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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Jul 25;103(31):11701–11706. doi: 10.1073/pnas.0602185103

Differential HIV-1 replication in neonatal and adult blood mononuclear cells is influenced at the level of HIV-1 gene expression

Vasudha Sundaravaradan 1,*, Shailendra K Saxena 1,*,†, Rajesh Ramakrishnan 1,*,‡, Venkat R K Yedavalli 1,§, David T Harris 1, Nafees Ahmad 1,
PMCID: PMC1544233  PMID: 16868088

Abstract

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 macrophages (MDM) from seven different donors. We found that HIV-1 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 replication did not depend on differences in cell proliferative capabilities, 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 expression in cord T lymphocytes compared with adult T lymphocytes and 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 that the increased replication of HIV-1 in cord blood compared with 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.

Keywords: AIDS, differential HIV-1 gene expression, pediatric AIDS, cord blood mononuclear cells, neonatal HIV-infection

HIV disease in neonates and infants has a more rapid and fatal course than seen in infected adults (1, 2). In infants 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 information related to the immunopathogenesis of AIDS has been gained from HIV-1 infection of primary adult M/M and T lymphocytes (11). However, the role of neonatal M/M and T lymphocytes in the immunopathogenesis of pediatric AIDS has not been fully elucidated. Because R5 viruses are involved in vertical transmission (12, 13), its interaction with monocyte-derived macrophages (MDM) and CD4+/CCR5+ T cells may play an important role in the establishment of HIV-1 infection and disease progression. It is likely that increased replication of HIV-1 in neonatal mononuclear cells may contribute to a high level of viremia and faster disease progression. Therefore, we sought to compare HIV-1 replication kinetics between neonatal and adult blood mononuclear cells (PBMCs) and determine the mechanisms of HIV-1 replication in these cell types. We have used cord blood in place of neonatal blood because, like neonatal blood, it has more CD45RA+ T cells and less CD45RO+ T cells, is immature compared with adult blood (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 capabilities, 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 lymphocytes 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.

Results

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 infected cord and adult blood T lymphocytes and MDM from seven different donors with HIV-1. An equal number of lymphocytes and MDM isolated as described in Materials and Methods were infected with an equal amount [5–15 × 104 reverse transcriptase (RT) cpm] of HIV-1BaL and HIV-1NL4–3 (lab adapted R5 and X4 HIV-1, 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 lymphocytes are shown in Fig. 1A and in MDM in Fig. 1B. The data clearly demonstrates that HIV-1BaL replicated 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-L replicated 9-fold better in cord MDM compared with adult MDM (Fig. 1B). We also found that HIV-1NL4–3 replicated 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, although some variability in fold differences in HIV-1 replication was 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-1NL4–3 in 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.

Fig. 1.

Fig. 1.

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Replication of HIV-1BaL in neonatal (cord) and adult blood T lymphocytes (A) and MDM (B), and HIV-1NL4–3 in T lymphocytes (C). T lymphocytes (1 × 106) and MDM (0.5 × 106) obtained from cord and adult blood were infected with 1 × 105 RT counts of HIV-1Ba-L and HIV-1NL4–3. At different time periods, the virus production was measured in the culture supernatant by RT assay, and the two peak days are shown. The results are expressed as counts per million per milliliter ± SD. of triplicate experiments. These experiments have been performed from seven different donors with the similar statistically significant results. The P values for HIV-1Ba-L in MDM were <0.001, <0.0001 for HIV-1Ba-L in T lymphocytes, and <0.0003 for HIV-1NL4–3 in T lymphocytes (n = 7).

HIV-1 Primary Isolates (R5 and X4) Display Differential Replication Kinetics in Cord and Adult Blood MDM and T Lymphocytes.

Six HIV-1 primary isolates (2099, 2101, 2449, 2758, 2759, and 5441) were compared for their infectivity and replication efficiency in cord and adult blood MDM and T lymphocytes. Equal numbers of lymphocytes 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 measured every 3 days by RT assay. As shown in Fig. 2B, the R5 primary (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 lymphocytes but with a better efficiency in cord T lymphocytes than adult lymphocytes (Fig. 2A). These data show that the R5 primary isolates, like HIV-1BaL (Fig. 1B), replicated better in cord blood MDM and T lymphocytes than adult cells (Fig. 2A). In addition, X4 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 results.

Fig. 2.

Fig. 2.

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Replication of HIV-1 primary isolates in cord and adult blood T lymphocytes (A) and MDM (B). T lymphocytes (1 × 106) and MDM (0.5 × 106) obtained from adult and cord blood were infected with 1 × 105 RT counts of several R5, X4, and X4/R5 HIV-1 primary isolates. Virus production was measured by RT assay in culture supernatant and three peak days (D) are shown. The results were expressed as counts per million per milliliter ± SD of triplicate experiments. These experiments have been performed in MDM and T lymphocytes from seven different cord and adult blood donors with similar results.

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 expression of CD4, CXCR4, and CCR5 in cord and adult blood T lymphocytes and MDM from seven different donors. Cells expressing 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 results demonstrated that there was no significant difference in the expression of CD4, CXCR4, and CCR5 on cord blood lymphocytes 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).

Fig. 3.

Fig. 3.

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FACS analysis of HIV-1 receptor (CD4) and coreceptor (CXCR4 and CCR5) expression in cord (dotted lines) vs. adult (dashed lines) blood T lymphocytes 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.

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 not expected to proliferate or replicate efficiently (10, 11), increased proliferation of cord MDM compared with adult MDM has been shown to be associated with increased replication of HIV-1 (17). To determine the effect of cellular proliferative capabilities of cord and 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-1BaL as 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 [3H]thymidine uptake by adult blood T lymphocytes compared with 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]thymidine 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).

Fig. 4.

Fig. 4.

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Cell proliferation ([3H]thymidine) uptake assay in adult and cord lymphocyte and MDM (A) and postentry events of HIV-1 infection in MDM (B). T lymphocytes and MDM were incubated with [3H]thymidine mock and infected with HIV-1BaL as described in Materials and Methods. As can be seen (A), there was no difference in the proliferative abilities of cord blood MDM compared with adult blood MDM. T lymphocytes from adult blood proliferated 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 amplification 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, α-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.

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-1NL4–3 and HIV-1BaL) and two primary isolates (R5 and X4). were lysed 48 h after infection and analyzed 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. 4Bd) PCR products between cord and adult MDM. Although the R5 isolates (HIV-1BaL and 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, α-tubulin, 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 MDM.

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 compared with adult cells, we used a single-cycle replication competent pseudovirus, HIV-NL-Luc-E− (R+/R−) that measures transcriptional 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 HIV-1 gene expression was measured by luciferase activity (20). As shown in Fig. 5, there was a 3-fold increase in luciferase activity in cord blood T lymphocytes compared with adult T lymphocytes (Fig. 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 expression in cord vs. adult cells, with statistically significant higher levels of HIV-1 gene expression in cord vs. adult cells. The P values for HIV-1 gene expression between cord and adult T lymphocytes are <0.000001 and <0.01 for MDM (n = 7). The lower P values indicate less variability in fold differences in HIV-1 gene expression in cord vs. adult cells. We also performed a dose-dependent response of the amphotropic HIV-NL-Luc-R+E− virus (0.25–1.5 × 105 RT counts) on HIV-1 gene expression and found a linear relationship between increasing doses of HIV-NL-Luc-R+E− virus and luciferase activity (data not shown).

Fig. 5.

Fig. 5.

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HIV-1 gene expression in cord and adult T lymphocytes (A) and MDM (B) and HIV-1 LTR transcription in MDM (C). T lymphocytes (1 × 106) and MDM (0.5 × 106) were infected with 1 × 105 RT counts of HIV-NL-LucE−R+ (R+E−) and HIV-NL-Luc-E−R− (R−E−). Cultured cells were harvested 72 h later, and luciferase activity was assayed in cell lysate as described in Materials and Methods. The enzyme activity was normalized based on total cellular protein. The results are expressed as relative light units (RLU) ± SD of triplicate experiments. 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. α-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.

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.

Discussion

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 effect seen in MDM. More importantly, we show that differential HIV-1 replication in cord and adult MDM and T lymphocytes was significantly influenced at the level of HIV-1 gene expression 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 compared with adult MDM is consistent with earlier studies (9, 17), 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 not expected to proliferate (10), as also found in our study (Fig. 4A). 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 mononuclear cells does not depend on the cell surface expression of CD4 and CXCR4 and CCR5. The differential HIV-1 replication in these cell types also is not due to differences in cellular phenotypes (naïve-CD45RAhigh and memory-CD45RAlow) and cell activation, because T cell activation markers’ (CD25 and HLA-DR) expression 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 DNA that represents translocation of PIC into the nucleus between 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.

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 expression was up-regulated significantly in CBMCs compared with 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 HIV-1 seen in CBMCs is influenced at the level of gene expression, suggesting that HIV-1 LTR is being regulated differently in these cell types.

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. Recently, it was shown that HIV-1 LTR variability among different HIV-1 subtypes could affect LTR binding of either cellular or viral factors, influencing the level of transcription and disease progression (29).

Although the neonatal immune system is not fully developed and is unable to contain the virus (3, 4, 30), HIV-1 may interact differently with the immature immune cells and produce more HIV-1 than mature immune cells. It is likely that neonatal cells may 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 previously undescribed insights into the mechanisms of differential HIV-1 replication and disease progression in infants compared with adults.

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 anticoagulants (heparin sulfate). Cord and adult blood samples were collected around the same time and processed side by side. In general cord blood, samples are between 60 and 120 ml, yielding 300 to 600 million mononuclear cells. Cord blood yields more mononuclear 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 donors.

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 × 106 adult 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 μg/ml) for 24 to 48 h. The lymphocytes were washed and cultured in RPMI 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 culture for 7 days in macrophage media (RPMI medium 1640/15% human AB serum/penicillin-streptomycin/100 units/ml mononuclear 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 microbeads are stimulated by CD3 antibodies and, therefore, were not 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 differentiate into MDM as described above. The purity of T lymphocytes was confirmed by anti-CD3, -CD4, and -CD8 staining and MDM by esterase staining, anti-CD14 and -CD4. The MDM were found to be >97% pure. T lymphocytes and M/M purified from all 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 lymphocytes 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 Program. T Lymphocytes (1 × 106) were infected on day 2 and MDM (0.5 × 106) on day 7 after isolation with equal amounts of all viruses. Briefly, viruses were adsorbed on target cells for 2 h in RPMI medium 1640 without serum at 37°C in CO2 incubator (Forma Scientific, Marietta, OH). After adsorption, cells were washed to remove unbound virus and resuspended in 500 μl of appropriate culture media. The cells were fed every 3 days, and virus production 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 human CCR5 (R & D Systems, Minneapolis, MI) and mouse-IgG (isotype control for each color). Briefly the cells (1 × 106) were incubated with the conjugated antibodies, and unbound antibody was washed from the cells. After the final washes, cells were fixed in 1% paraformaldehyde, and cell surface expression was determined by FACS analysis with FACScan (Becton Dickinson, San Jose, CA). Data analysis was performed by using Cell Quest (Becton Dickinson).

Cell Proliferation Assay.

Cell proliferative capabilities of T lymphocytes and MDM from cord and adult blood were evaluated in triplicate experiments by methyl-[3H]thymidine incorporation (PerkinElmer, Boston, MA). We determined [3H]thymidine uptake in MDM and PHA-stimulated T lymphocytes from cord and adult blood in uninfected cells and 48 h after infection by HIV-1BaL 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 μCi 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 incorporated radioactivity was measured in a scintillation counter (Beckman-Coulter, Fullerton, CA). The results from triplicate experiments were expressed in counts per million per milliliter).

PCR Analysis of Reverse Transcription and Translocation of PIC into the Nucleus.

Sequential steps of HIV-1 reverse transcription (RU5 and RU3), first-strand DNA synthesis (gag), and translocation of PIC into the nucleus (2LTR) in T lymphocytes and MDM were analyzed by a modified PCR method (35). Because the accumulation of full-length viral DNA in infected cells reaches a peak between 36 and 48 h after infection (18), samples were collected at 48 h after infection. Cells were lysed and DNA extracted by using Qiagen (Valencia, CA) Blood DNA kit as per manufacturer’s instructions. PCR of α-tubulin gene in cell lysates was performed to standardize DNA recovery. PCR analysis of the postentry events was performed as described in refs. 19 and 35). The primers used in this analysis designed were based on HIV-1NL4–3 sequence (19, 35). One primer of each primer pair was end-labeled with [γ-32P]ATP. Each cycle of the PCR comprised of a denaturation step of 30 sec (94°C), a 30-sec annealing step (50°C), and a 1-min extension step (72°C) for 40 cycles. After agarose gel electrophoresis, amplified DNA was 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 in DMEM supplemented with 10% FBS at 37°C and 5% CO2. Cells (1.2 × 106) seeded the previous day in T 75 culture flasks were cotransfected with 10 to 15 μg pHIV-NLLuc-E−R+ or pHIV-NL-Luc E−R− and 10 to 15 μg of amphotrophic envelope glycoprotein expression vector pSV-A-MLV-env by electroporation at 276 V and 975 μF (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 infecting cord and adult blood T lymphocytes and MDM with a HIV-1 NL-Luc-E− virus, which is independent of CD4, CCR5, or CXCR4 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 μl of lysis buffer. The luciferase activity was determined in 50 μl of the lysate by using luciferase assay kit (Promega) and measuring the amount of light generated in a luminometer. The amount of luciferase activity in cells infected with this recombinant virus reflects the number of copies of integrated proviruses and their transcriptional activity (20).

Ribonuclease Protection Assay.

The ribonuclease protection assay was performed by using the Direct Protect lysate RPA kit (Ambion, Austin, TX) as per manufacturer’s instructions. An ≈305-nucleotide riboprobe was generated by transcription of ClaI-linearized pGEM-Luc plasmid with SP6 polymerase in the presence of [32P]UTP as per Riboprobe in vitro Transcription Systems (Promega) kit. Briefly, amphotropic HIV-1-NL-Luc-E− virus infected and control cells were pelleted by centrifugation and lysed in 1 ml of lysis/denaturation solution per 1 × 107 cells. 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 inactivation 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 figure.

Acknowledgments

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, and R. Mehta and B. Wellensiek for reviewing this manuscript. This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-40378-06 and Arizona Biomedical Research Commission Grant 7002 (to N.A.).

Abbreviations

CBMC

cord blood mononuclear cell

MDM

monocyte-derived macrophages

M/M

monocytes/macrophages

PBMC

adult blood mononuclear cell

PIC

preintegration complex

RPA

ribonuclease protection assay.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

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