaugh and the Mom-
basa cohort staff, who originally provided access to the samples from
which these sequences were derived and thus made this research pos-
This study was supported by NIH grant AI1077473 (M.S.), the
American Foundation for AIDS Research (amfAR) (M.S.), and a
Doris Duke Charitable Foundation Early Career Development Award
1. Alkhatib, G., M. Locati, P. E. Kennedy, P. M. Murphy, and E. A. Berger.
1997. HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines:
independence from G protein signaling and importance of coreceptor down-
modulation. Virology 234:340–348.
2. Baba, M., O. Nishimura, N. Kanzaki, M. Okamoto, H. Sawada, Y. Iizawa, M.
Shiraishi, Y. Aramaki, K. Okonogi, Y. Ogawa, K. Meguro, and M. Fujino.
1999. A small-molecule, nonpeptide CCR5 antagonist with highly potent and
selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA 96:5698–5703.
3. Ball, S. C., A. Abraha, K. R. Collins, A. J. Marozsan, H. Baird, M. E.
Quinones-Mateu, A. Penn-Nicholson, M. Murray, N. Richard, M. Lobritz,
P. A. Zimmerman, T. Kawamura, A. Blauvelt, and E. J. Arts. 2003. Com-
paring the ex vivo fitness of CCR5-tropic human immunodeficiency virus
type 1 isolates of subtypes B and C. J. Virol. 77:1021–1038.
4. Blaak, H., M. Brouwer, L. J. Ran, F. de Wolf, and H. Schuitemaker. 1998. In
vitro replication kinetics of human immunodeficiency virus type 1 (HIV-1)
variants in relation to virus load in long-term survivors of HIV-1 infection.
J. Infect. Dis. 177:600–610.
5. Brenchley, J. M., T. W. Schacker, L. E. Ruff, D. A. Price, J. H. Taylor, G. J.
Beilman, P. L. Nguyen, A. Khoruts, M. Larson, A. T. Haase, and D. C.
Douek. 2004. CD4? T cell depletion during all stages of HIV disease occurs
predominantly in the gastrointestinal tract. J. Exp. Med. 200:749–759.
6. Cann, A. J., J. A. Zack, A. S. Go, S. J. Arrigo, Y. Koyanagi, P. L. Green, Y.
Koyanagi, S. Pang, and I. S. Chen. 1990. Human immunodeficiency virus
type 1 T-cell tropism is determined by events prior to provirus formation.
J. Virol. 64:4735–4742.
7. Carrington, M., M. Dean, M. P. Martin, and S. J. O’Brien. 1999. Genetics of
HIV-1 infection: chemokine receptor CCR5 polymorphism and its conse-
quences. Hum Mol. Genet. 8:1939–1945.
8. Chackerian, B., L. M. Rudensey, and J. Overbaugh. 1997. Specific N-linked
and O-linked glycosylation modifications in the envelope V1 domain of
simian immunodeficiency virus variants that evolve in the host alter recog-
nition by neutralizing antibodies. J. Virol. 71:7719–7727.
9. Cheng-Mayer, C., A. Brown, J. Harouse, P. A. Luciw, and A. J. Mayer. 1999.
Selection for neutralization resistance of the simian/human immunodefi-
ciency virus SHIVSF33A variant in vivo by virtue of sequence changes in the
extracellular envelope glycoprotein that modify N-linked glycosylation. J. Vi-
10. Choisy, M., C. H. Woelk, J. F. Guegan, and D. L. Robertson. 2004. Com-
parative study of adaptive molecular evolution in different human immuno-
deficiency virus groups and subtypes. J. Virol. 78:1962–1970.
11. Clapham, P. R., and A. McKnight. 2002. Cell surface receptors, virus entry
and tropism of primate lentiviruses. J. Gen. Virol. 83:1809–1829.
12. Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic
variation, pathogenesis, and therapy. Science 267:483–489.
13. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,
and R. Coico. 1999. Saccharomyces cerevisiae. Wiley, New York, NY.
14. de Almeida, M. C., A. C. Silva, A. Barral, and M. Barral Netto. 2000. A
simple method for human peripheral blood monocyte isolation. Mem. Inst.
Oswaldo Cruz 95:221–223.
15. De Jong, J. J., A. De Ronde, W. Keulen, M. Tersmette, and J. Goudsmit.
1992. Minimal requirements for the human immunodeficiency virus type 1
V3 domain to support the syncytium-inducing phenotype: analysis by single
amino acid substitution. J. Virol. 66:6777–6780.
16. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, X. Wu, W. A. O’Brien, L.
Ratner, J. C. Kappes, G. M. Shaw, and E. Hunter. 2000. Sensitivity of human
immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by
coreceptor specificity defined by the V3 loop of gp120. J. Virol. 74:8358–
17. Di Marzio, P., J. Tse, and N. R. Landau. 1998. Chemokine receptor regu-
lation and HIV type 1 tropism in monocyte-macrophages. AIDS Res. Hum.
18. Dunfee, R. L., E. R. Thomas, P. R. Gorry, J. Wang, J. Taylor, K. Kunstman,
S. M. Wolinsky, and D. Gabuzda. 2006. The HIV Env variant N283 enhances
macrophage tropism and is associated with brain infection and dementia.
Proc. Natl. Acad. Sci. USA 103:15160–15165.
19. Fouchier, R. A., M. Brouwer, S. M. Broersen, and H. Schuitemaker. 1995.
Simple determination of human immunodeficiency virus type 1 syncytium-
inducing V3 genotype by PCR. J. Clin. Microbiol. 33:906–911.
20. Fouchier, R. A., M. Groenink, N. A. Kootstra, M. Tersmette, H. G. Huisman,
F. Miedema, and H. Schuitemaker. 1992. Phenotype-associated sequence
variation in the third variable domain of the human immunodeficiency virus
type 1 gp120 molecule. J. Virol. 66:3183–3187.
21. Frost, S. D., T. Wrin, D. M. Smith, S. L. Kosakovsky Pond, Y. Liu, E.
9706ETEMAD ET AL.J. VIROL.
Paxinos, C. Chappey, J. Galovich, J. Beauchaine, C. J. Petropoulos, S. J.
Little, and D. D. Richman. 2005. Neutralizing antibody responses drive the
evolution of human immunodeficiency virus type 1 envelope during recent
HIV infection. Proc. Natl. Acad. Sci. USA 102:18514–18519.
22. Gorry, P. R., J. Taylor, G. H. Holm, A. Mehle, T. Morgan, M. Cayabyab, M.
Farzan, H. Wang, J. E. Bell, K. Kunstman, J. P. Moore, S. M. Wolinsky, and
D. Gabuzda. 2002. Increased CCR5 affinity and reduced CCR5/CD4 depen-
dence of a neurovirulent primary human immunodeficiency virus type 1
isolate. J. Virol. 76:6277–6292.
23. Gray, L., J. Sterjovski, M. Churchill, P. Ellery, N. Nasr, S. R. Lewin, S. M.
Crowe, S. L. Wesselingh, A. L. Cunningham, and P. R. Gorry. 2005. Uncou-
pling coreceptor usage of human immunodeficiency virus type 1 (HIV-1)
from macrophage tropism reveals biological properties of CCR5-restricted
HIV-1 isolates from patients with acquired immunodeficiency syndrome.
24. Hahn, B. H., G. M. Shaw, M. E. Taylor, R. R. Redfield, P. D. Markham, S. Z.
Salahuddin, F. Wong-Staal, R. C. Gallo, E. S. Parks, and W. P. Parks. 1986.
Genetic variation in HTLV-III/LAV over time in patients with AIDS or at
risk for AIDS. Science 232:1548–1553.
25. Ho, D. D., A. U. Neumann, A. S. Perelson, W. Chen, J. M. Leonard, and M.
Markowitz. 1995. Rapid turnover of plasma virions and CD4 lymphocytes in
HIV-1 infection. Nature 373:123–126.
26. Huang, W., S. H. Eshleman, J. Toma, S. Fransen, E. Stawiski, E. E. Paxinos,
J. M. Whitcomb, A. M. Young, D. Donnell, F. Mmiro, P. Musoke, L. A. Guay,
J. B. Jackson, N. T. Parkin, and C. J. Petropoulos. 2007. Coreceptor tropism
in human immunodeficiency virus type 1 subtype D: high prevalence of
CXCR4 tropism and heterogeneous composition of viral populations. J. Vi-
27. Jansson, M., M. Popovic, A. Karlsson, F. Cocchi, P. Rossi, J. Albert, and H.
Wigzell. 1996. Sensitivity to inhibition by beta-chemokines correlates with
biological phenotypes of primary HIV-1 isolates. Proc. Natl. Acad. Sci. USA
28. Jensen, M. A., F. S. Li, A. B. van ’t Wout, D. C. Nickle, D. Shriner, H. X. He,
S. McLaughlin, R. Shankarappa, J. B. Margolick, and J. I. Mullins. 2003.
Improved coreceptor usage prediction and genotypic monitoring of R5-
to-X4 transition by motif analysis of human immunodeficiency virus type 1
env V3 loop sequences. J. Virol. 77:13376–13388.
29. Kaleebu, P., I. L. Nankya, D. L. Yirrell, L. A. Shafer, J. Kyosiimire-
Lugemwa, D. B. Lule, D. Morgan, S. Beddows, J. Weber, and J. A. Whit-
worth. 2007. Relation between chemokine receptor use, disease stage, and
HIV-1 subtypes A and D: results from a rural Ugandan cohort. J. Acquir.
Immune Defic. Syndr. 45:28–33.
30. Karlsson, I., L. Antonsson, Y. Shi, M. Oberg, A. Karlsson, J. Albert, B. Olde,
C. Owman, M. Jansson, and E. M. Fenyo. 2004. Coevolution of RANTES
sensitivity and mode of CCR5 receptor use by human immunodeficiency
virus type 1 of the R5 phenotype. J. Virol. 78:11807–11815.
31. Kimata, J. T., L. Kuller, D. B. Anderson, P. Dailey, and J. Overbaugh. 1999.
Emerging cytopathic and antigenic simian immunodeficiency virus variants
influence AIDS progression. Nat. Med. 5:535–541.
32. Kimpton, J., and M. Emerman. 1992. Detection of replication-competent
and pseudotyped human immunodeficiency virus with a sensitive cell line on
the basis of activation of an integrated beta-galactosidase gene. J. Virol.
33. Koning, F. A., D. Kwa, B. Boeser-Nunnink, J. Dekker, J. Vingerhoed, H.
Hiemstra, and H. Schuitemaker. 2003. Decreasing sensitivity to RANTES
(regulated on activation, normally T cell-expressed and -secreted) neutral-
ization of CC chemokine receptor 5-using, non-syncytium-inducing virus
variants in the course of human immunodeficiency virus type 1 infection.
J. Infect. Dis. 188:864–872.
34. Kwa, D., J. Vingerhoed, B. Boeser, and H. Schuitemaker. 2003. Increased in
vitro cytopathicity of CC chemokine receptor 5-restricted human immuno-
deficiency virus type 1 primary isolates correlates with a progressive clinical
course of infection. J. Infect. Dis. 187:1397–1403.
35. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A.
Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in
complex with the CD4 receptor and a neutralizing human antibody. Nature
36. Lederman, M. M., R. S. Veazey, R. Offord, D. E. Mosier, J. Dufour, M.
Mefford, M. Piatak, Jr., J. D. Lifson, J. R. Salkowitz, B. Rodriguez, A.
Blauvelt, and O. Hartley. 2004. Prevention of vaginal SHIV transmission in
rhesus macaques through inhibition of CCR5. Science 306:485–487.
37. Lewin, S. R., S. Sonza, L. B. Irving, C. F. McDonald, J. Mills, and S. M.
Crowe. 1996. Surface CD4 is critical to in vitro HIV infection of human
alveolar macrophages. AIDS Res. Hum. Retroviruses 12:877–883.
38. Li, S., J. Juarez, M. Alali, D. Dwyer, R. Collman, A. Cunningham, and H. M.
Naif. 1999. Persistent CCR5 utilization and enhanced macrophage tropism
by primary blood human immunodeficiency virus type 1 isolates from ad-
vanced stages of disease and comparison to tissue-derived isolates. J. Virol.
39. Li, Y., J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn.
1991. Molecular characterization of human immunodeficiency virus type 1
cloned directly from uncultured human brain tissue: identification of repli-
cation-competent and -defective viral genomes. J. Virol. 65:3973–3985.
40. Lobritz, M. A., A. J. Marozsan, R. M. Troyer, and E. J. Arts. 2007. Natural
variation in the V3 crown of human immunodeficiency virus type 1 affects
replicative fitness and entry inhibitor sensitivity. J. Virol. 81:8258–8269.
41. Long, E. M., S. M. Rainwater, L. Lavreys, K. Mandaliya, and J. Overbaugh.
2002. HIV type 1 variants transmitted to women in Kenya require the CCR5
coreceptor for entry, regardless of the genetic complexity of the infecting
virus. AIDS Res. Hum. Retroviruses 18:567–576.
42. Malim, M. H., and M. Emerman. 2001. HIV-1 sequence variation: drift,
shift, and attenuation. Cell 104:469–472.
43. Marozsan, A. J., and E. J. Arts. 2003. Development of a yeast-based recom-
bination cloning/system for the analysis of gene products from diverse hu-
man immunodeficiency virus type 1 isolates. J. Virol. Methods 111:111–120.
44. Marozsan, A. J., D. M. Moore, M. A. Lobritz, E. Fraundorf, A. Abraha, J. D.
Reeves, and E. J. Arts. 2005. Differences in the fitness of two diverse wild-
type human immunodeficiency virus type 1 isolates are related to the effi-
ciency of cell binding and entry. J. Virol. 79:7121–7134.
45. Mehandru, S., M. A. Poles, K. Tenner-Racz, A. Horowitz, A. Hurley, C.
Hogan, D. Boden, P. Racz, and M. Markowitz. 2004. Primary HIV-1 infec-
tion is associated with preferential depletion of CD4? T lymphocytes from
effector sites in the gastrointestinal tract. J. Exp. Med. 200:761–770.
46. Mkrtchyan, S. R., R. M. Markosyan, M. T. Eadon, J. P. Moore, G. B.
Melikyan, and F. S. Cohen. 2005. Ternary complex formation of human
immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured
as an intermediate of membrane fusion. J. Virol. 79:11161–11169.
47. Moore, J. P. 1997. Coreceptors: implications for HIV pathogenesis and
therapy. Science 276:51–52.
48. Morner, A., A. Bjorndal, J. Albert, V. N. Kewalramani, D. R. Littman, R.
Inoue, R. Thorstensson, E. M. Fenyo, and E. Bjorling. 1999. Primary human
immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, fre-
quently use CCR5 but show promiscuity in coreceptor usage. J. Virol. 73:
49. Olshevsky, U., E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski.
1990. Identification of individual human immunodeficiency virus type 1
gp120 amino acids important for CD4 receptor binding. J. Virol. 64:5701–
50. Ometto, L., M. Zanchetta, A. Cabrelle, G. Esposito, M. Mainardi, L. Chieco-
Bianchi, and A. De Rossi. 1999. Restriction of HIV type 1 infection in
macrophages heterozygous for a deletion in the CC-chemokine receptor 5
gene. AIDS Res. Hum. Retroviruses 15:1441–1452.
51. Overbaugh, J., and C. R. Bangham. 2001. Selection forces and constraints on
retroviral sequence variation. Science 292:1106–1109.
52. Page, K. A., N. R. Landau, and D. R. Littman. 1990. Construction and use of
a human immunodeficiency virus vector for analysis of virus infectivity.
J. Virol. 64:5270–5276.
53. Pantophlet, R., and D. R. Burton. 2006. GP120: target for neutralizing
HIV-1 antibodies. Annu. Rev. Immunol. 24:739–769.
54. Pastore, C., G. R. Picchio, F. Galimi, R. Fish, O. Hartley, R. E. Offord, and
D. E. Mosier. 2003. Two mechanisms for human immunodeficiency virus
type 1 inhibition by N-terminal modifications of RANTES. Antimicrob.
Agents Chemother. 47:509–517.
55. Patterson, B. K., A. Landay, J. Andersson, C. Brown, H. Behbahani, D.
Jiyamapa, Z. Burki, D. Stanislawski, M. A. Czerniewski, and P. Garcia.
1998. Repertoire of chemokine receptor expression in the female genital
tract: implications for human immunodeficiency virus transmission. Am. J.
56. Peden, K., M. Emerman, and L. Montagnier. 1991. Changes in growth
properties on passage in tissue culture of viruses derived from infectious
molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virology 185:
57. Peeters, M. (ed.). 2002. Recombinant HIV sequences: their role in the global
epidemic. Theoretical Biology and Biophysics Group, Los Alamos, NM.
58. Perelson, A. S., A. U. Neumann, M. Markowitz, J. M. Leonard, and D. D. Ho.
1996. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span,
and viral generation time. Science 271:1582–1586.
59. Peters, P. J., M. J. Duenas-Decamp, W. M. Sullivan, R. Brown, C. Ankghua-
mbom, K. Luzuriaga, J. Robinson, D. R. Burton, J. Bell, P. Simmonds, J.
Ball, and P. R. Clapham. 2008. Variation in HIV-1 R5 macrophage-tropism
correlates with sensitivity to reagents that block envelope: CD4 interactions
but not with sensitivity to other entry inhibitors. Retrovirology. 5:5.
60. Peters, P. J., W. M. Sullivan, M. J. Duenas-Decamp, J. Bhattacharya, C.
Ankghuambom, R. Brown, K. Luzuriaga, J. Bell, P. Simmonds, J. Ball, and
P. R. Clapham. 2006. Non-macrophage-tropic human immunodeficiency vi-
rus type 1 R5 envelopes predominate in blood, lymph nodes, and semen:
implications for transmission and pathogenesis. J. Virol. 80:6324–6332.
61. Platt, E. J., J. P. Durnin, and D. Kabat. 2005. Kinetic factors control effi-
ciencies of cell entry, efficacies of entry inhibitors, and mechanisms of ad-
aptation of human immunodeficiency virus. J. Virol. 79:4347–4356.
62. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998.
Effects of CCR5 and CD4 cell surface concentrations on infections by mac-
VOL. 83, 2009USE OF CCR5 BY HIV-1 V1-V5 VARIANTS OVER TIME9707
rophagetropic isolates of human immunodeficiency virus type 1. J. Virol.
63. Poss, M., and J. Overbaugh. 1999. Variants from the diverse virus popula-
tion identified at seroconversion of a clade A human immunodeficiency virus
type 1-infected woman have distinct biological properties. J. Virol. 73:5255–
64. Pugach, P., A. J. Marozsan, T. J. Ketas, E. L. Landes, J. P. Moore, and S. E.
Kuhmann. 2007. HIV-1 clones resistant to a small molecule CCR5 inhibitor
use the inhibitor-bound form of CCR5 for entry. Virology 361:212–228.
65. Rangel, H. R., J. Weber, B. Chakraborty, A. Gutierrez, M. L. Marotta, M.
Mirza, P. Kiser, M. A. Martinez, J. A. Este, and M. E. Quinones-Mateu.
2003. Role of the human immunodeficiency virus type 1 envelope gene in
viral fitness. J. Virol. 77:9069–9073.
66. Reeves, J. D., S. A. Gallo, N. Ahmad, J. L. Miamidian, P. E. Harvey, M.
Sharron, S. Pohlmann, J. N. Sfakianos, C. A. Derdeyn, R. Blumenthal, E.
Hunter, and R. W. Doms. 2002. Sensitivity of HIV-1 to entry inhibitors
correlates with envelope/coreceptor affinity, receptor density, and fusion
kinetics. Proc. Natl. Acad. Sci. USA 99:16249–16254.
67. Reeves, J. D., J. L. Miamidian, M. J. Biscone, F. H. Lee, N. Ahmad, T. C.
Pierson, and R. W. Doms. 2004. Impact of mutations in the coreceptor
binding site on human immunodeficiency virus type 1 fusion, infection, and
entry inhibitor sensitivity. J. Virol. 78:5476–5485.
68. Repits, J., M. Oberg, J. Esbjornsson, P. Medstrand, A. Karlsson, J. Albert,
E. M. Fenyo, and M. Jansson. 2005. Selection of human immunodeficiency
virus type 1 R5 variants with augmented replicative capacity and reduced
sensitivity to entry inhibitors during severe immunodeficiency. J. Gen. Virol.
69. Reynes, J., P. Portales, M. Segondy, V. Baillat, P. Andre, B. Reant, O.
Avinens, G. Couderc, M. Benkirane, J. Clot, J. F. Eliaou, and P. Corbeau.
2000. CD4? T cell surface CCR5 density as a determining factor of virus
load in persons infected with human immunodeficiency virus type 1. J. Infect.
70. Richman, D. D., T. Wrin, S. J. Little, and C. J. Petropoulos. 2003. Rapid
evolution of the neutralizing antibody response to HIV type 1 infection.
Proc. Natl. Acad. Sci. USA 100:4144–4149.
71. Rizzuto, C. D., R. Wyatt, N. Hernandez-Ramos, Y. Sun, P. D. Kwong, W. A.
Hendrickson, and J. Sodroski. 1998. A conserved HIV gp120 glycoprotein
structure involved in chemokine receptor binding. Science 280:1949–1953.
72. Rudensey, L. M., J. T. Kimata, E. M. Long, B. Chackerian, and J. Over-
baugh. 1998. Changes in the extracellular envelope glycoprotein of variants
that evolve during the course of simian immunodeficiency virus SIVMne
infection affect neutralizing antibody recognition, syncytium formation, and
macrophage tropism but not replication, cytopathicity, or CCR-5 coreceptor
recognition. J. Virol. 72:209–217.
73. Rusert, P., H. Kuster, B. Joos, B. Misselwitz, C. Gujer, C. Leemann, M.
Fischer, G. Stiegler, H. Katinger, W. C. Olson, R. Weber, L. Aceto, H. F.
Gunthard, and A. Trkola. 2005. Virus isolates during acute and chronic
human immunodeficiency virus type 1 infection show distinct patterns of
sensitivity to entry inhibitors. J. Virol. 79:8454–8469.
74. Sagar, M., E. Kirkegaard, L. Lavreys, and J. Overbaugh. 2006. Diversity in
HIV-1 envelope V1-V3 sequences early in infection reflects sequence diver-
sity throughout the HIV-1 genome but does not predict the extent of se-
quence diversity during chronic infection. AIDS Res. Hum. Retroviruses
75. Sagar, M., L. Lavreys, J. M. Baeten, B. A. Richardson, K. Mandaliya, B. H.
Chohan, J. K. Kreiss, and J. Overbaugh. 2003. Infection with multiple
human immunodeficiency virus type 1 variants is associated with faster dis-
ease progression. J. Virol. 77:12921–12926.
76. Sagar, M., X. Wu, S. Lee, and J. Overbaugh. 2006. HIV-1 V1-V2 envelope
loop sequences expand and add glycosylation sites over the course of infec-
tion and these modifications affect antibody neutralization sensitivity. J. Vi-
77. Shankarappa, R., J. B. Margolick, S. J. Gange, A. G. Rodrigo, D. Upchurch,
H. Farzadegan, P. Gupta, C. R. Rinaldo, G. H. Learn, X. He, X. L. Huang,
and J. I. Mullins. 1999. Consistent viral evolutionary changes associated with
the progression of human immunodeficiency virus type 1 infection. J. Virol.
78. Starcich, B. R., B. H. Hahn, G. M. Shaw, P. D. McNeely, S. Modrow, H. Wolf,
E. S. Parks, W. P. Parks, S. F. Josephs, R. C. Gallo, et al. 1986. Identification
and characterization of conserved and variable regions in the envelope gene
of HTLV-III/LAV, the retrovirus of AIDS. Cell 45:637–648.
79. Trkola, A., S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan,
P. Pugach, S. Xu, L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S.
McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore. 2002. HIV-1
escape from a small molecule, CCR5-specific entry inhibitor does not involve
CXCR4 use. Proc. Natl. Acad. Sci. USA 99:395–400.
80. Troyer, R. M., K. R. Collins, A. Abraha, E. Fraundorf, D. M. Moore, R. W.
Krizan, Z. Toossi, R. L. Colebunders, M. A. Jensen, J. I. Mullins, G. Van-
ham, and E. J. Arts. 2005. Changes in human immunodeficiency virus type 1
fitness and genetic diversity during disease progression. J. Virol. 79:9006–
81. Tsibris, A. M., M. Sagar, R. M. Gulick, Z. Su, M. Hughes, W. Greaves, M.
Subramanian, C. Flexner, F. Giguel, K. E. Leopold, E. Coakley, and D. R.
Kuritzkes. 2008. In vivo emergence of vicriviroc resistance in a human
immunodeficiency virus type 1 subtype C-infected subject. J. Virol. 82:8210–
82. Tuttle, D. L., C. B. Anders, M. J. Aquino-De Jesus, P. P. Poole, S. L. Lamers,
D. R. Briggs, S. M. Pomeroy, L. Alexander, K. W. Peden, W. A. Andiman,
J. W. Sleasman, and M. M. Goodenow. 2002. Increased replication of non-
syncytium-inducing HIV type 1 isolates in monocyte-derived macrophages is
linked to advanced disease in infected children. AIDS Res. Hum. Retrovi-
83. Veazey, R. S., R. J. Shattock, M. Pope, J. C. Kirijan, J. Jones, Q. Hu, T.
Ketas, P. A. Marx, P. J. Klasse, D. R. Burton, and J. P. Moore. 2003.
Prevention of virus transmission to macaque monkeys by a vaginally applied
monoclonal antibody to HIV-1 gp120. Nat. Med. 9:343–346.
84. Wei, X., J. M. Decker, H. Liu, Z. Zhang, R. B. Arani, J. M. Kilby, M. S. Saag,
X. Wu, G. M. Shaw, and J. C. Kappes. 2002. Emergence of resistant human
immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20)
monotherapy. Antimicrob. Agents Chemother. 46:1896–1905.
85. Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-
Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A.
Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutral-
ization and escape by HIV-1. Nature 422:307–312.
86. Wei, X., S. K. Ghosh, M. E. Taylor, V. A. Johnson, E. A. Emini, P. Deutsch,
J. D. Lifson, S. Bonhoeffer, M. A. Nowak, B. H. Hahn, et al. 1995. Viral
dynamics in human immunodeficiency virus type 1 infection. Nature 373:
87. Westby, M., C. Smith-Burchnell, J. Mori, M. Lewis, M. Mosley, M. Stock-
dale, P. Dorr, G. Ciaramella, and M. Perros. 2007. Reduced maximal inhi-
bition in phenotypic susceptibility assays indicates that viral strains resistant
to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry.
J. Virol. 81:2359–2371.
88. Zhang, L., T. He, A. Talal, G. Wang, S. S. Frankel, and D. D. Ho. 1998. In
vivo distribution of the human immunodeficiency virus/simian immunodefi-
ciency virus coreceptors: CXCR4, CCR3, and CCR5. J. Virol. 72:5035–5045.
9708ETEMAD ET AL. J. VIROL.