University of Washington School of Medicine, Virology Division, Retrovirology Laboratory, Seattle, Washington
Correspondence: Dr. Scott J. Brodie, University of Washington School of Medicine, Department of Laboratory Medicine, Vaccine/Virology Division, Room T293X, Seattle, WA 98195.
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) within these sites
contained tat fusion transcripts, a product of
post-transcriptional splicing and a correlate of productive infection.
When examining explant cultures of M and FDC, only M harbored HIV
tat mRNA and only M demonstrated budding retroviral
particles. Hence, germinal center FDC in secondary lymphoid tissues are
key reservoirs of immune-complexed HIV RNA and are likely to contribute
to AIDS-associated lymphoproliferations; however, these cells do not
support HIV replication, and failure to do so results from a
post-transcriptional block in the virus life cycle. Moreover, gut and
pulmonary M represent a lineage of cells that are permissive to HIV
replication and contribute significantly to the high viral load in
children with severe CD4+ T cell depletion. It will be
important to identify the molecular mechanisms that allow for these
highly productive infections of M.
Key Words: BALT • GALT • macrophages • follicular dendritic cells • peripheral blood lymphocytes
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Four potential cellular reservoirs of HIV replication thus far have
been identified: activated CD4+ T lymphocytes [11
, 14
, 15
];
blood monocytes [16
, 17
]; macrophages (M) of various lineage [8
, 18
19
], characterized by their long life-span [20
] and ability to express
HIV for prolonged periods [21
]; and follicular dendritic cells (FDC),
which can store large quantities of virus [22
23
24
25
26
]. It is clear that
FDC are important in maintaining HIV infection of lymph node germinal
centers and can transmit virus to activated CD4+ T cells
[27
28
29
30
31
]. It is not clear whether FDC support HIV replication in vivo
[32
33
34
35
36
], because it is difficult to demonstrate by in situ
hybridization and by transmission electron microscopy the exact
location of virus within these cells. Few studies have examined virus
replication in mature explant-derived FDC and none in the context of
alternatively spliced viral transcripts as correlates of productive
infection. Here, we present a possible explanation of the pathobiology
of persistent HIV replication, based on observations that some children
with severe depletion of CD4+ T cells maintain high plasma
viral load and recent demonstrations that combination antiretroviral
therapy effectively extinguishes FDC reservoirs of virus [37
38
39
] yet
virus replication persists in most individuals [40
41
42
43
].
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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View this table: [in a new window] |
Table 1. Persistent reservoirs of HIV replication
|
Plasma antigen and virus isolation
Plasma was assessed for HIV RNA using the bDNA assay (Chiron,
Norwood, MA), which has a lower limit of detection of 500 HIV RNA
copies/ml [49
]. Isolation of infectious HIV and quantitative
microculture was performed as described previously [50
]. A minimum of
107 cells were applied to phytohemagglutinin-stimulated and
interleukin-2 (IL-2)-treated HIV-negative donor PBMC (Table 1)
.
Serological tests to detect antibodies to human herpesvirus 8 (HHV-8)
used an immunofluorescence assay for lytic cycle cytoplasmic antigen
and were performed as described previously [10
, 51
, 52
].
Cell purification
Peripheral blood and tissue lysates were applied to
discontinuous density gradients, washed, and resuspended in RPMI 1640,
followed by a 1-h incubation (37°C and 5% CO2) in
fibronectin-coated flasks (20 µg/ml), whereby adherent cells were
removed. The nonadherent lymphocyte-enriched population was labeled
with magnetic bead-conjugated monoclonal antibodies (mAbs) (4:1
bead:cell ratio; Dynal, Great Neck, NY) to CD8, CD14, CD16, CD19, CD20,
and CD21 to enrich for CD4+ T cells. The CD4+
cells were then sorted (FACS, fluorescence activated cell sorting) for
CD45RO+ memory T cells. Tissue FDC were isolated from lung
explants in the presence of GM-CSF, TNF-
, and IL-4 [53
, 54
]. FDC
were further purified using negative selection and magnetic bead
elutriation [33
] until an immunomorphologically pure CD1a+,
B7/BB1+, HLA-2+, and CD4+ (low)
population was obtained. FDC were nonadherent, nonphagocytic, and had a
veiled surface appearance. Tissue M were initially isolated by
adherence, enriched using magnetic bead elutriation (anti-CD68, clone
EBM11, DAKO), and then sorted to high-purity. By combining magnetic
bead elutriation and FACS, >99% of isolated cells were
immunophenotypically compatible with memory (CD45RO+) PBL,
CD68+ (esterase+) M, and CD1a+
FDC, respectively. Unless otherwise indicated, all mAbs were products
of PharMingen (San Diego, CA).
Quantitative fluorescent probe polymerase chain reaction (TaqMan)
DNA and RNA were extracted (QIAmp DNA and RNA kits; Qiagen,
Santa Clarita, CA) from cultured cells as described previously [55
].
HIV tat RNA and tat fusion mRNA were
reverse-transcribed in a solution containing 1.25 mM MgCl2,
10 mM dithiothreitol, 10 mM Tris-HCl pH 8.3), 50 mM KCl, 2.0 mM of each
dNTP, 10 pM of specific antisense primer (below), 200 U Superscript II
reverse transcriptase (RT) (Gibco BRL, Grand Island, NY), and 4 µl (2
µg) of RNA in a 20-µl reaction. High-purity DNA and cDNA were then
applied at a final concentration of 0.5 µg per well and TaqMan
performed on samples in quadruplicate. The amplification mix and
polymerase chain reaction (PCR) conditions for a 50-µl reaction
consisted of 1X PCR buffer II, 8% glycerol, 1.0 U AmpErase (uracil
glycoslase; Epicenter, Madison, WI), 2.5 U AmpliTaq DNA polymerase
(Perkin-Elmer), 4 mM MgCl2, 200 µM dATP, dGTP, dCTP, and 400 µM dUTP, 30 µmol of forward MF5869
(5'-GCGAATTCATGGAKCCAGTAGATCCTAGACTA-3' [nt 5869–5886]) and reverse
MF8760 (GCTCTAGACTACTGTCCCCTCAGCTACTGCTATGG-3' [nt 8760–8733]) of
HIV strain HXB2, respectively, and 20 µmol TaqMan probe (MF5945,
5'-ATTGTAAAAAGTGTT-splice-GCTWTCATTGC-3'). The designation K refers to
the single-code letter for G or C, and W refers to A or T for
degenerative bases. Tat plasmids were provided by Dr.
Manohar Furtado and have been described previously [56
]. TaqMan primers
and probes for HIV gag have been described [48
]. HIV DNA and
RNA concentrations in test samples were determined by coamplification
of ß-actin and GADPH (Perkin-Elmer), respectively. Target and control
DNA/cDNA were subjected to 50°C for 2 min and 95°C for 10 min,
followed by 42 cycles of amplification at 95°C for 20 s and
60°C for 1 min, using ABI PRISM 7700 sequence detector
(Perkin-Elmer). Assay controls consisted of ACH-2 cell DNA and armored
RNA (Ambion, Austin, TX) as standards [48
], as well as nucleic acid
derived from PBL, M, and FDC of HIV-seronegative children [10
].
PCR in situ hybridization
PCR in situ hybridization (PCR-ISH) was used to localize HIV DNA
to cultured cells and to cells within tissues. Tissue sections were
deparaffinized, rehydrated in Tris-buffered saline (TBS; 0.1 M Tris
[pH 7.5], 0.1 M NaCl), digested with proteinase K (20 (g/ml, 37°C,
30 min; Sigma, St. Louis, MO), and washed in DEPC water. Fifty µl of
a solution containing 1X PCR buffer, 4 mM MgCl2, 0.01%
gelatin, 200 µM dNTPs, 50 pM of HIV gag-specific primers
[55
] and Taq polymerase (0.15 U/µl) was applied to
tissues. Thermocycling conditions were the same as described previously
[47
, 55
]. The PCR product was detected by ISH using a cocktail of 3 HIV
gag-specific oligonucleotides labeled with
digoxigenin-11-dUTP (DIG, Boehringer Mannheim, Indianapolis, IN), and
all were in sense orientation and internal to the PCR primer-binding
sequences (SK102, 5'-GAGACCATCAATGAGGAAGCTGCAGAATGGGAT; SK19,
5'-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC; and SJB9I,
5'-CTGTTCCTGGTTTCCTTGGGAAATCTCTGA). Hybridization conditions and
controls were as described previously [47
, 55
]. The presence of HIV DNA
was indicated by a purple cell-associated precipitate and visualized by
incident light microscopy. Images from 10 representative regions of the
tissue were transmitted to a computer equipped with a digital imaging
board and an average viral load was determined [48
].
Combined immunohistochemistry and in situ hybridization
Immunohistochemistry was used in combination with ISH to
identify cells expressing viral RNA (Fig. 2), as described previously
[10
]. For fresh cells and frozen tissues, CD45RO+ PBL,
CD19+ B cells, CD68+ M, and
CD1a+ FDC were localized in the context of HIV
gag-pol, EBV EBER1, and HHV-8 orf 26 RNA expression. RNA
encoding HIV gag p24 (0.8 kb) and pol (0.7 kb) was cloned
into transcription vectors under the control of T7 and SP6 RNA
polymerase promoters (pGEM; Promega, Madison, WI), using methods
described previously [47
, 55
]. RNA-encoding EBER1 and orf 26 were as
described previously [10
, 51
, 52
]. The constructs were linearized and
used as templates for in vitro transcription to which DIG-11-UTP
(Boehringer Mannheim) was incorporated. Tissue preparation and
hybridization procedures were as described previously [10
, 51
, 52
]. The
presence of viral nucleic acid was indicated by a purple/brown
cell-associated precipitate and visualized by incident light
microscopy. Tissues labeled with fluorescent probes were visualized
directly with an ACAS 570 laser confocal microscope [48
]. The lower
limit of detection was 
20 RNA copies per cell [51
, 52
].
A separate in situ hybridization method was used to detect immune-complexed HIV RNA associated with germinal center FDC. 35S-labeled CTP (Amersham, Arlington Heights, IL) was incorporated into gag and pol riboprobes (described above), which were then applied to protease K-treated tissues (5 µg/ml, 37°C, 30 min; Sigma), as described [10 ]. This technique can identify cells with as few as 5 gag-pol RNA copies and can also be used as means to gauge the amount of germinal center HIV RNA when examined using low-power dark-field microscopy.
Transmission electron microscopy
FDC and M derived from tissue explants were fixed in
half-strength Karnovsky’s fixative, washed in 0.1 M cacodylate buffer,
postfixed in 1% osmium tetroxide, dehydrated, treated with propylene
oxide, and embedded in Epon. Embedded cells were cut in ultrathin
sections, stained with uranyl acetate and lead citrate, and analyzed
with a JOEL 100S transmission electron microscope.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 View larger version (27K): [in a new window] |
Figure 1. Quantification of HIV RNA in postmortem tissues using fluorescent probe
PCR. Results represent the mean and SEM of 4 replicate
samples and are expressed as HIV gag RNA copies per ng of
total cellular DNA (1 ng cellular DNA (150 cells). Tissues include
periventricular white matter, caudal lung lobe, spleen, caudal
mediastinal lymph node, and distal colon. Open characters identify
subjects with AIDS-associated opportunistic lymphoproliferations.
Closed characters identify children with severe late-stage lymphoid.
Bars indicate the median gag RNA copy number where
statistically relevant.
|
expressing HIV RNA (Fig. 2AII
and BII
) and viral
core proteins (Fig. 2AIII
) localized peripheral to
hyperplastic BALT and GALT. Large accumulations of germinal center HIV
RNA were detected in conjunction with proteolytic digestion (Fig. 2AV
and BIII
), indicating that virus bound to FDC
was protein-coated (immune-complexed). A high proportion (5–20%) of
germinal center B cells expressed EBV EBER1 RNA (Fig. 2E
).
However, there was no evidence of opportunistic herpesvirus infections
of GALT or in any of the other tissues examined. Only 1 child (Child 8)
demonstrated CNS disease. A thorough histolopathological evaluation
revealed multifocal granulomatous lesions (Fig. 2CI
)
containing gag p24 immunoreactive multinucleated giant cells
(Fig. 2CII
) within periventricular white matter.
 View larger version (99K): [in a new window] |
Figure 2. Representative histomorphology (AI, BI, CI) and sites HIV
(AII, BII, BIII) and EBV (E) RNA localization in
sections of caudal right lung (AI–V, E) and distal colon
(BI–III) from a child (Child 2) with severe multicentric
lymphoid hyperplasia, and periventricular white matter (CI,
II) from a child (Child 8) with severe lymphoid atrophy. Note that
there is marked expansion of bronchiole-associated lymphoid tissue and
;250q;;25>diffuse thickening of the pulmonary interstitium
(AI, H, E). Cells harboring gag RNA (panel
AII, 35S-riboprobes) and gag proteins
(panel AIII, anti-p24 HPO) were morphologically compatible
with interstitial lymphocytes and M (arrows). Large amounts of HIV
RNA aggregated within lymphoid germinal centers (panel AIV,
anti-CD21 HPO) and localized to FDC (panel AV,
35S-riboprobes, dark-field). Gut-associated lymphoid tissue
(BI, H, E) contained high numbers of mononuclear cells
harboring HIV RNA (BII) within perifollicular and
intervillous spaces (DIG-riboprobes, anti-DIG-AP). Large aggregates of
HIV RNA were present within lymphoid germinal centers (panel
BIII, 35S-riboprobes, dark-field). Only 1 child
(Child 8) demonstrated lesions within the periventricular white matter
characteristic of AIDS-associated granulomatous encephalitis (CI,
H, E). Note the high number of gag p24 immunoreactive
multinucleated giant cells (panel CII, anti-p24 HPO). A
detailed ultrastructural study of pulmonary FDC revealed HIV particles
to be adherent to the cell body and dendritic processes
(DI), whereas pulmonary M demonstrated both
intracytoplasmic and budding retroviral particles (DII)
(-50,000). Pulmonary lymphoid hyperplasia was invariably associated
with a high proportion of germinal center B cells expressing EBV EBER1
RNA (panel E, DIG-riboprobes, anti-DIG-AP). (Bars = 100
µm).
|
-expressing gag-pol RNA in
histologic sections of distal colon (Table 1)
. Tissues that would
normally be lymphocyte-rich, such as the pulmonary interstitium (Fig. 3A
) and mediastinal lymph node (Fig. 3D
) were
severely hypocellular. A thorough evaluation of these tissues using a
combination of immunohistochemical and in situ hybridization procedures
showed that lung, and to a lesser extent gut, harbored high numbers of
cells expressing gag-pol RNA (Fig. 3Ci
).
Dual-staining techniques, when applied to histologic sections of lung,
showed that these RNA-positive cells were unequivocally alveolar and
interstitial macrophages (Fig. 3Cii
). Similar observations
were made within sections of distal colon; only mucosal M harbored
HIV RNA in tissues demonstrating severe lymphoid atrophy. FDC
reservoirs of virus were not observed in children with lymphoid
atrophy. Moreover, protease treatment neither increased the number of
HIV RNA-positive cells nor did it reveal areas of residual
FDC-associated HIV. Rather, viral expression localized exclusively to
M.
 View larger version (117K): [in a new window] |
Figure 3. Representative histomorphology (A, D) and sites of
localization of HIV gag DNA (B, E) and
gag-pol RNA (C, F) in sections of caudal right
lung (A–C) and caudal mediastinal lymph node
(D–F) from a child (Child 7) with severe lymphocyte
depletion and AIDS. Note the hypocellularity of the pulmonary
interstitium (A) and mediastinal lymph node (D, H,
E). gag DNA localized to round cells with lymphocyte
morphology (open arrows) and to stromal cells with M-like features
(closed arrows) in both lung (B) and lymph node
(E) (PCR-ISH, anti-DIG-AP). Cells expressing
gag-pol RNA were only detected in the lung (panel
Ci; ISH, anti-DIG-AP) and not in the accompanying
mediastinal lymph node, and were shown by dual staining to be
exclusively alveolar and interstitial macrophages (panel
Cii; IHC and ISH, PE-anti-CD68 and
FITC-anti-gag-pol). Controls consisted of plasma clot
preparations of HIV-infected (G, arrows) and uninfected
(H, arrows) MT2 cells, lung tissue from an HIV-seronegative
child (I, J), and lung from a child with high titers of HIV
RNA (K, L) [10
]. Control tissues were hybridized with sense
(I, K) and antisense (G, H, J) gag-pol
riboprobes, and with nonsense bluetongue virus RNA probes [47
]
(L) (anti-DIG-AP). (Bars = 100 µm).
|
was the best
predictor of plasma viremia in these severely lymphopenic children
(Table 2)
. M and FDC derived from explant cultures of secondary lymphoid
tissues and PBL were assayed for spliced and unspliced HIV
tat RNA. All cells harbored unspliced tat, but
only PBL and M contained tat fusion transcripts, products
of post-transcriptional splicing and a correlate of productive
infection [15
, 41
]. The ratio of spliced to unspliced tat
varied per cell type and per disease state. FDC contained the highest
proportion of unspliced tat RNA (Table 2)
, levels that
corresponded with concentrations found in germinal centers (Table 1)
;
still, these FDC showed no evidence of fusion mRNA. These relatively
high concentrations of unspliced HIV RNA were likely the result of
virion RNA bound to the cell surface and/or harbored within
intracytoplasmic vesicles that were not completely removed by protease
treatment. Still, based on a variety of assays that detected only HIV
DNA, including PCR in situ hybridization, virus was able to enter FDC
and at least partially reverse transcribe. Moreover, after >3 weeks in
culture and multiple treatments with trypsin, the cultivated FDC still
harbored unspliced tat transcripts, but at a 10-fold
reduction in concentration (not shown), indicating that these cells
were permissive to infection and virus transcription. Because these
cells showed no evidence of spliced tat mRNA and did not
produce infectious virus (Table 2)
, the defect in virus replication
most likely occurred at the level of post-transcriptional ligation. In
contrast, pulmonary M from lymphopenic children produced high titers
of tat fusion mRNA, levels much higher than children with
lymphoproliferative disease (P = 0.016, unpaired
t-test) (Table 2)
. When comparing viral titers in PBL and
M from children with opportunistic lymphoproliferations, there was
no difference in the amount of unspliced (P = 0.1,
unpaired t-test) and spliced (P = 0.7,
unpaired t-test) tat RNA, with mean ratios of
5:1 and 7:1 of unspliced to spliced RNA in PBL and M,
respectively. In contrast, M from lymphopenic children contained
much higher concentrations of unspliced tat RNA
(P = 0.003, unpaired t-test), with a mean
ratio of 27:1 of unspliced to spliced RNA. In these T cell depleted
children, there was no evidence of tat fusion transcripts or
cultivable virus in PBL (Table 2)
. Only M harbored was infectious
virus in this population. |
View this table: [in a new window] |
Table 2. Quantification of HIV RNA in peripheral blood lymphocytes, pulmonary follicular dendritic cells, and pulmonary macrophages during late-stage pediatric AIDS
|
demonstrated both intracytoplasmic and budding
retroviral particles (Fig. 2Dii
), evidence of productive
infection.
Latent reservoirs
HIV gag DNA localized to cells morphologically
compatible with FDC, M, and lymphocytes in all tissues examined by
PCR-ISH, including a few residual lymphocytes within the pulmonary
interstitium (Fig. 3B
), lymph nodes (Fig. 3E
),
and spleen from children with severe lymphoid atrophy. With the
exception of lung, the numbers of cells harboring gag DNA
were at least 20-fold greater than those expressing gag RNA,
indicating that the majority of infections were nonproductive.
Conversely, lung tissue contained a high proportion of cells
transcriptionally active for HIV, with a ratio of 2:1 DNA- to
RNA-positive cells, respectively. In children with late-stage disease,
these transcriptionally active cells were exclusively alveolar and
interstitial M.
Opportunistic lymphoproliferation
Because gamma herpesviruses have been linked to
opportunistic lymphoproliferations in children [10
, 57
, 58
] and animals
[59
, 60
] with chronic retroviral infections, we examined mucosal
lymphoid tissues for viral transcripts indicative of EBV and HHV-8
infections. All children with lymphoid hyperplasia expressed EBV RNA,
with as many as 20% of germinal center B cells in BALT harboring EBV
EBER-1 RNA (Fig. 2E
). Interestingly, EBV was not detected in
GALT, although these tissues showed similar levels of lymphoid
expansion as lung. Lung tissues from children with widely disseminated
EBV showed high numbers of HIV-infected cells with abundant virus. The
HIV-positive M in these samples far exceeded the number of
productively infected lymphocytes, a finding characteristic of
hyperplastic lymph nodes without opportunistic infection. Surprisingly,
children with lymphoid atrophy had similar numbers of HIV-infected
M. EBV was not detected in tissues from children with lymphoid
atrophy and HHV-8 was not present in any tissues examined. A
retrospective analysis of serum showed that none of the subjects
possessed measurable antibodies to HHV-8.
Collectively, these data suggest that the lung and gut, and to a much lesser extent the CNS, are primary sites of HIV replication in children with late-stage disease, with and without opportunistic infections.
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The role of long-lived mononuclear phagocytes and dendritic cells as a
source of HIV replication has been considered minimal, as few if any of
these cells show productive infection in circulation or in lymphoid
tissue. This is especially true during asymptomatic infection and
during effective antiretroviral treatment [18
, 23
, 37
, 39
, 61
]. Here,
we show high numbers of HIV-infected M in the lung and colon from
children during late-stage AIDS and a high proportion of these
RNA-positive cells contained tat fusion transcripts,
indicating there was post-transcriptional splicing and ligation,
processes which have been associated with productive infection [15
, 41
56
]. Others have shown CD4+ M, in addition to expressing
a wide variety of HIV co-receptors essential for virus entry [8
, 62
63
64
], are the principle HIV-infected cells in the CNS of patients
with AIDS dementia [1
, 2
, 18
], and adults and children with
opportunistic pneumonia [7
, 8
, 10
], and possibly some gastrointestinal
disorders [9
, 13
]. Moreover, their numbers are maintained or increased
with disease progression and, unlike lymphocytes, are capable of
producing large amounts of both intracellular and extracellular virus
without succumbing to the lethal effects of productive viral infection
[7
].
FDC are capable of trapping large amounts of HIV on their cell surfaces in the form of antigen-antibody complexes and holding these complexes for long periods [22 23 24 25 26 ]. As such, FDC-bound virus is likely to serve as a source of new infection for activated CD4+ T cells trafficking through germinal centers congested with FDC-bound HIV [27 28 29 30 31 61 ]. Still, it is yet unclear if FDC are capable of supporting HIV replication in vivo, because there are many conflicting reports that support and refute their role as a source of HIV replication [32 33 34 35 36 , 65 ]. These discrepancies may be explained, in part, by differences in the ontogeny of dendritic cells; many FDC are morphologically similar but can be derived from several unrelated pathways of differentiation [25 , 32 , 66 ]. Moreover, because dendritic cells can be derived from a wide variety of different progenitor cells, it is likely that if productive infection occurs in this cell type it is restricted to specific lineages of FDC and/or perhaps occurs only within certain differentiation/maturation states [53 , 54 , 67 ].
Because FDC can proliferate and form extensive networks within secondary lymphoid tissues of individuals with AIDS and concurrent EBV infection [10 , 37 , 68 ], we took advantage of this as a means to examine FDC in situ, as well as harvest these cells from tissues and examine them at high-purity in vitro. Few studies have examined HIV replication in mature explant-derived FDC or in the context of dual in situ labeling. In vitro infection of monocyte-derived dendritic cells with lymphocytotropic and monocytotropic HIV showed that viral RNA was reverse transcribed to complete DNA provirus [53 ]. However, the infection was nonproductive, as judged from measuring the activity encoded by reverse transcriptase in culture supernatant. Thus, HIV infection of dendritic cells was restricted at a step post-entry. We show that HIV could enter mucosal FDC, reverse transcribe, and initiate transcription of at least the tat gene, but failed to process transcripts into translatable mRNA. Furthermore, we could not detect intracytoplasmic or budding viral particles after an exhaustive search using transmission electron microscopy, again suggesting that these cells did not support HIV replication.
A variety of herpesviruses have been implicated in pediatric
lymphoproliferations [10
, 57
, 58
]. We show that EBV localized to
germinal center B lymphoblasts in tissues with hyperplastic BALT. This
high incidence of EBV infection and virus replication may result from
defective regulation of EBV in patients with AIDS or AIDS-related
disorders [69
]. Patients with opportunistic lymphoproliferations
typically show high numbers of EBV-infected B cells in circulation as a
consequence of profound defects in T-cell immunity [69
, 70
].
Co-infection of B cells with HIV and EBV has also been described as a
possible co-factor in the progression from polyclonal B cell
proliferation to lymphoma, because both the upregulation of
c-myc and activation of EBV can occur as a result of HIV
infection [71
]. We have shown previously that FDC in secondary lymphoid
tissues are major sites for EBV replication and that proliferation of
germinal center B cells in children with opportunistic
lymphoproliferations results from EBV infection of B cells and/or their
association with viral antigens bound to and/or expressed by FDC [10
].
Others have shown that EBV can infect and transform FDC, although
infection in vivo was rare as were FDC tumors [72
73
74
75
]. Collectively,
these data show that EBV is an important opportunist in germinal center
hyperplasia, although the mechanisms of these events are not clear.
Moreover, because M from children without opportunistic
lymphoproliferations produced similar amounts of HIV, it is unlikely
that co-viral interactions, such as EBV-mediated transactivation of the
HIV LTR was responsible for the increases in HIV replication that occur
during late-stage AIDS.
This study focuses attention on the persistence of HIV replication in
children with chronic infection. We demonstrate that germinal center
FDC in secondary lymphoid tissues are key reservoirs of
immune-complexed HIV RNA and are likely to contribute to
AIDS-associated lymphoproliferations; however, these cells do not
support HIV replication, and failure to do so results from a
post-transcriptional block in the virus life cycle. Moreover, gut and
pulmonary M represent a lineage of cells that are permissive to HIV
replication and contribute significantly to the high viral load seen in
some children with severe CD4+ T cell depletion. It will be
important to identify the molecular mechanisms that allow for these
highly productive infections of M.
My thanks to Kurt Diem, Larry Stensland, and Lisa Glazatcheff for technical assistance, and to my colleagues Dr. Lawrence Corey, Dr. Andrew Johnson, and Dr. Bruce Patterson for helpful discussions and critical review of this article.
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