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Journal of Virology, January 2001, p. 251-259, Vol. 75, No. 1
Laboratoire de Recherche Antivirale, INSERM
U-82,1 and INSERM U-332
ICGM,3 Paris, France, and Unit of
Immunobiology of HIV, DIBIT San Raffaele Scientific Institute, Milan,
Italy2
Received 8 August 2000/Accepted 11 October 2000
We developed a recombinant virus technique to determine the
coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from
plasma samples, the source expected to represent the most actively
replicating virus population in infected subjects. This method is not
subject to selective bias associated with virus isolation in culture, a
step required for conventional tropism determination procedures. The
addition of a simple subcloning step allowed semiquantitative
evaluation of virus populations with a different coreceptor (CCR5 or
CXCR4) usage specificity present in each plasma sample. This procedure
detected mixtures of CCR5- and CXCR4-exclusive virus populations as
well as dualtropic viral variants, in variable proportions. Sequence
analysis of dualtropic clones indicated that changes in the V3 loop are
necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5.
This convenient technique can greatly assist the study of virus
evolution and compartmentalization in infected individuals.
Human immunodeficiency virus type 1 (HIV-1) entry into target cells relies on a complex interaction between
viral and cellular proteins, eventually leading to viral and plasma
membrane lipid mixing. The binding of the viral gp120 envelope
glycoprotein to the cellular CD4 molecule induces a
conformational change in the gp120 protein, contributing to the
exposure of the binding site for a coreceptor (reviewed in reference
77). Several chemokine receptors can serve as
coreceptors for HIV-1 entry (1, 11, 20-23, 28, 46, 58,
60) (see references 3, 5, 12, 13, and
50 for reviews), but the relevant coreceptors used in vivo seem limited to CCR5 and CXCR4 (78, 79) and viral isolates that use alternative chemokine receptors also use either CCR5
or CXCR4. The initial phases of HIV-1 infection in vivo involve viral
strains characterized by the ability to grow both in stimulated peripheral blood mononuclear cells (PBMCs) and in macrophages, but not in established T-cell lines (63, 64, 80). These strains are now known to use CCR5 as a coreceptor and are hence named
R5 viruses (4). Later on in the course of disease
progression, some of the HIV-1-infected population harbor virus strains
capable of productive infection of established T-cell lines as well as stimulated PBMCs (17, 64). These viral strains grow more
rapidly than R5 viruses (16, 71) and use CXCR4 as a
coreceptor; therefore, they are called X4 strains (4). A
fraction of the CXCR4-using viruses can also use CCR5 and are called
dualtropic or R5X4 viruses (15, 22, 68).
R5 strains are most commonly transmitted in vivo (29, 64, 72, 75,
80), suggesting that a poorly understood selective force acts
against X4 strains at the level of virus transmission independently of
the route of transmission. Examples of X4 virus suppression after
blood-borne infection also supports this hypothesis (18,
44). Accordingly, despite the fact that few (two or three) mutations in the third variable loop (V3 loop) of the envelope gene can
confer the ability to use CXCR4 (14, 30, 37), X4 viruses
appear only late after infection, indicating that these strains are
selected against by a competent immune system (9, 53). On
the other hand, the association of the emergence of X4 viruses with the
marked decline in CD4 T-cell counts (39, 41, 59), together
with the observation of increased cytopathogenicity of these viruses in
experimental systems (31, 38, 54, 57), may support the
alternative hypothesis that disease progression could be the
consequence, rather than the cause, of the development of X4 strains.
The pathogenic potential of R5 viruses, however, is proven, given that
most individuals who die from AIDS-related complications appear to
harbor only R5 viruses (59).
Current understanding of the precise kinetics of the appearance of X4
and dualtropic viruses is hampered by technical difficulties and by the
selective bias that is associated with virus isolation, a step required
for the determination of coreceptor usage. Virus isolation in culture
(47) typically relies on cultivating patient PBMCs in the
presence of interleukin-2 after stimulation by phytohemagglutinin or
anti-CD3 antibodies. Fresh donor PBMCs are added to provide new target
cells for the production of a virus culture with a sufficient titer for
testing on indicator cells. Extensive virus culturing may lead to the
selection of viruses adapted to culturing conditions that misrepresent
the original virus source. In vitro culturing of PBMCs may alter the
level of chemokine receptor expression on the surface of the cells
(6, 8), generally favoring the expression of CXCR4 with
respect to CCR5. In addition, virus isolation from patient PBMCs may
lead to the production of virus that does not necessarily reflect the
actively replicating population, because activation of infected PBMCs
can induce the expression of archived integrated proviral genomes.
We describe here a new method to determine primary HIV-1 coreceptor
usage that does not require virus propagation in tissue culture. This
method allows the study of plasma virus, which best represents the
actively replicating virus population in the patient (25, 55,
73). We compared and validated our method by analyzing sequential series of plasma samples and viral isolates from pediatric patients that had been previously characterized by conventional phenotypic procedures. We found that R5 viruses persisted in the plasma
of patients throughout the observation period and were paralleled by,
but not replaced by, the emergence of X4 viruses. Using a subcloning
procedure, we could determine that viral populations found after the
acquisition of CXCR4 usage capacity consisted of R5 virus mixed with
X4-exclusive and/or authentic dualtropic viruses. Sequence analysis of
the primary dualtropic viruses showed that CXCR4 usage is associated
with an increase in the positive charge of the V3 loop, as previously
described for X4 (exclusive) viruses. In addition, the finding that
some X4 and dualtropic primary viruses share an identical V3 loop
sequence suggests that domains within V1-V2 and the second conserved
region (C2), located immediately upstream of V3, contain the
determinants for the expanded tropism of dualtropic viruses.
Vector construction.
The SalI-BamHI
fragment from pNL4.3 (from nucleotide 5785 to 8465) containing most of
the envelope gene sequence was subcloned in pBluescript II SK(+)
(Stratagene) to provide a convenient vector (SK-SB) for site-directed
mutagenesis. The sequence spanning the V1-V2 loops, the C2 domain, and
V3 loop (nucleotides 6610 to 7250) was excised by
oligonucleotide-directed mutagenesis using the Quick change mutagenesis
kit (Stratagene) with the following oligonucleotides: Delta V+,
5'-CCCCACTCTGTGTTAGTTTTAAGTGCTAGCAAATTAAGAGAAC-3', and Delta
V Virus RNA amplification.
Viral RNA was isolated from frozen
patient plasma samples or (where indicated) from PBMC culture
supernatants, using the Roche Amplicor kit (Roche Diagnostics). An
initial reverse transcription-PCR (RT-PCR) amplification was carried
out using the following primers: E00,
5'-TAGAAAGAGCAGAAGACAGTGGCAATGA-3' (nucleotides 6196 to 6224 of pNL4.3), and ES8B, 5'-CACTTCTCCAATTGTCCCTCA-3'
(nucleotides 7638 to 7662 of pNL4.3). An aliquot of the RT-PCR
product was then used in a nested PCR with the following primers: E20,
5'-GGGCCACACATGCCTGTGTACCCACAG-3' (nucleotides 6426 to 6452 of pNL4.3), and E115, 5'-AGAAAAATTCCCCTCCACAATTAA-3' (nucleotides 7341 to 7364 of pNL4.3). By this approach,
900-bp-long products that span the V1-V3 region deleted from the
43- Cell culture and tropism recombinant test (TRT).
293-T cells
and U373MG-CD4 cells (34) were cultivated in Dulbecco
modified Eagle medium supplemented with 10% fetal calf serum (FCS) and
antibiotics. U373MG-CD4 cells stably transfected with an expression
vector for the chemokine receptor CCR5 or CXCR4 (43) were
cultured in the presence of 10 µg of puromycine/ml and 100 µg of
hygromycin-B. The three U373MG-CD4-derived cell lines (373, 373-CCR5,
and 373-CXCR4) contain an HIV-1 long terminal repeat (LTR)-LacZ
cassette that allows the detection of single cycle infection by a
colorimetric assay based on Tat-induced expression of
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.251-259.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Determination of Coreceptor Usage of Human Immunodeficiency Virus
Type 1 from Patient Plasma Samples by Using a Recombinant
Phenotypic Assay
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, 5'-GTTCTCTTAATTTGCTAGCACTTTAAACTAACACAGAGTGGGG-3'. The natural NheI site just downstream of the V3 loop was
preserved and used in combination with SalI to transfer the
V1-V3 deleted fragment of the envelope gene back into a variant of
pNL4.3 (43XCS [49]), creating the final construct
43-
V. The same NheI site can be used to linearize the
43-V vector between the envelope domains C1 and C3.
V vector were obtained, with approximately 150-bp extensions on
each side to allow homologous recombination during transfection. PCR
products were verified by agarose gel electrophoresis and were column
purified (Qiagen) prior to use in transfection.
-galactosidase
(-Gal) (43).
V vector DNA and 1 µg
of PCR-amplified DNA from patient samples, using the calcium phosphate
precipitation method. Virus-containing supernatant (3 ml) was collected
36 h after transfection and clarified by centrifugation.
Triplicates of 100 µl were used to infect subconfluent target cells
cultured in 96-well plates in the presence and in the absence of 2 µg
of DEAE-dextran/ml. Parallel cultures of 373, 373-CCR5, and 373-CXCR4 cells were used as target cells in all assays. At 24 to 36 h after infection, virus infectivity was determined in target cell cultures by
measurement of -Gal activity in cell lysates, using a colorimetric assay based on cleavage of
chlorophenolred--D-galactopyranoside (CPRG) by -Gal
(termed here CPRG assay), adapted from a study by Eustice et al.
(24). Briefly, following elimination of the supernatant,
target cells were lysed in 100 µl of lysis buffer (5 mM
MgCl2, 0.1% NP-40 in phosphate-buffered saline). After
incubation for 5 min at room temperature, 100 µl of reaction buffer
(6 mM CPRG in lysis buffer) was added to the cell lysates and incubated for between 5 min and 2 h at 37°C. Optical densities in the
reaction wells were read at 570 nm with a reference filter set at 690 nm. The CCR5 or CXCR4 coreceptor usage of recombinant viruses was determined by measurement of CPRG in the different target cells and
comparison with cells in wells exposed to supernatant produced by
transfection in the absence of PCR products. Optical density values
greater than twice the background value were considered positive.
Values between 2 and 10 times the background value were confirmed in at
least two independent experiments.
PCR product subcloning and sequence analysis. RT-PCR-amplified material from patient plasma samples and from PBMC culture supernatants were cloned using the Topo-TA Cloning kit (Invitrogen) according to the manufacturer's instructions. Low-cycle PCR was conducted using the primers E20 and E115 directly on single colonies from Luria-Bertani agar plates, and 500 ng of PCR product was used in the recombinant virus assay to determine the tropism associated with single envelope sequences. Nucleotide sequencing of the V3 loop of the env gene was performed from the subcloned V1-V3 RT-PCR product. Samples of 4 µl of PCR product were used for cycle sequencing (Thermo sequenase fluorescent labelling primer cycle sequencing kit; Amersham Pharmacia Biotech, Little Chalfont Buckinghamshire, United Kingdom) with CY5-labeled J53Y primer (5'-AATTTCTGGGTCCCCTCCTG-3') according to the manufacturer's protocol. The generated fragments were analyzed in a 6% polyacrylamide gel with an automated laser fluorescent sequencing apparatus (Pharmacia Biotech, Uppsala, Sweden).
Phylogenetic analysis was conducted with Phylogeny Inference Package (PHYLIP) version 3.57c programs (26, 27) as previously described (61). Briefly, nucleotide distances were estimated by means of the maximum-likelihood model. Phylogenies were reconstructed by both the neighbor-joining method and the Fitch-Margoliash distance method in order to increase confidence in the reconstructed phylogenies. Bootstrap resampling (200 replicates) was applied to the neighbor-joining trees to assess the strength of support for each branch.Nucleotide sequence accession numbers. The sequences reported here have been deposited in the GenBank database under the following accession numbers: for patient 136 samples, no. AF284505 to AF284526, and for patient 145 samples, no. AF284527 to AF284552.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Vector construction and experimental procedure.
The region
spanning the three N-terminal variable loops of the gp120 surface
glycoprotein contain the main determinants for HIV-1 coreceptor usage
(2, 7, 14, 32, 33, 35, 36, 48, 51, 52, 66, 69, 74, 76). To
generate a vector for the phenotypic determination of cellular tropism
of primary viruses, we deleted the entire V1-V3 region (600 bp) of the
envelope from a previously described pNL4-3 derivative, thus obtaining the vector 43-V. The natural and unique NheI restriction
site just downstream of the deletion was used to linearize the vector. Recombinant viruses were produced by complementation of the
43-V-linearized vector with PCR products from patients' samples
encompassing the deleted region and short (100 to 150 nucleotides)
overlaps that allowed homologous recombination. In our experimental
system, 900-bp-long amplification products were produced by RT-PCR from plasma virus. As controls, PCR products from plasmids encoding HXB-2 or
ADA envelope, an X4 viral clone and an R5 viral clone, respectively,
were also used (Fig. 1).
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-Gal expression. As shown in
Fig. 1, complementation of the 43-V vector with PCR product from the
HXB-2 envelope sequence resulted in a strong and specific signal in
target cells expressing CXCR4 but not CCR5, consistent with the known
coreceptor specificity of this virus. Conversely, vector
complementation with a PCR product from the ADA envelope tested
positive only in cells expressing CCR5. This finding indicates that the
V1-V3 region of the envelope glycoprotein is sufficient for the
prediction of virus tropism using the recombinant virus assay described
here, in agreement with several observations pointing to this region as
the major tropism determinant. As expected, supernatant from cells
transfected with the 43-V-linearized vector only, in the absence of
a PCR product, did not induce -Gal expression in target cells (Fig.
1, mock wells). As a control, 373-CD4 cells that carried the LTR-LacZ
cassette but that did not express a coreceptor molecule always tested
negative when exposed to virus-containing supernatant (data not shown).
Figure 1 also shows the result obtained when RT-PCR products from
representative plasma samples were used to complement the 43-V
vector. Some samples tested positive exclusively for CCR5 usage
(samples P1) while others tested positive both on CCR5- and on
CXCR4-expressing 373 cells (samples P2 and P3). These data show that
the TRT described here allows the determination of coreceptor usage of
viruses from plasma samples, without the need for virus isolation in culture.
Comparison of TRT with current phenotyping procedures.
We
compared our method with conventional virus tropism assays, testing
consecutive plasma samples from three selected perinatally infected
patients (Table 1) whose viruses
underwent a phenotypic change from R5 to X4 during disease progression
(62). Emergence of X4 plasma virus was detected by the TRT
in all three patients at the same time points as conventional
phenotyping procedures based on MT-2 syncytium scoring and on virus
replication in U87-CD4+ cells expressing CXCR4 (Table 1).
Interestingly, TRT analysis indicated that the ability of plasma
viruses to use CCR5 was preserved throughout the follow-up (Table 1).
In contrast, the R5 virus population was not detected using the
CCR5-expressing U87-CD4+ indicator cells at months 48 and
67 for patient 145 (Table 1). This discrepancy between the two
detection methods may be due to the different virus sources or may
reflect the replicative advantage of X4 strains in the PBMC cultures
used for the U87-CD4+ assay (see below).
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Coreceptor usage of single virus clones from a mixed plasma virus
population.
The ability to use both CCR5 and CXCR4 could be the
result of two nonexclusive phenomena: (i) the expanded coreceptor usage capacity conferred by a single envelope sequence or (ii) the presence of a mixed virus population in which different envelope sequences allow
the use of different coreceptors. To analyze the composition of plasma
virus samples from patient 145 at different time points, we subcloned
the V1-V3 RT-PCR product. Individual clones were then screened for
coreceptor specificity by TRT, using plasmid DNA as a PCR substrate.
Clones obtained from an authentic dualtropic virus are expected to test
positive on both 373-CCR5 and 373-CXCR4 cells, while clones from R5 and
X4 viruses can infect only one of the target cell lines. As shown in
Table 2, for the sample obtained at month
5 from patient 145, which was determined to be R5-exclusive both by our
method and by current procedures, all tested clones (11 out of 11)
harbored R5-specific envelope sequences when analyzed by TRT. At month
48, the first available time point for which X4 viruses were detected
for patient 145, an analysis of the clones showed that the plasma virus
was composed of a mixed population, with an equivalent proportion of X4
and dualtropic isolates (4 out of 9 clones, each) and a minority of R5
isolates (1 out of 9 clones). The capacity of the mixed plasma virus
population to infect target cells using both coreceptors was thus due
to the coexistence of all three possible virus populations in this
plasma sample: R5-exclusive, X4-exclusive, and dualtropic viruses. At
67 months, plasma virus from patient 145 again tested positive on
indicator cells expressing either coreceptor (Table 1). This sample
harbored similar proportions of R5-exclusive and dualtropic viruses (5 and 6 clones, respectively, out of 11 tested), while the X4-exclusive
virus population was no longer detected (Table 2).
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Virus culture biases the coreceptor usage determination of virus replicating in patients. To determine why the R5 virus population detected by our recombinant virus technique (Tables 1 and 2) was not recognized in the phenotypic assay using cultured virus (Table 1), we further analyzed the case of patient 145. Viral RNA was extracted from the supernatants of the patient PBMC cultures used to infect U87-CD4 cells, and PCR amplification products were cloned as described above. Clones were tested for coreceptor usage by TRT and compared to clones from plasma samples obtained at the same time points (Table 2). Month 5 clones from plasma and culture supernatants were all R5 exclusive, in agreement with previous characterization on MT2 cells and U87-CD4 cells (62). Interestingly, for subsequent time points (months 48 and 67), no R5-exclusive virus was detected in the culture supernatant samples, in contrast to plasma samples obtained at the same time points (Table 2). Dualtropic and X4-exclusive viruses were instead found in both supernatants (Table 2). A comparison of virus population with different tropisms in plasma and PBMC culture supernatants was performed also for the available samples from patient 136 (months 60 and 64; Table 2). The majority of clones from PBMC supernatant consisted of X4 and dualtropic clones (Table 2), while in the corresponding plasma samples, the large majority of the clones were R5 (Table 2). These results suggest that either the source of the virus (PBMCs versus plasma) or selection against R5-exclusive variants in culture may have caused the misrepresentation of plasma virus population by tropism assays that rely on virus propagation in culture.
Genetic determinants of virus tropism.
An additional advantage
offered by the subcloning approach used here consists of the ability to
correlate virus tropism with the amino acid sequences of the cloned
fragments. The V3 loop has been shown to contain signature sequences
typical of either R5 or X4 viruses, consisting both of specific amino
acid substitutions and of a change in the overall charge of the domain
(10, 19, 30, 36, 42, 67, 70). We analyzed the amino acid
sequence of the V3 loop for clones obtained from selected plasma
samples from patients 136 and 145 (Fig. 2A and
B, respectively). Six pre-switch sequences were analyzed for both patients 136 and 145 and were found to
be quite homogeneous within each patient, differing at most by one
residue. For patient 136, after the phenotypic switch, the majority of
the clones corresponded to R5 viruses (Table 2), and their sequences
were still highly conserved (Fig. 2A). The single X4-exclusive clone
detected displayed a marked increase of the positive charge of the V3
domain and a characteristic positively charged residue at position 11 (Fig. 2A). Four months later, all R5-exclusive clones were still
identical, while several differences were observed in the sequences of
the clones able to use CXCR4, consistent with active replication and
diversification of X4 viruses. Interestingly, the single dualtropic
clone obtained from this sample displayed a V3 loop amino acid sequence
identical to that of some of the X4-exclusive clones from the same
patient (Fig. 2A). Concerning patient 145, only one clone from the
month 48 plasma sample was R5 exclusive, and it harbored several amino acid differences with respect to pre-switch R5 clones (Fig. 2B), a
likely consequence of the relatively long time elapsed between the
collection of these samples. Clones shown to use CXCR4 that were from
the month 48 plasma sample displayed a net increase in the positive
charge of the V3 domain associated with the replacement of a positively
charged residue at position 11, among other changes. Some variability
was observed between single clones, and again some of the dualtropic
clones had a V3 loop sequence identical to that of some X4-exclusive
clones. At month 67, R5-exclusive clones displayed increasing
variability, while minor changes were detected in the dualtropic clones
at this time point.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Determination of the coreceptor usage of HIV-1 in vivo is relevant both to the prediction of disease progression and to the precise understanding of the dynamic process of virus evolution in infected subjects. Faster disease progression rates (39, 41, 59) as well as increased replication kinetics and pathogenicity in vitro (31, 38, 54, 57) are associated with the capacity of HIV-1 strains to use CXCR4 as a coreceptor. Although phenotypic characterization of the cellular tropism of patient virus populations has led to major advances in the understanding of the infectious process in vivo, it remains unclear whether the appearance of X4 viruses in patients is a cause or a consequence of immunodeficiency. Arguments can be found to support both views, and it is only by frequently sampling large patient populations by using a sensitive technique that the temporal relationship between these two events will be elucidated. Extensive studies in this field have been hampered by the fact that conventional procedures are time-consuming and involve complex sample manipulation. Most of the viruses characterized by current phenotypic procedures originate from PBMC cocultivation followed by in vitro culture to achieve a sufficient infectious titer for testing on selective target cells (47). Phenotyping techniques based on virus culture suffer from some drawbacks: they are difficult to standardize due to primary cell variability, HIV-1 genomes integrated in host DNA can be reactivated in stimulated PBMCs, and the virus produced can differ from the population replicating in the patient, which is best approximated by plasma virus (25, 55, 73). In addition, culture conditions exert a selective pressure that may also lead to misrepresentation of the virus population, even after a limited number of passages.
To overcome some of these technical difficulties, we have developed an assay that determines the coreceptor usage of primary HIV-1 viruses which does not rely on virus isolation in culture, thus providing an additional tool for the study of the kinetics and equilibrium of virus populations in infected subjects. This approach eliminates the selective bias associated with virus culture, allows easy monitoring of large patient cohorts, and was used here to study the tropism of virus populations found in plasma. We evaluated the tropism of virus from three infected children for which sequential samples preceding and following the emergence of X4 strains were available. These viruses had been previously characterized with tropism assays using both MT-2 syncytium scoring and U87-CD4+ cells expressing a single coreceptor (62). For all three patients, our recombinant method detected viruses able to use CXCR4 (X4 or dualtropic) at the same time points they were first detected by assays based on MT-2 and U87 cells. Besides, the addition of a simple step, consisting of the subcloning of PCR products, allowed semiquantitative analysis of the complexity of the patient virus population. We could thus identify different proportions of R5- and X4-exclusive viral components as well as authentic primary dualtropic viruses present in plasma samples. A more precise quantification of viruses with different tropisms that are present in a population would require the screening of very large number of clones. Even then, the quantification could suffer from the possibility that some primary sequences may not be functional in the context of the NL4-3 vector used here.
Primary dualtropic viruses have been previously obtained by limiting dilution of PBMC culture supernatants from two patients (68). In that study, no X4-exclusive viruses were found, suggesting that the syncytium-inducing phenotype of the overall isolate reflected CXCR4 usage by dualtropic clones (68). Accordingly, some of the virus populations analyzed here by TRT were a mixture of R5-specific and dualtropic viruses (Table 2, patient 145, month 67). Nevertheless, we unambiguously showed that X4-exclusive viruses can be found in some plasma samples (Table 2, patient 136, month 64, and patient 145, month 48). Our finding that X4 viruses are present in plasma samples shows that evolution towards the exclusive usage of CXCR4 is a relevant phenomenon in patients and not only the consequence of in vitro virus culture on established T-cell lines. Dualtropic isolates were proposed to represent a transitional state in the evolution from R5 to X4 viruses (15, 22). The finding that dualtropic viruses displayed reduced replication kinetics, with respect to X4 viruses, in primary cells expressing only the CXCR4 coreceptor (56) also supports the hypothesis that they precede X4 viruses. Improved CXCR4 usage would then confer to X4 viruses an increased colonization potential, given that this molecule is more widely expressed in primary cells than CCR5 (see reference 77 and references within). An alternative hypothesis is that R5 viruses could switch to an X4 phenotype by just a few changes in their envelope sequence, and dualtropism may represent a subsequent evolutionary adaptation with peculiar biological properties. The understanding of the temporal relationship between these forms would provide valuable information on the selective forces at play and on the viral replication strategy.
Our study also establishes that primary dualtropic viruses have V3 loop sequence characteristics similar to those of X4 viruses, with comparable values of net positive charge, the same substitutions at specific positions with respect to R5 viruses, and, in some cases, complete amino acid sequence identity with X4 viruses from the same patient. V3 loop sequences from dualtropic and X4 plasma viruses from the same patient clustered together, separate from all R5 sequences analyzed here. These findings suggest that modification of the V3 loop sequence is required for the use of CXCR4 as a coreceptor, while the possibility to maintain the capacity to use CCR5, in addition to CXCR4, depends on the overall context of the V1-V3 region. The molecular determinants responsible for the expanded tropism of dualtropic primary viruses are not well established, and the study of a larger cohort is required before we can generalize on the implication of specific residues or domains. As mentioned above, losing the capacity to use CCR5 could represent the evolutionary cost for specialized CXCR4-mediated entry.
Interestingly, we showed here that the R5 viral population persisted after the emergence of X4 and/or dualtropic viruses in all three patients. The lack of detection of the R5 component in one of the three patients (patient 145) using U87 cells, was due to the differences in the virus source and technical approach. Persistent CCR5 utilization was previously shown for both blood- and tissue-derived isolates, with the additional observation that isolates obtained from late stages of disease displayed increased replication ability in macrophages (45). Similarly, R5 viruses isolated from patients with AIDS also displayed faster replication kinetics and increased T-cell depletion in SCID-hu mice with respect to viruses isolated from the same patients at earlier time points (65). These reports suggest that for HIV, as shown for simian immunodeficiency virus (SIV) (40), virus pathogenicity increases with time in an infected individual independently of coreceptor usage change. The analysis of sequential R5 viruses with a recombinant virus technique similar to the one described here could determine whether changes in the envelope sequence are solely responsible for the different biological characteristics of late R5 viruses. These could be the consequence of increased affinity for or increased ability to use CCR5. Alternatively, faster virus replication kinetics and pathogenicity could result from the slower process of coevolution of several viral genes. Such a study would provide important information on the relationship between HIV and the host.
Persistence of the R5 virus population indicates that the environmental conditions that favor X4 and dualtropic virus replication in vivo are not necessarily more restrictive for R5 viruses. Accordingly, the emergence of X4 viruses would represent the colonization of new target cells in an expanding infectious process, rather than the appearance of virus population directly competing with R5 viruses. The development of X4 and dualtropic strains could reflect the colonization of a tissue compartment in which the usage of CXCR4 confers an advantage either by allowing increased replication kinetics or because the local immune surveillance is decreased. Although the present study does not analyze HIV tropism in specific tissue compartments, the molecular approach used might provide important insights on this issue. We envisage the use of samples from different compartments, feasible as long as a PCR product can be generated, to compare the kinetics of appearance of X4 viruses and the complexity of the virus isolates.
Virus tropism can be expected to evolve also under the selective pressure of antiviral molecules aimed at inhibiting virus entry by competing for coreceptor binding. Natural and synthetic molecules that block coreceptors and exert strong inhibition of virus infection in culture are being evaluated for their potential use in the treatment of HIV-1 infection, and a few have progressed to clinical trials (5). Blocking one coreceptor may select for variants that use alternative coreceptors, leading to a shift in the virus population. Monitoring the coreceptor usage of viruses from patients treated with entry inhibitors can be facilitated by our assay. Overall, we believe that the study of the kinetics and of the conditions under which viruses with different coreceptor specificity evolve in patients can be greatly assisted by the recombinant virus approach described here.
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ACKNOWLEDGMENTS |
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We thank Allan Hance and Esther Race for critically reading the manuscript.
This work was supported in part by a grant from the Agence Nationale de Recherche sur le SIDA (ANRS). F.S. was the recipient of a fellowship from Istituto Superiore di Sanità, Rome, Italy.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Recherche Antivirale, IMEA/INSERM Hôpital Bichat-Claude Bernard, 46 rue H. Huchard, 75018 Paris, France. Phone: 33-1-4025 6359. Fax: 33-1-4025 6370. E-mail: mammano{at}bichat.inserm.fr.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1998[Abstract]. |
| 2. |
Andeweg, A. C.,
P. Leeflang,
A. D. Osterhaus, and M. L. Bosch.
1993.
Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation.
J. Virol.
67:3232-3239 |
| 3. | Berger, E. A. 1997. HIV entry and tropism: the chemokine receptor connection. AIDS 11:S3-S16. |
| 4. | Berger, E. A., R. W. Doms, E. M. Fenyo, B. T. Korber, D. R. Littman, J. P. Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss. 1998. A new classification for HIV-1. Nature 391:240[CrossRef][Medline]. |
| 5. | Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657-700[CrossRef][Medline]. |
| 6. |
Bleul, C. C.,
L. Wu,
J. A. Hoxie,
T. A. Springer, and C. R. Mackay.
1997.
The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA
94:1925-1930 |
| 7. |
Boyd, M. T.,
G. R. Simpson,
A. J. Cann,
M. A. Johnson, and R. A. Weiss.
1993.
A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism.
J. Virol.
67:3649-3652 |
| 8. |
Carroll, R. G.,
J. L. Riley,
B. L. Levine,
Y. Feng,
S. Kaushal,
D. W. Ritchey,
W. Bernstein,
O. S. Weislow,
C. R. Brown,
E. A. Berger,
C. H. June, and D. C. St. Louis.
1997.
Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells.
Science
276:273-276 |
| 9. |
Chan, S. Y.,
R. F. Speck,
C. Power,
S. L. Gaffen,
B. Chesebro, and M. A. Goldsmith.
1999.
V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1.
J. Virol.
73:2350-2358 |
| 10. |
Chesebro, B.,
K. Wehrly,
J. Nishio, and S. Perryman.
1992.
Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.
J. Virol.
66:6547-6554 |
| 11. | Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148[CrossRef][Medline]. |
| 12. | Clapham, P. R. 1997. HIV and chemokines: ligands sharing cell-surface receptors. Trends Cell Biol. 7:264-268[Medline]. |
| 13. | Clapham, P. R., J. D. Reeves, G. Simmons, N. Dejucq, S. Hibbitts, and A. McKnight. 1999. HIV coreceptors, cell tropism and inhibition by chemokine receptor ligands. Mol. Membr. Biol. 16:49-55[CrossRef][Medline]. |
| 14. | Cocchi, F., A. L. DeVico, A. Garzino-Demo, A. Cara, R. C. Gallo, and P. Lusso. 1996. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nat. Med. 2:1244-1247[CrossRef][Medline]. |
| 15. |
Collman, R.,
J. W. Balliet,
S. A. Gregory,
H. Friedman,
D. L. Kolson,
N. Nathanson, and A. Srinivasan.
1992.
An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.
J. Virol.
66:7517-7521 |
| 16. |
Connor, R. I.,
H. Mohri,
Y. Cao, and D. D. Ho.
1993.
Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.
J. Virol.
67:1772-1777 |
| 17. |
Connor, R. I.,
K. E. Sheridan,
D. Ceradini,
S. Choe, and N. R. Landau.
1997.
Change in coreceptor use coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185:621-628 |
| 18. | Cornelissen, M., G. Mulder-Kampinga, J. Veenstra, F. Zorgdrager, C. Kuiken, S. Hartman, J. Dekker, L. van der Hoek, C. Sol, R. Coutinho, and J. Goudsmit. 1995. Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population. J. Virol. 69:1810-1818[Abstract]. |
| 19. |
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 |
| 20. | Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R. Littman, and N. R. Landau. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666[CrossRef][Medline]. |
| 21. | Deng, H. K., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997. Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388:296-300[CrossRef][Medline]. |
| 22. | Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158[CrossRef][Medline]. |
| 23. | Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673[CrossRef][Medline]. |
| 24. |
Eustice, D.,
P. Feldman,
A. Colberg-Poley,
R. Buckery, and R. Neubauer.
1991.
A sensitive method for the detection of -galactosidase in transfected mammalian cells.
BioTechniques
11:739-743.
|
| 25. | Feinberg, M. B. 1996. Changing the natural history of HIV disease. Lancet 348:239-246[CrossRef][Medline]. |
| 26. | Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791[CrossRef]. |
| 27. | Felsenstein, J. 1993. PHYLIP manual version 3.25c. Berkeley University Herbarium, University of California, Berkeley. |
| 28. | Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877[Abstract]. |
| 29. | Fenyo, E. M., J. Fiore, A. Karlsson, J. Albert, and G. Scarlatti. 1994. Biological phenotypes of HIV-1 in pathogenesis and transmission. Antibiot. Chemother. 46:18-24[Medline]. |
| 30. |
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 |
| 31. | Glushakova, S., J. C. Grivel, W. Fitzgerald, A. Sylwester, J. Zimmerberg, and L. B. Margolis. 1998. Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency. Nat. Med. 4:346-349[CrossRef][Medline]. |
| 32. |
Groenink, M.,
A. C. Andeweg,
R. A. Fouchier,
S. Broersen,
R. C. van der Jagt,
H. Schuitemaker,
R. E. de Goede,
M. L. Bosch,
H. G. Huisman, and M. Tersmette.
1992.
Phenotype-associated env gene variation among eight related human immunodeficiency virus type 1 clones: evidence for in vivo recombination and determinants of cytotropism outside the V3 domain.
J. Virol.
66:6175-6180 |
| 33. |
Groenink, M.,
R. A. Fouchier,
S. Broersen,
C. H. Baker,
M. Koot,
A. B. van't Wout,
H. G. Huisman,
F. Miedema,
M. Tersmette, and H. Schuitemaker.
1993.
Relation of phenotype evolution of HIV-1 to envelope V2 configuration.
Science
260:1513-1516 |
| 34. |
Harrington, R. D., and A. P. Geballe.
1993.
Cofactor requirement for human immunodeficiency virus type 1 entry into a CD4-expressing human cell line.
J. Virol.
67:5939-5947 |
| 35. | Hoffman, T. L., and R. W. Doms. 1999. HIV-1 envelope determinants for cell tropism and chemokine receptor use. Mol. Membr. Biol. 16:57-65[CrossRef][Medline]. |
| 36. |
Hwang, S. S.,
T. J. Boyle,
H. K. Lyerly, and B. R. Cullen.
1992.
Identification of envelope V3 loop as the major determinant of CD4 neutralization sensitivity of HIV-1.
Science
257:535-537 |
| 37. |
Hwang, S. S.,
T. J. Boyle,
H. K. Lyerly, and B. R. Cullen.
1991.
Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1.
Science
253:71-74 |
| 38. |
Kaneshima, H.,
L. Su,
M. L. Bonyhadi,
R. I. Connor,
D. D. Ho, and J. M. McCune.
1994.
Rapid-high, syncytium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse.
J. Virol.
68:8188-8192 |
| 39. | Keet, I. P., P. Krijnen, M. Koot, J. M. Lange, F. Miedema, J. Goudsmit, and R. A. Coutinho. 1993. Predictors of rapid progression to AIDS in HIV-1 seroconverters. AIDS 7:51-57[Medline]. |
| 40. | 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[CrossRef][Medline]. |
| 41. |
Koot, M.,
I. P. Keet,
A. H. Vos,
R. E. de Goede,
M. T. Roos,
R. A. Coutinho,
F. Miedema,
P. T. Schellekens, and M. Tersmette.
1993.
Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS.
Ann. Intern. Med.
118:681-688 |
| 42. | Korber, B., and G. Myers. 1992. Signature pattern analysis: a method for assessing viral sequence relatedness. AIDS Res. Hum. Retrovir. 8:1549-1560[Medline]. |
| 43. |
Labrosse, B.,
A. Brelot,
N. Heveker,
N. Sol,
D. Schols,
E. De Clercq, and M. Alizon.
1998.
Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100.
J. Virol.
72:6381-6388 |
| 44. | Lathey, J. L., R. D. Pratt, and S. A. Spector. 1997. Appearance of autologous neutralizing antibody correlates with reduction in virus load and phenotype switch during primary infection with human immunodeficiency virus type 1. J. Infect. Dis. 175:231-232[Medline]. |
| 45. |
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 advanced stages of disease and comparison to tissue-derived isolates.
J. Virol.
73:9741-9755 |
| 46. |
Liao, F.,
G. Alkhatib,
K. W. Peden,
G. Sharma,
E. A. Berger, and J. M. Farber.
1997.
STRL33, A novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1.
J. Exp. Med.
185:2015-2023 |
| 47. | Liesnard, C., M. L. Delforge, M. Tchetcheroff, V. De Maertelaer, C. M. Farber, and J. P. Van Vooren. 1997. Importance of method in the determination of syncytium-inducing phenotype of human immunodeficiency virus type 1 clinical isolates. J. Virol. Methods 64:137-145[CrossRef][Medline]. |
| 48. | Malykh, A., M. S. Reitz, Jr., A. Louie, L. Hall, and F. Lori. 1995. Multiple determinants for growth of human immunodeficiency virus type 1 in monocyte-macrophages. Virology 206:646-650[CrossRef][Medline]. |
| 49. |
Mammano, F.,
V. Trouplin,
V. Zennou, and F. Clavel.
2000.
Retracing the evolutionary pathways of human immunodeficiency virus type 1 resistance to protease inhibitors: virus fitness in the absence and in the presence of drug.
J. Virol.
74:8524-8531 |
| 50. | Moore, J. P., A. Trkola, and T. Dragic. 1997. Co-receptors for HIV-1 entry. Curr. Opin. Immunol. 9:551-562[CrossRef][Medline]. |
| 51. | O'Brien, W. A., Y. Koyanagi, A. Namazie, J. Q. Zhao, A. Diagne, K. Idler, J. A. Zack, and I. S. Chen. 1990. HIV-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the CD4-binding domain. Nature 348:69-73[CrossRef][Medline]. |
| 52. | Palmer, C., P. Balfe, D. Fox, J. C. May, R. Frederiksson, E. M. Fenyo, and J. A. McKeating. 1996. Functional characterization of the V1V2 region of human immunodeficiency virus type 1. Virology 220:436-449[CrossRef][Medline]. |
| 53. | Pantaleo, G., O. J. Cohen, T. Schacker, M. Vaccarezza, C. Graziosi, G. P. Rizzardi, J. Kahn, C. H. Fox, S. M. Schnittman, D. H. Schwartz, L. Corey, and A. S. Fauci. 1998. Evolutionary pattern of human immunodeficiency virus (HIV) replication and distribution in lymph nodes following primary infection: implications for antiviral therapy. Nat. Med. 4:341-345[CrossRef][Medline]. |
| 54. |
Penn, M. L.,
J. C. Grivel,
B. Schramm,
M. A. Goldsmith, and L. Margolis.
1999.
CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue.
Proc. Natl. Acad. Sci. USA
96:663-668 |
| 55. | 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[Abstract]. |
| 56. | Picchio, G. R., R. J. Gulizia, and D. E. Mosier. 1997. Chemokine receptor CCR5 genotype influences the kinetics of human immunodeficiency virus type 1 infection in human PBL-SCID mice. J. Virol. 71:7124-7127[Abstract]. |
| 57. |
Picchio, G. R.,
R. J. Gulizia,
K. Wehrly,
B. Chesebro, and D. E. Mosier.
1998.
The cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in SCID mice reconstituted with human peripheral blood leukocytes.
J. Virol.
72:2002-2009 |
| 58. |
Pleskoff, O.,
C. Treboute,
A. Brelot,
N. Heveker,
M. Seman, and M. Alizon.
1997.
Identification of a chemokine receptor encoded by human cytomegalovirus as a cofactor for HIV-1 entry.
Science
276:1874-1878 |
| 59. | Richman, D. D., and S. A. Bozzette. 1994. The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J. Infect. Dis. 169:968-974[Medline]. |
| 60. | Rucker, J., A. L. Edinger, M. Sharron, M. Samson, B. Lee, J. F. Berson, Y. Yi, B. Margulies, R. G. Collman, B. J. Doranz, M. Parmentier, and R. W. Doms. 1997. Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses. J. Virol. 71:8999-9007[Abstract]. |
| 61. | Salvatori, F., S. Masiero, C. Giaquinto, C. M. Wade, A. J. Brown, L. Chieco-Bianchi, and A. De Rossi. 1997. Evolution of human immunodeficiency virus type 1 in perinatally infected infants with rapid and slow progression to disease. J. Virol. 71:4694-4706[Abstract]. |
| 62. | Scarlatti, G., E. Tresoldi, A. Bjorndal, R. Fredriksson, C. Colognesi, H. K. Deng, M. S. Malnati, A. Plebani, A. G. Siccardi, D. R. Littman, E. M. Fenyo, and P. Lusso. 1997. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. Nat. Med. 3:1259-1265[CrossRef][Medline]. |
| 63. | Schuitemaker, H. 1994. Macrophage-tropic HIV-1 variants: initiators of infection and AIDS pathogenesis? J. Leukoc. Biol. 56:218-224[Abstract]. |
| 64. |
Schuitemaker, H.,
M. Koot,
N. A. Kootstra,
M. W. Dercksen,
R. E. de Goede,
R. P. van Steenwijk,
J. M. Lange,
J. K. Schattenkerk,
F. Miedema, and M. Tersmette.
1992.
Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population.
J. Virol.
66:1354-1360 |
| 65. |
Scoggins, R. M.,
J. R. Taylor, Jr.,
J. Patrie,
A. B. van't Wout,
H. Schuitemaker, and D. Camerini.
2000.
Pathogenesis of primary R5 human immunodeficiency virus type 1 clones in SCID-hu mice.
J. Virol.
74:3205-3216 |
| 66. | Shioda, T., J. A. Levy, and C. Cheng-Mayer. 1991. Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene. Nature 349:167-169[CrossRef][Medline]. |
| 67. |
Shioda, T.,
J. A. Levy, and C. Cheng-Mayer.
1992.
Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1.
Proc. Natl. Acad. Sci. USA
89:9434-9438 |
| 68. | Simmons, G., D. Wilkinson, J. D. Reeves, M. T. Dittmar, S. Beddows, J. Weber, G. Carnegie, U. Desselberger, P. W. Gray, R. A. Weiss, and P. R. Clapham. 1996. Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry. J. Virol. 70:8355-8360[Abstract]. |
| 69. | Speck, R. F., K. Wehrly, E. J. Platt, R. E. Atchison, I. F. Charo, D. Kabat, B. Chesebro, and M. A. Goldsmith. 1997. Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop. J. Virol. 71:7136-7139[Abstract]. |
| 70. |
Takeuchi, Y.,
M. Akutsu,
K. Murayama,
N. Shimizu, and H. Hoshino.
1991.
Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene.
J. Virol.
65:1710-1718 |
| 71. |
van't Wout, A. B.,
H. Blaak,
L. J. Ran,
M. Brouwer,
C. Kuiken, and H. Schuitemaker.
1998.
Evolution of syncytium-inducing and non-syncytium-inducing biological virus clones in relation to replication kinetics during the course of human immunodeficiency virus type 1 infection.
J. Virol.
72:5099-5107 |
| 72. | van't Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, and H. Schuitemaker. 1994. Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J. Clin. Investig. 94:2060-2067. |
| 73. | 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 373:117-122[CrossRef][Medline]. |
| 74. |
Westervelt, P.,
D. B. Trowbridge,
L. G. Epstein,
B. M. Blumberg,
Y. Li,
B. H. Hahn,
G. M. Shaw,
R. W. Price, and L. Ratner.
1992.
Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.
J. Virol.
66:2577-2582 |
| 75. |
Wolinsky, S. M.,
C. M. Wike,
B. T. Korber,
C. Hutto,
W. P. Parks,
L. L. Rosenblum,
K. J. Kunstman,
M. R. Furtado, and J. L. Munoz.
1992.
Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.
Science
255:1134-1137 |
| 76. | Wu, L., N. P. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, A. A. Cardoso, E. Desjardin, W. Newman, C. Gerard, and J. Sodroski. 1996. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384:179-183[CrossRef][Medline]. |
| 77. |
Wyatt, R., and J. Sodroski.
1998.
The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.
Science
280:1884-1888 |
| 78. |
Zhang, L.,
T. He,
Y. Huang,
Z. Chen,
Y. Guo,
S. Wu,
K. J. Kunstman,
R. C. Brown,
J. P. Phair,
A. U. Neumann,
D. D. Ho, and S. M. Wolinsky.
1998.
Chemokine coreceptor usage by diverse primary isolates of human immunodeficiency virus type 1.
J. Virol.
72:9307-9312 |
| 79. |
Zhang, Y. J.,
T. Dragic,
Y. Cao,
L. Kostrikis,
D. S. Kwon,
D. R. Littman,
V. N. KewalRamani, and J. P. Moore.
1998.
Use of coreceptors other than CCR5 by non-syncytium-inducing adult and pediatric isolates of human immunodeficiency virus type 1 is rare in vitro.
J. Virol.
72:9337-9344 |
| 80. | Zhu, T., H. Mo, N. Wang, D. S. Nam, Y. Cao, R. A. Koup, and D. D. Ho. 1993. Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 261:1179-1181. |
Journal of Virology, January 2001, p. 251-259, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.251-259.2001
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[Abstract] [Full Text] -
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(2007). Significant Genetic and Antigenic Variability within the env Gene of Systemic Human Immunodeficiency Virus Type 1 Group O Populations during the Natural Course of a Heterosexual Infection: a Pilot Study. J. Clin. Microbiol.
45: 1319-1321
[Abstract] [Full Text] -
Whitcomb, J. M., Huang, W., Fransen, S., Limoli, K., Toma, J., Wrin, T., Chappey, C., Kiss, L. D. B., Paxinos, E. E., Petropoulos, C. J.
(2007). Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism. Antimicrob. Agents Chemother.
51: 566-575
[Abstract] [Full Text] -
Lambele, M., Labrosse, B., Roch, E., Moreau, A., Verrier, B., Barin, F., Roingeard, P., Mammano, F., Brand, D.
(2007). Impact of Natural Polymorphism within the gp41 Cytoplasmic Tail of Human Immunodeficiency Virus Type 1 on the Intracellular Distribution of Envelope Glycoproteins and Viral Assembly. J. Virol.
81: 125-140
[Abstract] [Full Text] -
Delobel, P., Nugeyre, M.-T., Cazabat, M., Sandres-Saune, K., Pasquier, C., Cuzin, L., Marchou, B., Massip, P., Cheynier, R., Barre-Sinoussi, F., Izopet, J., Israel, N.
(2006). Naive T-cell depletion related to infection by x4 human immunodeficiency virus type 1 in poor immunological responders to highly active antiretroviral therapy.. J. Virol.
80: 10229-10236
[Abstract] [Full Text] -
Legoff, J., Bouhlal, H., Gresenguet, G., Weiss, H., Khonde, N., Hocini, H., Desire, N., Si-Mohamed, A., de Dieu Longo, J., Chemin, C., Frost, E., Pepin, J., Malkin, J.-E., Mayaud, P., Belec, L.
(2006). Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV. J. Clin. Microbiol.
44: 423-432
[Abstract] [Full Text] -
Skrabal, K., Saragosti, S., Labernardiere, J.-L., Barin, F., Clavel, F., Mammano, F.
(2005). Human Immunodeficiency Virus Type 1 Variants Isolated from Single Plasma Samples Display a Wide Spectrum of Neutralization Sensitivity. J. Virol.
79: 11848-11857
[Abstract] [Full Text] -
Nobile, C., Petit, C., Moris, A., Skrabal, K., Abastado, J.-P., Mammano, F., Schwartz, O.
(2005). Covert Human Immunodeficiency Virus Replication in Dendritic Cells and in DC-SIGN-Expressing Cells Promotes Long-Term Transmission to Lymphocytes. J. Virol.
79: 5386-5399
[Abstract] [Full Text] -
Kuhmann, S. E., Pugach, P., Kunstman, K. J., Taylor, J., Stanfield, R. L., Snyder, A., Strizki, J. M., Riley, J., Baroudy, B. M., Wilson, I. A., Korber, B. T., Wolinsky, S. M., Moore, J. P.
(2004). Genetic and Phenotypic Analyses of Human Immunodeficiency Virus Type 1 Escape from a Small-Molecule CCR5 Inhibitor. J. Virol.
78: 2790-2807
[Abstract] [Full Text] -
Labrosse, B., Labernardiere, J.-L., Dam, E., Trouplin, V., Skrabal, K., Clavel, F., Mammano, F.
(2002). Baseline Susceptibility of Primary Human Immunodeficiency Virus Type 1 to Entry Inhibitors. J. Virol.
77: 1610-1613
[Abstract] [Full Text] -
Vicenzi, E., Panina-Bodignon, P., Vallanti, G., Di Lucia, P., Poli, G.
(2002). Restricted replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not in Th1 CD4+ T cells. J. Leukoc. Biol.
72: 913-920
[Abstract] [Full Text]
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