Differential HIV-1 replication in neonatal and adult
blood mononuclear cells is influenced at the level
of HIV-1 gene expression
Vasudha Sundaravaradan*, Shailendra K. Saxena*†, Rajesh Ramakrishnan*‡, Venkat R. K. Yedavalli§, David T. Harris,
and Nafees Ahmad¶
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724
Edited by Robert C. Gallo, University of Maryland, Baltimore, MD, and approved June 16, 2006 (received for review March 17, 2006)
The majority of HIV-1-infected neonates and infants have a higher
level of viremia and develop AIDS more rapidly than infected
adults, including differences seen in clinical manifestations. To
determine the mechanisms of HIV-1 infection in neonates vs.
adults, we compared the replication kinetics of HIV-1 in neonatal
(cord) and adult blood T lymphocytes and monocyte-derived mac-
replicated 3-fold better in cord blood T lymphocytes compared
with adult blood T lymphocytes and 9-fold better in cord MDM
than adult MDM. We also show that this differential HIV-1 repli-
cation did not depend on differences in cell proliferative capabil-
ities, cell surface expression of CD4, CXCR4, and CCR5, or in the
amount of PCR products of reverse transcription, DNA synthesis,
and translocation of preintegration complex into the nucleus in
cord and adult T lymphocytes and MDM. Furthermore, using a
single-cycle replication competent HIV-1-NL4–3-Env?luciferase
amphotropic virus, which measures HIV-1 transcriptional activity
independent of receptor and coreceptor expression, we found
there was a 3-fold increase of HIV-1 LTR-driven luciferase expres-
10-fold in cord MDM than in adult MDM. The HIV-1 LTR-driven
luciferase expression correlated with HIV-1 LTR transcription, as
measured by ribonuclease protection assay. These data suggest
adult blood mononuclear cells is regulated at the level of HIV-1
gene expression, resulting in a higher level of viremia and faster
disease progression in neonates than adults.
AIDS ? differential HIV-1 gene expression ? pediatric AIDS ?
cord blood mononuclear cells ? neonatal HIV-infection
with AIDS, encephalopathy, bacterial infections, and a unique
type of lymphocytic pneumonia occur more frequently than in
adults (2–4). Most infected infants become symptomatic within
the first few months of life, however, a subset of infants remains
asymptomatic with immune abnormalities for years (2–4). In
contrast to X4 viruses associated with AIDS progression in
adults (5, 6), rapidly progressing infected infants generally
harbor R5 viruses associated with a high viral load (7, 8). In
contrast to HIV-1-infected adults where initial infection results
in an acute retroviral syndrome with a high level of viremia
followed by a set point (5, 6), infected infants have a higher level
of viremia than infected adults that is sustained over a long
period (3, 4). The pathogenesis of pediatric AIDS is not clearly
understood but may be partially explained by relative immaturity
of the immune system in early infancy.
Different isolates of HIV-1 infect not only T lymphocytes but
also other cells of the immune system, particularly monocytes and
their mature form, macrophages (9). The monocytes?macrophages
(M?M) are relatively refractory to the cytopathic effects of HIV-1
and may serve as a major reservoir for the virus (10). Much
IV disease in neonates and infants has a more rapid and
fatal course than seen in infected adults (1, 2). In infants
information related to the immunopathogenesis of AIDS has been
gained from HIV-1 infection of primary adult M?M and T lym-
phocytes (11). However, the role of neonatal M?M and T lympho-
cytes in the immunopathogenesis of pediatric AIDS has not been
fully elucidated. Because R5 viruses are involved in vertical trans-
mission (12, 13), its interaction with monocyte-derived macro-
phages (MDM) and CD4??CCR5?T cells may play an important
role in the establishment of HIV-1 infection and disease progres-
sion. It is likely that increased replication of HIV-1 in neonatal
mononuclear cells may contribute to a high level of viremia and
in these cell types. We have used cord blood in place of neonatal
blood because, like neonatal blood, it has more CD45RA?T cells
(14), and is available in a larger volume than neonatal blood.
Here we show that HIV-1 replicates more efficiently in cord
blood MDM and T lymphocytes compared with adult blood cells.
There was no significant difference in the cell proliferative capa-
bilities, the levels of HIV-1 receptor (CD4) and coreceptors
(CXCR4 and CCR5) for virus entry, and the levels of postentry
events [reverse transcription and translocation of preintegration
complex (PIC) into the nucleus] of cord blood mononuclear cells
(CBMCs) vs. PBMCs. However, there was a significant up-
regulation in HIV-1 gene expression in cord MDM and T lympho-
cytes compared with adult cells, suggesting that the differential
HIV-1 replication in cord and adult target cells is regulated at the
level of HIV-1 gene expression.
HIV-1 Replicates More Efficiently in Neonatal (Cord) Blood MDM and
T Lymphocytes Compared with Adult Blood Cells. To determine the
replication efficiency of HIV-1 in cord and adult target cells, we
MDM isolated as described in Materials and Methods were infected
with an equal amount [5–15 ? 104reverse transcriptase (RT) cpm]
of HIV-1BaL and HIV-1NL4–3 (lab adapted R5 and X4 HIV-1,
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CBMC, cord blood mononuclear cell; MDM, monocyte-derived macro-
phages; M?M, monocytes?macrophages; PBMC, adult blood mononuclear cell; PIC, prein-
tegration complex; RPA, ribonuclease protection assay.
*V.S., S.K.S., and R.R. contributed equally to this work.
†Present address: Center for Cellular and Molecular Biology, Hyderabad 500 007, India.
‡Present address: Department of Molecular Virology and Microbiology, Baylor College of
Medicine, Houston, TX 77030.
§Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and
Infectious Diseases, Bethesda, MD 20892.
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
August 1, 2006 ?
vol. 103 ?
no. 31 ?
respectively). Culture supernatants were replaced every three days
and assayed for virus production by measuring RT activity (12, 15).
The results of HIV-1BaL replication in cord and adult blood T
clearly demonstrates that HIV-1BaLreplicated 3-fold better in cord
T lymphocytes compared with adult T lymphocytes at peak RT
production (Fig. 1A). Moreover, this effect was more profound in
MDM, where HIV-1Ba-Lreplicated 9-fold better in cord MDM
compared with adult MDM (Fig. 1B). We also found that HIV-
1NL4–3replicated 3-fold better in cord T lymphocytes compared
with adult T lymphocytes (Fig. 1C). The HIV-1 replication kinetics
in cord and adult blood T lymphocytes and MDM has been
performed from seven different donors with similar results, al-
observed from various donor sets. However, the magnitude of
HIV-1 replication was higher in cord lymphocytes and MDM
compared with adult cells with statistically significant results. The
P values for HIV-1BaL in MDM were ?0.001, HIV-1BaL in T
lymphocytes ?0.0001, and HIV-1NL4–3in T lymphocytes ?0.0003
(n ? 7). These data demonstrate that there was an increased
replication efficiency of HIV-1 in both cord T lymphocytes and
MDM compared with adult cells, with a more profound effect seen
in MDM than T lymphocytes.
HIV-1 Primary Isolates (R5 and X4) Display Differential Replication
Kinetics in Cord and Adult Blood MDM and T Lymphocytes. SixHIV-1
primary isolates (2099, 2101, 2449, 2758, 2759, and 5441) were
adult blood MDM and T lymphocytes. Equal numbers of lympho-
cytes and CD14?selected monocytes that differentiated into
macrophages from cord and adult blood were infected with an
equal amount of HIV-1 isolates, and virus production was mea-
(2449 and 2759) and dual-tropic X4?R5 (2101) isolates replicated
more efficiently in cord MDM compared with adult MDM,
whereas primary X4 (2758) isolate could not replicate in MDM
probably because of postentry blocks (16). On the contrary, all R5
(2449 and 2759) and X4 (2758 and 5441) isolates replicated in T
adult lymphocytes (Fig. 2A). These data show that the R5 primary
isolates, like HIV-1BaL(Fig. 1B), replicated better in cord blood
primary isolates along with HIV-1NL4–3(Fig. 1C) replicated better
in cord T lymphocytes than adult T lymphocytes (Fig. 2A). These
experiments have been performed in MDM and T lymphocytes
from seven different cord and adult blood donors with similar
No Significant Difference in Expression of CD4 Receptor and CXCR4
and CCR5 Coreceptors Between Cord and Adult Blood MDM and T
Lymphocytes. To establish a correlation between HIV-1 replication
and coreceptor expression, we determined the cell surface expres-
sion of CD4, CXCR4, and CCR5 in cord and adult blood T
ing CD4, CXCR4, and CCR5 become stained, with the intensity of
staining directly proportional to the density of CD4, CXCR4, and
CCR5. The isotype controls did not show any background staining,
whereas anti-CD4, -CXCR4, and -CCR5 showed specific staining.
the expression of CD4, CXCR4, and CCR5 on cord blood lym-
phocytes and MDM compared with adult lymphocytes and MDM
(Fig. 3). Therefore, expression levels of CD4, CXCR4, and CCR5
cannot explain the enhanced replication of HIV-1 in cord MDM
and T lymphocytes compared with adult MDM and lymphocytes
(Figs. 1 and 2).
Increased Replication of HIV-1 in Cord Blood MDM and T Lymphocytes
Compared with Adult Blood Cells Is Not Due to Increased Cell
Proliferation. Although MDM are nondividing resting cells and are
106) and MDM (0.5 ? 106) obtained from cord and adult blood were infected
with 1 ? 105RT counts of HIV-1Ba-Land HIV-1NL4–3.At different time periods,
the two peak days are shown. The results are expressed as counts per million
per milliliter ? SD. of triplicate experiments. These experiments have been
results. The P values for HIV-1Ba-Lin MDM were ?0.001, ?0.0001 for HIV-1Ba-L
in T lymphocytes , and ?0.0003 for HIV-1NL4–3in T lymphocytes (n ? 7).
Replication of HIV-1BaLin neonatal (cord) and adult blood T lympho-
lymphocytes (A) and MDM (B). T lymphocytes (1 ? 106) and MDM (0.5 ? 106)
obtained from adult and cord blood were infected with 1 ? 105RT counts of
several R5, X4, and X4?R5 HIV-1 primary isolates. Virus production was mea-
sured by RT assay in culture supernatant and three peak days (D) are shown.
experiments. These experiments have been performed in MDM and T lym-
phocytes from seven different cord and adult blood donors with similar
Replication of HIV-1 primary isolates in cord and adult blood T
www.pnas.org?cgi?doi?10.1073?pnas.0602185103Sundaravaradan et al.
proliferation of cord MDM compared with adult MDM has been
adult cells on HIV-1 replication, we performed [3H]thymidine
uptake in MDM and T lymphocytes from cord and adult blood in
uninfected cells and 48 h after infection by HIV-1BaLas described
in ref. 17. There was no evidence of increased proliferation in cord
blood T lymphocytes and MDM (Fig. 4A) compared with adult
blood lymphocytes and MDM. In contrast, we found an increase in
cord blood T lymphocytes in control uninfected cells, although
there was very little uptake in MDM because they are nondividing
and nonproliferative cells (10, 11). The slight increase in [3H]thy-
midine in infected T lymphocytes and MDM compared with
uninfected cells could be due to HIV-1 DNA synthesis. This
experiment suggests that the difference in viral replication kinetics
between cord and adult blood cells (Figs. 1 and 2) is not due to a
difference in cell proliferative capabilities (Fig. 4A).
No Significant Difference in Postentry Events of HIV-1 Infection (R5
and X4 isolates) Between Cord and Adult Blood MDM. To determine
whether postentry events influence increased HIV-1 replication
in cord vs. adult MDM, we performed a comparative analysis of
the postentry events. These events include R?U5, the initial step
of reverse transcription (18), R?U3 of first-strand transfer, and
2LTR DNA circles, a marker for nuclear translocation of the
HIV-1 DNA (16). We infected MDM from cord and adult with
two lab-adapted (HIV-1NL4–3and HIV-1BaL) and two primary
by PCR according to a model of retroviral reverse transcription
(16, 19), including R?U5, R?U3, and 2LTR. The results of these
experiments are shown in Fig. 4B. The data demonstrate that the
reverse transcription steps (R?U5) were positive for all R5 and
X4 isolates (Fig. 4Ba) and there was no difference between cord
and adult MDM, suggesting that the same amount HIV-1
isolates entered in both cord and adult MDM. In addition, there
was no significant difference in the synthesis of first-strand
transfer (R?U3) (Fig. 4Bb), LTR?gag (Fig. 4Bc), and 2LTR (Fig.
R5 isolates (HIV-1BaLand primary R5) were able to synthesize
2LTR DNA, the X4 isolates could not make 2LTR DNA (Fig.
4Bd). Fig. 4Be shows the PCR for a housekeeping gene, ?-tu-
bulin, for DNA standardization. These results suggest that
postentry events of HIV-1 infection may not contribute to
increased viral replication in cord MDM compared with adult
Significant Up-Regulation of HIV-1 Gene Expression in Cord Blood
MDM and T Lymphocytes Compared with Adult Blood Cells. To
determine whether HIV-1 gene expression influences increased
HIV-1 replication in cord blood T lymphocytes and MDM com-
pared with adult cells, we used a single-cycle replication competent
pseudovirus, HIV-NL-Luc-E?(R??R?) that measures transcrip-
tional activity of HIV-1 LTR (20). Equal amounts of (1 ? 105) RT
counts of the NL-Luc-E?(R??R?) viruses were used to infect T
lymphocytes and MDM isolated from adult and cord blood, and
shown in Fig. 5, there was a 3-fold increase in luciferase activity in
5A) and a 10-fold increase in cord blood MDM compared with
adult MDM (Fig. 5B). The gene expression data here correlated
with the data of HIV-1 replication kinetics (Fig. 1), suggesting that
the increased gene expression of HIV-1 may contribute to an
accelerated viral replication in cord blood target cells compared
with adult cells. These experiments were done in triplicate and
normalized with the amount of protein in the cells and performed
from seven different cord and adult blood donors. We observed
donor-specific variability in the magnitude of HIV-1 gene expres-
sion in cord vs. adult cells, with statistically significant higher levels
of HIV-1 gene expression in cord vs. adult cells. The P values for
CCR5) expression in cord (dotted lines) vs. adult (dashed lines) blood T lym-
phocytes and MDM. The x axis represents the expression of the receptor?
coreceptor, whereas y axis represents cell count. FACS analysis has been
performed on lymphocytes and MDM from seven different donors with no
significant difference in the levels of CD4, CXCR4, and CCR5.
FACS analysis of HIV-1 receptor (CD4) and coreceptor (CXCR4 and
T lymphocytes and MDM were incubated with [3H]thymidine mock and in-
there was no difference in the proliferative abilities of cord blood MDM
compared with adult blood MDM. T lymphocytes from adult blood prolifer-
ated better than cord T lymphocytes. The P values for MDM are ?0.01 (n ? 4)
and ?0.05 for lymphocytes (n ? 5). (B) Comparative analysis of PCR amplifi-
cation products of HIV-1 postentry events, including reverse transcription,
DNA synthesis, and translocation of PIC into the nucleus in adult and cord
MDM. Ba, R?U5; Bb, R?U3; Bc, gag; Bd, 2LTR; Be, ?-tubulin. Lanes: 1, BaL; 2,
primary R5 isolate; 3, NL 4–3; 4, primary X4 isolate, 5, Mock. There was no
significant difference in postentry events. In 2LTR, the X4 isolates bands are
much weaker than R5 because of the block at 2LTR level. These experiments
were performed in MDM from seven different cord and adult blood donors
with similar results.
Cell proliferation ([3H]thymidine) uptake assay in adult and cord
Sundaravaradan et al.
August 1, 2006 ?
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no. 31 ?
HIV-1 gene expression between cord and adult T lymphocytes are
?0.000001 and ?0.01 for MDM (n ? 7). The lower P values
in cord vs. adult cells. We also performed a dose-dependent
105RT counts) on HIV-1 gene expression and found a linear
and luciferase activity (data not shown).
Up-Regulation of HIV-1 Gene Expression in Cord MDM Compared with
Adult MDM Correlates with Increased Transcription. To determine
whether the up-regulation of HIV-1 gene expression in cord cells
compared with adult cells is due to increased transcription, we
performed ribonuclease protection assay (RPA) on luciferase
mRNA from cord and adult blood MDM infected with NL-Luc-
E?R?and NL-Luc-E?R?viruses. As shown in Fig. 5C, there was
a significant increase (?10-fold) in luciferase mRNA driven by
LTR in cord MDM compared with adult MDM (densitogram not
shown). The RPA results correlated with the gene expression data
(Fig. 5B). These experiments have been performed in MDM and
T lymphocytes (data not shown) from five different donors of cord
and adult blood with P ? 0.001.
We have compared HIV-1 replication kinetics in neonatal
(cord) and adult blood MDM and T lymphocytes and found that
HIV-1 replicated better in both cord blood MDM and T
lymphocytes compared with adult cells, with a more profound
HIV-1 replication in cord and adult MDM and T lymphocytes
was significantly influenced at the level of HIV-1 gene expres-
sion and not at the level of entry and postentry events or cells’
proliferative capabilities. The efficient replication of HIV-1 in
cord MDM further supports our previous finding that R5 viruses
are transmitted from mother to infant and initially maintained
with the same properties (12), which may be critical for the
establishment of HIV-1 infection in infants. Furthermore, the
increased HIV-1 gene expression and replication in cord cells
compared with adult cells may contribute to a higher and
sustained viral load (21, 22) and faster disease progression in
neonates and infants than adults (1, 2).
Our data on increased HIV-1 replication in cord blood MDM
but these studies offered no mechanisms. In addition, our study
used several primary isolates and showed that HIV-1 not only
replicates better in cord MDM than adult MDM, but also in cord
T lymphocytes compared with adult T lymphocytes isolated from
seven different donors. Although the increased HIV-1 replication
in cord vs. adult MDM has been attributed to differences in cell
proliferative capabilities (17), MDM are nondividing cells and are
On the contrary, adult mononuclear cells proliferated better than
CBMCs in response to virus or mitogens (23). These data suggest
that cell proliferative capabilities of cord and adult MDM or T
lymphocytes are not responsible for differential HIV-1 replication
in these cells.
HIV-1 entry into target cells, including T lymphocytes and
MDM, depends on the expression of CD4 and CXCR4 or CCR5.
Our FACS analysis of cord and adult T lymphocytes and MDM
(Fig. 3) demonstrates that there was no significant difference in
CD4, CXCR4, and CCR5 expression in these cell types. Similar
results on CD4, CXCR4, and CCR5 expression on cord and adult
lymphocytes and M?M have been reported before (14). These data
suggest that differential HIV-1 replication in cord vs. adult mono-
cell types also is not due to differences in cellular phenotypes
(naı ¨ve-CD45RAhighand memory-CD45RAlow) and cell activation,
are not significantly different between cord and PBMCs (14).
After HIV-1 enters target cells, postentry events including
reverse transcription and transport of PIC into the nucleus, might
play a crucial role in the establishment of infection (16, 19). We
found that the R?U5 step of reverse transcription was positive for
all HIV-1 isolates tested (Fig. 4Ba) with no significant difference
between cord and adult blood MDM, suggesting that the same
amount of virus entered in both cell types. This result was expected
because lymphocytes and MDM from cord and adult expressed
both coreceptors, CXCR4 and CCR5, at the same levels (Fig. 3).
The two intermediary steps of reverse transcription, R?U3 and
LTR?gag synthesis, showed no significant difference between cord
and adult MDM. Furthermore, we found no difference in 2LTR
cord and adult MDM. However, there was a significant difference
between X4 and R5 viruses with R5 significantly higher for 2LTR
DNA than X4 virus, suggesting postentry blocks for X4 viruses in
MDM (16). These results suggest that postentry events of HIV-1
infection may not significantly contribute to the differential HIV-1
replication in cord vs. adult MDM.
(B) and HIV-1 LTR transcription in MDM (C). T lymphocytes (1 ? 106) and MDM
HIV-NL-Luc-E?R?(R?E?). Cultured cells were harvested 72 h later, and lucif-
The enzyme activity was normalized based on total cellular protein. The
results are expressed as relative light units (RLU) ? SD of triplicate experi-
ments. These experiments have been performed in MDM (P ? 0.01) and T
lymphocytes (P ? 0.000001) from seven different donors of cord and adult
blood. (C) Ribonuclease protection assay of luciferase mRNA transcription.
Cord and adult blood MDM were infected with equal amounts of HIV-NL-Luc-
E?R?(R?E?) and HIV-NL-Luc-E?R?(R?E?) amphotropic viruses, and lysates
were hybridized with luciferase antisense RNA probe and digested with
RNase. Protected bands were analyzed on urea-PAGE. ?-tubulin was used as
internal control. NS, nonspecific RNA. The band in the mock is similar to
nonspecific RNA. The densitometric analysis was done on RPA from five
donors of cord and adult MDM (data not shown) with P values of P ? 0.001.
HIV-1 gene expression in cord and adult T lymphocytes (A) and MDM
www.pnas.org?cgi?doi?10.1073?pnas.0602185103Sundaravaradan et al.
Upon integration, HIV-1 genes are expressed from HIV-1 LTR
(promoter) by using cellular factors (24). We used a single-cycle
replication competent amphotropic pseudovirus, HIV-NL-Luc-
E?, that measures transcriptional activity of HIV-1 LTR (20)
independent of CD4 and CCR5 or CXCR4 levels. HIV-1 gene
adult blood cells. We observed a 3-fold increase in HIV-1 LTR
driven luciferase expression in cord lymphocytes and a 10-fold
increase in cord MDM compared with adult lymphocytes and
MDM, respectively (Fig. 5 A and B). Moreover, the up-regulation
of HIV-1 gene expression in cord MDM compared with adult
MDM correlated with increased transcription as determined by
RPA (Fig. 5C). We conclude that the increased replication of
suggesting that HIV-1 LTR is being regulated differently in these
HIV-1 gene expression is controlled in part by the dynamic
interplay of viral and cellular transcription factors with the HIV-1
LTR sequences (24, 25). HIV-1 LTR-directed transcription is
regulated through several pathways involving various factors that
serve as components of the basal transcriptional machinery and
transcription factors that act through protein–nucleic acid (26),
protein–protein (27), and protein–ligand (28) interactions. Recen-
influencing the level of transcription and disease progression (29).
is unable to contain the virus (3, 4, 30), HIV-1 may interact
differently with the immature immune cells and produce more
express higher levels of transcriptional factors (26), lower level of
repressors, and?or differential levels of cellular factors than adult
cells, which may be responsible for an increased HIV-1 gene
expression in neonatal cells. The data presented in this paper on
increased HIV-1 infection in neonatal target cells may contribute
to higher levels of viremia (21, 22) and faster disease progression in
infants compared with adults (1, 2). These results provide previ-
ously undescribed insights into the mechanisms of differential
Materials and Methods
Cord and Adult Blood Donors. We collected cord blood and adult
blood from seven donors representing various ethnic and racial
backgrounds in Arizona under similar conditions and same anti-
coagulants (heparin sulfate). Cord and adult blood samples were
collected around the same time and processed side by side. In
to 600 million mononuclear cells. Cord blood yields more mono-
nuclear cells than adult blood (31, 32), so to obtain a comparable
number of PBMCs, we collected 150–200 ml of adult blood. This
study was approved by the University of Arizona Human Subjects
Committee, and written informed consent was obtained from all
Isolation and Culture of CBMCs and PBMCs. We isolated adult
mononuclear cells from adult blood and CBMCs from cord blood
by a single-step Ficoll-Hypaque method (12, 33). To separate
lymphocytes from M?M, we used two methods. In some initial
experiments, we plated 4 ? 106adult mononuclear cells and
CBMCs onto multiwell plates in RPMI 1640 medium with 15%
human serum. After 12 to 16 h, M?M adhered to the plates and
lymphocytes were separated and stimulated with PHA (5 ?g?ml)
1640 medium?10% FBS?penicillin-streptomycin?10 units?ml of
IL-2. The M?M after day 1 of isolation contain mostly immature
monocytes (34) and were allowed to differentiate into MDM after
human AB serum?penicillin-streptomycin?100 units?ml mononu-
clear phagocyte colony-stimulating factor). In some experiments,
we separated T lymphocytes on anti-CD3 microbeads (Miltenyi
Biotech, Auburn, CA). T lymphocytes isolated on anti-CD3 mi-
activated by PHA but cultured in RPMI medium 1640?10%
FBS?IL-2 for 2 days before infection. We also isolated M?M on
anti-CD14 microbeads and cultured CD14 selected M?M to dif-
ferentiate into MDM as described above. The purity of T lympho-
cytes was confirmed by anti-CD3, -CD4, and -CD8 staining and
MDM by esterase staining, anti-CD14 and -CD4. The MDM were
these methods gave similar results. In most of the experiments, we
have used CD14?-selected M?M (?99% pure). Freshly collected
cord and adult blood samples yielded a better quality of lympho-
cytes and monocytes that gave reproducible results with respect to
HIV-1 replication and gene expression.
Viruses and Infection. HIV-1 isolates (laboratory-adapted HIV-
1NL4–3 and HIV-1BaL), primary isolates [2758, 5441 (X4), 2101
(X4?R5), 2099, 2449, 2759 (R5)], cell lines (COS-1), and other
HIV-related reagents used have been obtained from the National
Institutes of Health AIDS Research and Reference Reagent Pro-
gram. T Lymphocytes (1 ? 106) were infected on day 2 and MDM
Briefly, viruses were adsorbed on target cells for 2 h in RPMI
medium 1640 without serum at 37°C in CO2incubator (Forma
Scientific, Marietta, OH). After adsorption, cells were washed to
remove unbound virus and resuspended in 500 ?l of appropriate
was measured in culture supernatant by RT assay (12, 15). All
infection experiments were performed in triplicate.
Flow Cytometry. The expression of CD4, CXCR4, and CCR5 on
normal cord and adult lymphocytes and MDM was evaluated by
flow cytometry as described in ref. 34. Cells were labeled with
CyC-anti human CD4, PE-anti human CXCR4, FITC-anti hu-
man CCR5 (R & D Systems, Minneapolis, MI) and mouse-IgG
(isotype control for each color). Briefly the cells (1 ? 106) were
was washed from the cells. After the final washes, cells were fixed
in 1% paraformaldehyde, and cell surface expression was de-
termined by FACS analysis with FACScan (Becton Dickinson,
San Jose, CA). Data analysis was performed by using Cell Quest
Cell Proliferation Assay. Cell proliferative capabilities of T lympho-
cytes and MDM from cord and adult blood were evaluated in
triplicate experiments by methyl-[3H]thymidine incorporation
(PerkinElmer, Boston, MA). We determined [3H]thymidine up-
take in MDM and PHA-stimulated T lymphocytes from cord and
as described in ref. 17. Briefly, T lymphocytes (1 ? 106) or MDM
(0.5 ? 106) were plated in 48-well plates, and cells were infected
with HIV-1BaL. The uninfected and infected cells were incubated
with [3H]thymidine at a concentration of 1 ?Ci per well (1 Ci ? 37
GBq) 48 h after infection. Cultured cells were harvested 12 h after
[3H]thymidine incubation as described in ref. 17, and the inco-
rporated radioactivity was measured in a scintillation counter
(Beckman-Coulter, Fullerton, CA). The results from triplicate
experiments were expressed in counts per million per milliliter).
Nucleus. Sequential steps of HIV-1 reverse transcription (RU5 and
RU3), first-strand DNA synthesis (gag), and translocation of PIC
by a modified PCR method (35). Because the accumulation of
full-length viral DNA in infected cells reaches a peak between 36
Sundaravaradan et al.
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no. 31 ?
and 48 h after infection (18), samples were collected at 48 h after Download full-text
infection. Cells were lysed and DNA extracted by using Qiagen
(Valencia, CA) Blood DNA kit as per manufacturer’s instructions.
PCR of ?-tubulin gene in cell lysates was performed to standardize
DNA recovery. PCR analysis of the postentry events was per-
formed as described in refs. 19 and 35). The primers used in this
primer of each primer pair was end-labeled with [?-32P]ATP. Each
a 30-sec annealing step (50°C), and a 1-min extension step (72°C)
fixed in a methanol-acetic acid bath for 30 min to 1 h and blotted
onto Nytran membrane by using an electroblotter. The Nytran
membrane was exposed to autoradiograph.
DNA Transfections. Transfections were done to generate a single-
cycle replication competent amphotrophic HIV-NL-Luc-E?virus
for gene expression studies (20). Briefly, COS-1 cells were cultured
(1.2 ? 106) seeded the previous day in T 75 culture flasks were
cotransfected with 10 to 15 ?g pHIV-NLLuc-E?R?or pHIV-NL-
Luc E?R?and 10 to 15 ?g of amphotrophic envelope glycoprotein
975 ?F (12) to generate luciferase reporter viruses. The amount of
amphotropic recombinant virus generated was measured by RT
assay in the culture supernatant.
Gene Expression. HIV-1 gene expression was determined by infect-
ing cord and adult blood T lymphocytes and MDM with a HIV-1
levels for entry and by measuring luciferase activity as described in
ref. 20. Briefly, the cultures were harvested 72 h after infection by
lysing the cells in 200 ?l of lysis buffer. The luciferase activity was
determined in 50 ?l of the lysate by using luciferase assay kit
(Promega) and measuring the amount of light generated in a
this recombinant virus reflects the number of copies of integrated
proviruses and their transcriptional activity (20).
Ribonuclease Protection Assay. The ribonuclease protection assay
Austin, TX) as per manufacturer’s instructions. An ?305-
nucleotide riboprobe was generated by transcription of ClaI-
of [32P]UTP as per Riboprobe in vitro Transcription Systems
(Promega) kit. Briefly, amphotropic HIV-1-NL-Luc-E?virus in-
fected and control cells were pelleted by centrifugation and lysed in
1 ml of lysis?denaturation solution per 1 ? 107cells. The probe was
hybridized with the lysate containing target RNA for 15 h at 37°C.
Unhybridized probe was removed by RNase mixture (RNase
A?RNase T1) treatment for 30 min at 37°C. After RNase inacti-
vation and precipitation of the protected species, the pellets were
dissolved in gel-loading buffer, heated for 3 min at 95°C, and
electrophoresed on a 7% polyacrylamide 7 M urea gel in TBE
buffer (90 mM Tris?64.6 mM boric acid?2.5 mM EDTA, pH 8.3).
The bands were visualized by autoradiography of the dried gel and
quantified by densitometric analysis of autoradiographs.
Statistical Analysis. All of the experiments were done in triplicate at
least three times, and a representative experiment is illustrated in
Results. The data were analyzed by using Student’s t test, and a P
value (two-tailed) of ?0.05 was considered significant. Lower P
values indicate less variance in fold differences as shown in each
We thank N. Landau (The Salk Institute, La Jolla, CA) for providing
pHIV-NL-E?R?Luc and pSV-A-MLV-env plasmids, AIDS Reference
and Reagent Program (Germantown, MD) for providing HIV-1 isolates,
was supported by National Institute of Allergy and Infectious Diseases
Grant AI-40378-06 and Arizona Biomedical Research Commission
Grant 7002 (to N.A.).
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www.pnas.org?cgi?doi?10.1073?pnas.0602185103 Sundaravaradan et al.