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J Virol, January 1998, p. 488-496, Vol. 72, No. 1
Department of Clinical Viro-Immunology,
Received 21 April 1997/Accepted 14 October 1997
We studied the temporal relationship between human immunodeficiency
type 1 (HIV-1) quasispecies in tissues and in peripheral blood
mononuclear cells (PBMC) of infected individuals. Sequential PBMC and
tissue samples from various organs obtained at autopsy from three
patients who died of AIDS-related complications were available for
analysis. Biological HIV-1 clones were isolated from PBMC samples, and
cellular tropism and syncytium-inducing (SI) capacity were determined.
Genomic DNA was isolated from 1 cm3 of organ tissue, and
proviral DNA was amplified by means of PCR and cloned with the PGEM-T
vector system. A 185-bp region encompassing the third variable domain
of the virus envelope, known to influence HIV-1 biological properties,
was sequenced. HIV-1 could be amplified from all PBMC and organ
samples, except from liver tissue for two patients. Both SI and
non-syncytium-inducing (NSI) genotypes could be detected in the
different tissues. Tissue-specific quasispecies were observed in brain,
lung, and testis. Lymphoid tissues, such as bone marrow, lymph node,
and spleen, harbored several different variants similar to those
detected in blood in the last PBMC samples. In general, only tissues in
which macrophages are likely to be the main target cell for HIV-1
harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI
sequences that clustered with sequences from late-stage PBMC were
present in other tissues, which may indicate that the presence of HIV-1
in those tissues is secondary to lymphocyte infiltration rather than to
tissue tropism of HIV-1 itself. These data suggest that the viral
reservoir may be limited, which will have important implications for
the success of HIV-1 eradication.
The human immunodeficiency virus
type 1 (HIV-1) variants can differ with respect to syncytium-inducing
(SI) capacity, replication rate, and cellular tropism (2, 7, 51,
52). In asymptomatic individuals, slowly replicating,
preferentially macrophage-tropic nonsyncytium-inducing (NSI) HIV-1
variants predominant (4, 10, 42, 45, 54, 58). With
progression of disease, more rapidly replicating, preferentially
T-cell-tropic viruses emerge, in 50% of individuals coinciding with
the emergence of SI HIV-1 variants (28, 45, 52). The
presence of preferentially macrophage-tropic HIV-1 in the asymptomatic
phase, when host immunity is relatively intact, has led to the
suggestion that macrophage tropism is a mechanism for HIV-1 variants to
escape immune surveillance (45). Alternatively or in
addition, the persistence of macrophage-tropic NSI variants during all
stages of disease may point to a reservoir function for HIV-1-infected
macrophages (46) from which new variants are generated. The
presence of SI HIV-1 in lymph nodes in the absence of this phenotype in
peripheral blood indeed indicates that new virus variants may be
generated in lymphoid tissues (34, 50). The lymphoid tissues
have been shown to be active sites of replication throughout HIV
infection also during the asymptomatic phase (14, 39). In
situ hybridization, however, showed that only a very few macrophages in
lymph nodes were infected (23). This can mean either that
macrophages constitute a reservoir at another body site or that this
cell type does not constitute a major reservoir in HIV-1 infection.
With the availability of potent treatment regimens, the eradication of
HIV-1 seems feasible. To achieve this, however, all compartments
relevant for virus replication should be accessible to the
antiretroviral compounds. The exact identification of the viral
reservoir in the body is therefore of the utmost importance. The
presence of HIV-1 in nonlymphoid tissues may be attributed to infected
macrophages. These cells could thus constitute a separate compartment
of viral replication, with subsequent separate evolution of HIV-1
quasispecies. Alternatively, the presence of HIV-1 in these nonlymphoid
tissues may result only from lymphocyte infiltration secondary to
infections with opportunistic pathogens coinciding with the onset of
AIDS (13). If the first scenario were true, HIV-1
quasispecies in nonlymphoid tissues would probably be more closely
related to the quasispecies that were present in peripheral blood
mononuclear cells (PBMC) early in infection than to those late in
infection, as the two compartments would have evolved separately from a
common precursor. Conversely, close resemblance of the HIV-1
quasispecies in the nonlymphoid tissues to the peripheral quasispecies
after the onset of AIDS would indicate similar virus turnover with
continuous exchange in tissues and periphery or very late spread of the
virus to the nonlymphoid tissues. The latter case, although mediated
through a different mechanism, would not influence the outcome of
antiretroviral intervention, as the spread to the nonlymphoid tissues
would be limited.
To investigate the relation between virus variants in periphery and in
lymphoid and nonlymphoid tissues and to identify the reservoir function
of macrophages for HIV-1 in tissues, we analyzed HIV-1 populations in
sequential blood samples and several organ tissues obtained at autopsy.
Phylogenetic analyses were performed to study the relation between
infectious cellular quasispecies present in PBMC over time and virus
variants present in tissue compartments at the time of death.
Patient samples.
Tissues from various lymphoid and
nonlymphoid organs were obtained at autopsy, carried out within 2 days
of death, from three individuals, one female and two males, who died of
AIDS-related complications. Clinical, immunological, and viral load
information for the years prior to death are summarized in Table
1 and Fig. 1. Samples of brain, lung, liver, kidney,
spleen, lymph node, bone marrow, and/or testis were washed with
phosphate-buffered saline-10% trinatrium citrate, dissected into
1-cm3 pieces, and stored at Analysis of CD4+ T-cell counts.
T-lymphocyte
immunophenotyping for CD4+ and CD8+ T cells was
carried out at 3-month intervals by flow cytofluorometry. PBMC were
stained with CD4 or CD8 monoclonal antibody according to standard
procedures for fluorescence-activated cell sorting analysis.
Preparation of DNA from patient tissues.
Extraction of DNA
from the tissues was carried out by resuspending small pieces of tissue
in lysis buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 50 mM EDTA,
1% sodium-n-lauroylsarcosine, 100 µg of proteinase K per
ml) as described elsewhere (13). The digestion process was
allowed to continue for 2 to 3 h at 65°C. This was followed by
phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation
overnight. DNA pellets were dried and resuspended in 200 µl of
distilled water.
Cloning and sequencing of PCR products.
Envelope sequences
were amplified by PCR with primers V1V2-1
(5'-GCCTGTGTACCCCACAGACCCCAA-3', nucleotide position 6463, sense) (22) and PS-D (5'-ATTACAGTAGAAAAATTCCCC-3',
position 7381, antisense) (49) in the presence of 3 mM
MgCl2 in the first reaction and primers V1V2-2
(5'-GAGGATATAATCAGTTTATGGGAT-3', position 6562, sense)
(22) and PS-C (5'-CTGGGTCCCCTCCTGAGG-3', position
7331, antisense) (49) in the presence of 3 mM
MgCl2 in the nested reaction. For amplification, the
following PCR amplification cycles were used: 5 min at 95°C once and
1.5 min at 95°C, 1 min at 55°C, and 1 min at 72°C, repeated 25 times, followed by an extra 5 min of extension at 72°C and subsequent
cooling to 4°C. The resulting ±800-bp products from three to six
nested PCRs were pooled and purified with a Geneclean kit (Bio 101, Inc., Vista, Calif.). These purified products were cloned into PGEM-T
vector (Promega) according to the manufacturer's protocol. Fifty
microliters of competent Escherichia coli cells (strain
JM109) was then transformed with 4 to 7 µl of ligation mix. The
recombinants were plated on Luria broth agar with ampicillin (75 mg/ml), incorporating 50 µl of
5-bromo-4-chloro-3-indolyl- Isolation of biological virus clones from patient PBMC.
Limiting dilution cultures were performed as described elsewhere (45).
Briefly, participant PBMC (0.5 × 104 to 4 × 104 cells per well, 24 to 96 replicates per concentration)
or CSF samples (1-in-5 dilution series) were cocultivated with
PHA-stimulated healthy donor PBMC (105 per well) in 96-well
microtiter plates. Every week for 5 weeks, one-third of each culture
supernatant was collected for detection of p24 antigen by an in-house
p24 antigen capture enzyme-linked immunosorbent assay. At the same
time, one-third of the culture volume, containing half of the cells,
was transferred to new 96-well plates and 105 fresh
PHA-stimulated healthy donor PBMC were added to propagate the culture.
The proportion of productively infected CD4+ T cells was
estimated by the formula for Poisson distribution, F = Infection of monocyte-derived macrophages.
Virus-containing
culture supernatants derived from the CSF-derived virus clones from
patients HIVAms198 and ACH6052 and PBMC-derived virus
clones from patient ACH0208 were tested for their capacity to infect
monocyte-derived macrophages from different healthy blood donors as
described elsewhere (46, 54).
DNA isolation, PCR, and sequencing of biological virus
clones.
Total DNA from PBMC harboring the biological HIV-1 clones
was isolated as described elsewhere (6). Envelope V3
sequences were amplified by PCR as described elsewhere (49),
with primers PS-A and PS-H in the first reaction and PS-B and PS-G in
the nested reaction. The resulting ±700-bp products were purified with
a Geneclean kit (Bio 101, Inc.), and 185 bp were sequenced directly with sense primer PS-B (49) by the dideoxy chain termination method with Sequenase (U.S. Biochemical), both according to the instructions from the manufacturers.
Sequence data analysis.
Alignment of the sequences was
straightforward, done with the PILEUP program, and checked manually, to
ensure that codons remained intact. To check for contamination, a BLAST
search of GenBank HIV-1 sequences was performed for sequences with the
highest similarity. Phylogenetic analyses were done with the
neighbor-joining program (43) as implemented in the PHYLIP
package (16). For bootstrapping, the SEQBOOT, DNADIST,
NEIGHBOR, and CONSENSE programs from this package were used. PHYLIP's
DRAWTREE program was used to produce the plots. For direct comparison
of nucleotide sequences, Hamming distances were used. The distance
matrix input for the neighbor-joining analysis was generated with
Kimura's two-parameter estimation for nucleotides (27).
Estimation of the number of silent and nonsilent substitutions was done
according to Nei and Gojobori's method (37), as implemented
in MEGA (32). To evaluate possible associations between
certain amino acid changes and tissue tropisms, the following method
was used. The tissues were divided into two groups (one dominated by
T-cell-tropic HIV-1 [PBMC, lymph node, spleen, and bone marrow] and
the other dominated by macrophage-tropic HIV-1 [brain, CSF, lung, and
liver]). Kidney was regarded as a control for contamination with
variants from the blood. For each position in the sequences, a pairwise
comparison matrix consisting of zeros and ones was constructed,
indicating for each pair of sequences whether they contained identical
or different nucleotides at that position (position matrix). A second
matrix contained a similar same-different index indicating whether each
pair of sequences came from the same or a different tissue group (group matrix). A Pearson correlation coefficient was then calculated between
corresponding cells of the position matrix and the tissue group matrix,
yielding a position-wise indication of the association between sequence
differences and tissue group (31).
Nucleotide sequence accession numbers.
All newly generated
sequences have been deposited in GenBank (accession no.
AF021367-AF021476 [HIVAms198], AF021477-AF021683 [ACH6052], and AF021684-AF021790 [ACH0208]).
Proviral HIV-1 sequences (185 bp encompassing the V3 loop) were
obtained from all PBMC and tissue samples, except from the livers of
patients ACH0208 and ACH6052. PCR amplification of the HIV-1 V3 region
from DNA isolated from a liver tissue sample from patient
HIVAms198 yielded only 1 positive reaction (of 14), which was used for ligation. In all samples tested, the sequences from clonal
populations reflected bulk sequences from uncloned material (both PBMC
and tissues), i.e., neither cloning procedure preferentially expanded
rare variants (data not shown). SI capacity of biological clones
isolated from peripheral blood was determined by cocultivation with the
MT-2 cell line, in each case confirming the phenotype as predicted from
the genotype (19). The 98% correlation between V3 genotype
and HIV-1 biological phenotype with respect to SI capacity enabled us
to predict the phenotype from the virus clones in different tissues
based on their V3 sequence (18). In the phylogenetic tree
for each patient, sequences from the two other patients were included,
both as outgroups and to exclude contamination. In all instances, the
patient sequences clustered separately from the outgroup sequences in
100 of 100 trees (data not shown). The patient sequences were also
different from the laboratory strains available in our laboratory
(HIV-1 BaL, HIV-1 ADA, HIV-1 IIIb, ACH0320.2A5/2A6, and ACH0172.Ba-L).
Moreover, a BLAST similarity search of GenBank HIV-1 sequences did not
provide evidence for contamination with virus strains obtained from
other patients in the Amsterdam cohort (data not shown). Tissue
sequences from all three patients were further analyzed for signature
patterns pertaining to particular tissues, and for presumed
T-cell-tropic HIV-1-dominated tissues (PBMC, lymph node, bone marrow,
and spleen) versus compartments considered to be macrophage-tropic
HIV-1 dominated (lung, brain, CSF, and liver). Although specific
groupings were detected in each patient separately (see below), no
specific signature pattern for either of these comparisons was detected
(all Pearson correlations, Results per patient.
From patient HIVAms198, eight
different tissues and three different PBMC samples (collected 7 months
before and 6 and 15 months after AIDS diagnosis) were available for
analysis (Table 2). From all body sites, only NSI variants were
obtained, as judged from the MT-2 assay and/or predicted from V3
sequences. Alignment of all sequences relative to the consensus NSI
sequences from the first PBMC sample is shown in Fig.
2A. With these sequences, neighbor-joining trees with bootstrap resampling were constructed (Fig.
3A). Brain and CSF quasispecies were
highly homogeneous, clustered closely together, and were most closely
related to some of the latest available sequences obtained from PBMC
(15 months after AIDS diagnosis). In cell culture, these CSF variants
were shown to be highly macrophage tropic, able to infect
monocyte-derived macrophages from several different donors (data not
shown). The liver quasispecies also clustered closely together;
however, the liver sequences were all generated from only one positive
PCR, and the homogeneity is possibly a reflection of low proviral copy numbers. The quasispecies from the other tissues and PBMC samples were
heterogeneous and did not show a particular tissue- or time-specific clustering. Heterogeneity of the samples was further analyzed by
calculating pairwise Hamming distances (Fig.
4). Hamming distances were less than 2%
for brain, CSF, and liver but ranged up to 14% for sequences derived
from the other tissues and from PBMC.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of the Temporal Relationship between Human
Immunodeficiency Virus Type 1 Quasispecies in Sequential Blood
Samples and Various Organs Obtained at Autopsy
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C (Table
2). Because of the absence of HIV-related
neurological symptoms in patient ACH0208, no brain autopsy was
performed. From the two other patients, samples of cerebrospinal fluid
(CSF) were also obtained. These were cultured in a limiting dilution
fashion on phytohemagglutinin (PHA)-stimulated PBMC for 4 weeks (see
below). PBMC samples were available from all three individuals spanning
at least the period from AIDS onset to death (in subjects ACH6052 and
HIVAms198) or the time from seroconversion to death (in
subject ACH0208).
TABLE 1.
Characteristics of the three subjects under study
View larger version (21K):
[in a new window]
FIG. 1.
Changes in the numbers of CD4 ( 
) and CD8 (
) T
cells and cellular infectious load (
) relative to the moment of AIDS
diagnosis for the three subjects under study (HIVAms198
[top], ACH6052 [middle], and ACH0208 [bottom]). White, grey, and
black inverted triangles denote the moment of seroconversion, emergence
of SI variants, and death, respectively.
TABLE 2.
List of samples analyzed
-D-galactopyranoside (X-Gal; 40 mg/ml) and 20 µl of
isopropyl-3-D-thiogalactopyranoside (IPTG; 100 ng/ml), and
incubated overnight at 37°C. A total of 20 to 55 individual clones
were picked and expanded overnight in Luria broth at 37°C. Bacterial
cells were pelleted, DNA was extracted with the Vacman system (Qiagen),
and samples were digested with restriction enzymes SphI and
PstI (GIBCO-BRL) for 1 h at 37°C and checked for the
presence of insert on a 1% agarose gel. Clones containing insert were
sequenced directly with sense primer PS-B (49) by the
dideoxy chain termination method with Sequenase (U.S. Biochemical,
Cleveland, Ohio) according to the instructions from the manufacturer.
ln (F0), in which F0 is
the fraction of negative cultures. PBMC from wells testing positive
were transferred to 25-ml culture flasks containing 5 × 106 fresh PHA-stimulated PBMC in 5 ml of culture medium to
grow virus stocks. Virus-containing cell-free culture supernatant was
stored at 70°C until further use, cells were frozen, and
approximately 106 cells were used for isolation of DNA. SI
capacity of virus clones was determined by cocultivation with MT-2
cells (106). Biological virus clones from patient
HIVAms198 and patient ACH6052 were all isolated on target
cells from the same healthy blood donor. For patient ACH0208,
additional isolations were performed on target cells derived from three
additional healthy blood donors. In our experience, target cells
derived from different healthy blood donors do not influence the clonal
composition of the viruses isolated from one sample, except when the
healthy blood donor is heterozygous or homozygous for the 32-bp
deletion in CCR5 (data not shown).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
0.23). Moreover, no evidence for
previously described signature patterns for brain and/or CSF sequences
was observed (30, 35).
View larger version (71K):
[in a new window]
FIG. 2.
Deduced amino acid sequences of the V3 region. The
sequences are aligned with the consensus sequence of the NSI variants
present in the first sample for each patient. Amino acid positions
involved in SI capacity are marked by asterisks. Dashes indicate
identity with the reference sequence. Position 1 corresponds to amino
acid 296 of the HXB2 envelope protein. Mo., number of months relative
to AIDS diagnosis; #, number of virus clones with a specific sequence.
Virus clones were obtained from PBMC and tissue samples of patient
HIVAms198 (A), participant ACH6052 (B), and participant
ACH0208 (C).
View larger version (19K):
[in a new window]
FIG. 3.
Results of phylogenetic analysis of the V3 region
(neighbor-joining method, unrooted tree) from virus clones obtained
during the course of infection. Bootstrap values indicate the
percentages of trees showing the observed specific groupings. White and
black symbols depict NSI and SI PBMC-derived sequences,
respectively, and grey symbols depict tissue-derived sequences. Branch
lengths are drawn to scale. (A) Subject HIVAms198; (B)
subject ACH6052; (C) subject ACH0208.
View larger version (18K):
[in a new window]
FIG. 4.
Plots of Hamming distances between the V3 regions of the
virus clones obtained from PBMC in the course of HIV-1 infection and
those from several tissues obtained at autopsy from three subjects,
HIVAms198 (top), ACH6052 (middle), and ACH0208 (bottom).
Distances were calculated with DNADIST as implemented in the PHYLIP
program. Means and standard deviations of comparisons between NSI
variants ( ), between SI variants (
), and between SI and NSI
variants (
) from the same time point are shown.
7- and 3-month PBMC, lymph node, and testes (Fig.
2B).
Thirteen PBMC samples spanning the 74-month period between 6 months
after seroconversion and up to 14 months before death were available
from patient ACH0208, in addition to the five tissue samples obtained
after death (Table 2). None of the biological virus clones obtained
from PBMC samples were able to infect monocyte-derived macrophages
obtained from two healthy blood donors (data not shown). Of note, this
patient was shown to be heterozygous for a previously described 32-bp
deletion (
32) in the CCR5 coreceptor gene. After seroconversion,
this patient harbored only NSI variants for 15 months (=40 months
before AIDS diagnosis) before developing SI variants (see alignments in
Fig. 2C). Biological virus clones from the PBMC sample obtained 10 months after AIDS diagnosis were isolated on PHA-stimulated target
cells from a healthy blood donor, who was retrospectively identified to
be homozygous for the 32-bp deletion in CCR5, reflected by the
isolation of only SI variants from this sample. Two possible
intermediate NSI variants (so-called switch NSI) were detected 43 and
40 months before AIDS diagnosis with arginines at positions 10 and/or
25. Thereafter, the SI and NSI quasispecies seem to have continued to
evolve as separate groups (see neighbor-joining tree in Fig. 3C).
Again, no new SI variants generated from late NSI variants were
detected. Between 34 and 22 months before AIDS diagnosis, there was a
complete replacement of the SI virus population by SI variants with
glutamine at amino acid position 16, and between 22 and 10 months
before AIDS diagnosis, there was a replacement of the NSI virus
population by NSI variants with threonine at position 13. Pairwise
Hamming distances between SI and NSI sequences obtained from the same
sample (either tissue or PBMC) increased over time up to 10%, while
within the respective phenotypic groups Hamming distance was generally
less than 5% (Fig. 4). NSI clones isolated from 10 months before AIDS
diagnosis onwards may have resulted from a recombination of 5' NSI and
3' SI ends between SI and NSI virus clones found at the previous time
point (Fig. 2C).
All tissues harbored only SI variants that were also present in PBMC
between 10 and 19 months after AIDS diagnosis. The lymph node
quasispecies clustered with the last available SI PBMC sequences obtained 19 months after AIDS diagnosis. While the testis quasispecies were rather homogeneous (three main variants, Hamming distance within
2%), the spleen, lung, and kidney quasispecies were more diverse
(Hamming distances ranging from 0 to 8%).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, we analyzed the temporal relationship between the clonal composition of HIV-1 populations in sequential PBMC samples and various tissue samples obtained at autopsy. Using phylogenetic analysis, we tried to identify the PBMC sample containing virus variants most closely similar to the viral quasispecies in lymphoid and nonlymphoid tissues, since this could shed light on the moment in infection at which HIV-1 was disseminated into the nonlymphoid tissue compartment or, alternatively, at which moment HIV-1 was shed from tissue into the circulation. Viral quasispecies from most lymphoid and nonlymphoid tissues clustered with the viral sequences obtained from late PBMC samples. This can indicate either that migration of HIV-1 into the nonlymphoid tissues is a late event in the clinical course of infection (13) or that there is a relatively high turnover of viral quasispecies with an active interplay between replication in all tissues and peripheral blood. The lymph node sequences clustered most closely with very late PBMC sequences. This is in agreement not only with the assumptions that plasma virions are produced in lymph nodes and are responsible for PBMC infection (24, 55) but also with the close correlation between plasma virus load and cellular infectious load (5). The most likely explanation for the presence of HIV-1 in nonlymphoid tissues is migration of HIV-1-infected lymphocytes, secondary to late-stage pathology in these tissues. Alternatively, the migration could be a consequence of disturbed lymphocyte homing due to the HIV-1-related destruction of lymph node architecture (36, 40, 41).
The viral quasispecies from brain tissue were very homogeneous and clustered separately from all other sequences, but most closely with some of the late PBMC sequences. However, the brain-CSF populations were distinctly different from the PBMC variants despite their clustering. These results are more suggestive of compartmentalization and early spread of HIV-1 to the brain, which is in agreement with previous findings (1, 3, 8, 15, 21, 26, 30, 38, 48, 56). The fact that some of the late PBMC sequences are more similar to this compartment may actually reflect leakage of virus produced in the brain into the circulation.
One individual in our present study harbored only NSI HIV-1 variants, both in PBMC and in all tissues analyzed. In the other two individuals, SI variants were present at least at later stages of infection. The emergence of SI variants seemed to be a unique event, as no new SI variants that could have evolved from later-stage NSI variants were detected (53), nor did the SI populations in tissues cluster separately from the PBMC SI variants. Rather, the SI and NSI quasispecies in all tissue compartments seemed to evolve separately from a common precursor NSI sequence, which was indeed reflected by the increasing Hamming distance between the coexisting NSI and SI HIV-1 populations, up to 14%.
Within the quasispecies of either NSI or SI variants, Hamming distances did not exceed the 5% level. This may indicate fierce competition between coexisting NSI and SI virus populations, in which only the fittest variant in each phenotypic group evolves further, generating the new homogeneous virus population that is subsequently present. The detection of possible recombinants between SI and NSI variants in the two patients who harbored both variants may indicate that SI and NSI variants have some overlapping target cell populations. Although the recombinants detected in the tissues could be an artifact due to PCR recombination, the fact that the same recombinants were detected in tissue and biological virus clones (with a clonal input for the PCRs) suggests that they are not solely the result of a PCR artifact.
In individual HIVAms198 harboring only NSI variants, brain and CSF harbored very homogeneous virus populations, whereas other compartments contained highly heterogeneous HIV-1 quasispecies with Hamming distances ranging up to 14%. Apparently, in some compartments, more variation may occur if these NSI HIV-1 quasispecies are able to occupy the niches from which they otherwise would have been excluded by competing coexisting SI variants. The homogeneity in virus populations in brain and CSF might then be explained by the fact that SI HIV-1 lacks the capacity anyway to replicate in these compartments and that only limited variation in NSI is allowed in replication at these sites. Alternatively, the limited variation may reflect slower turnover of the virus populations and infected cells in this compartment compared to that in the lymphoid tissues, compatible with the slow resistance development detected in the brain (48, 56).
Although we know that SI HIV-1 evolves from NSI HIV-1, it is still unclear in which compartment the generation of SI variants occurs. The presence of SI sequences in lymph nodes of individuals who showed future SI conversion has been reported elsewhere (34, 50). This indeed points to the lymph nodes as the most active site for viral replication. Here, we observed the presence of both NSI and SI HIV-1 in all tissues, although a tissue-specific abundance of specific phenotypes could not be excluded. The clustering of tissue SI HIV-1 sequences with SI sequences from late-stage-obtained PBMC and not with the first SI sequences obtained from PBMC recently after phenotype conversion exclude, in our opinion, the possibility that new virus variants are generated in tissues other than lymph nodes and then shed into the circulation.
Patient ACH6052 showed an SI prevalence in lymph node and a predominance of NSI HIV-1 in lung and CSF and to a lesser extent in spleen and testis. The preferential presence of NSI variants in testis is in accordance not only with a previous study that compared virus variants in blood and semen (59) but also with the hypothesis that HIV-1 infection is preferentially established by macrophage-tropic NSI variants (20, 58), which might be due to compartmentalization of macrophage-tropic HIV-1 in the donor. Kidney tissue from patient ACH6052, which was used as a control for blood contamination, indeed harbored an NSI-SI distribution that reflected the clonal composition in peripheral blood.
Despite the presence of NSI HIV-1 in peripheral blood at least up to 14 months prior to death, patient ACH0208 harbored only SI HIV-1 variants
in all tissues analyzed. Although it may be that by the time of death
only SI variants were present in PBMC as well, other explanations are
also feasible. Interestingly, this individual was heterozygous for the
previously described 32-bp deletion in the CCR5 gene which encodes the
second receptor for primary HIV-1 (11, 44). PBMC from CCR5
32 heterozygotes show impaired support of NSI HIV-1 replication
(33), associated in vivo with lower levels of viral RNA load
in serum (12, 25). SI HIV-1 is able to use CXCR4 as an
alternative coreceptor and may therefore have a growth advantage over
NSI HIV-1 in CCR5 32 heterozygotes (17). However, if SI
and NSI variants indeed remained present up to the time of death,
tissue migration could be specific for CXCR4-expressing potential SI
target lymphocytes. The absence of NSI HIV-1, which in general is more
macrophage tropic (46), in tissues in which macrophages are
considered the important target cell, might directly be associated with
the impaired coreceptor expression in CCR5 32 heterozygotes. This
reduced expression together with the already low expression of CD4 on
primary macrophages (9) may be insufficient to support
replication even of macrophage-tropic NSI HIV-1. This latter
observation also leads to the intriguing possibility that 32
heterozygous individuals do not have an important macrophage reservoir
for HIV-1 replication. Indeed, none of the biological virus clones
obtained from ACH0208 either early or late in infection was able to
productively infect monocyte-derived macrophages.
Assuming that all subjects initially had a relatively homogeneous virus
population (54, 57, 58), considerable diversification has
occurred up to the moment of AIDS diagnosis. Thereafter, there seems to
be no significant further increase in variability in any of the
subjects. It has been postulated that on this V3 region of the viral
envelope a considerable pressure is exerted, which is reflected in a
higher rate of nonsynonymous than synonymous mutations (29, 47,
49). Assuming that at the moment of AIDS diagnosis the host
immune system has collapsed, our finding may suggest that the selection
pressure on V3 is predominantly exerted by immune control. However, the
strong segregation of central nervous system-lymphoid quasispecies in
both subjects where central nervous system virus could be evaluated and
the emergence and dominance (in tissues) of the SI type sequences when
32 CCR5 was present show the strong influence of host cell
availability on the V3 region.
In conclusion, apart from the brain, our study finds no evidence for the existence of true tissue reservoirs that are installed early in infection and from which new variants are continuously generated. Rather, the presence of HIV-1 in most nonlymphoid tissues seems to be a late-stage event and secondary to lymphocyte infiltration due to pathology. The absence of a large tissue reservoir for HIV-1 favors the possibility of completely eradicating HIV-1. The presence of a separate virus reservoir in the brain underscores, however, the importance of including in the combination of drugs used antiretroviral drugs which have good bioavailability in the brain.
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ACKNOWLEDGMENTS |
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These studies were performed as part of the Amsterdam Cohort Studies of AIDS, a collaboration among the Municipal Health Service, The Academic Medical Centre, and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. Proleukin (recombinant interleukin-2) was kindly provided by R. Rombouts, Chiron Benelux B.V., Amsterdam, The Netherlands. We are greatly indebted to Jan Weening, head of the Department of Pathology at the Academic Medical Centre, and his colleagues and especially to Iwan Nektar for outstanding assistance during the autopsy procedures; to Marijke Roos and colleagues for excellent technical assistance; and to Hetty Blaak, Frank van Engelenburg, Maarten Koot, and Frank Miedema for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Clinical Viro-Immunology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Phone: 31 20 512 3317. Fax: 31 20 512 3310. E-mail: clbkvi{at}xs4all.nl.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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