To study the mechanism of HIV-1 latency in the resting G0
lymphocytes that comprise a stable reservoir for HIV-1 in vivo, we purified resting CD4+
T lymphocytes from peripheral blood samples of infected individuals who exhibited long-term suppression of viral replication on HAART (Fig. ). A two-stage purification procedure gave preparations of resting CD4+
T lymphocytes that contained <0.1% contamination with activated cells. In previous studies, we and others have shown that replication-competent virus persists in this population of resting CD4+
T lymphocytes despite prolonged treatment with HAART (8
). After the initiation of HAART, the frequency of resting CD4+
T lymphocytes harboring latent HIV-1 decreases in a biphasic fashion (4
). The rapid initial decay within the first 3 months of treatment reflects the loss of virus in the labile preintegration state of latency (39
). The subsequent slower phase represents the persistence of lymphocytes with stably integrated provirus (9
). Therefore, to analyze HIV-1 transcription in resting CD4+
T lymphocytes in postintegration latency, we studied patients on HAART who exhibited suppression of plasma HIV-1 RNA levels to below the limit of detection (<50 copies of viral RNA/ml) for prolonged periods of time (23 to 54 months [Table ]).
FIG. 1. Quantitative measurements of HIV-1 proviral DNA present in highly purified resting CD4+ T lymphocytes. (A) Isolation of resting CD4+ T lymphocytes from patients on HAART. The results of flow cytometric analysis of CD4 and HLA-DR expression (more ...)
Characteristics of patients and HIV-1 DNA levels in purified resting CD4+ T lymphocytes
To determine the frequency of infected cells, we first measured proviral HIV-1 DNA in resting CD4+
T lymphocytes (Table and Fig. ). In the patients on long-term HAART, the median number of copies of HIV-1 DNA per 106
T lymphocytes was 100. For one subject (patient 8), the higher levels of HIV-1 DNA measured may have been the result of a blip in plasma HIV-1 RNA that may have allowed some new infection of resting CD4+
T cells. Using a previously described inverse PCR assay (9
), integration of HIV-1 DNA into the host cell genome was demonstrated in these samples (J. D. Siliciano et al., unpublished data). Replication-competent virus was rescued from resting CD4+
T lymphocytes at the same frequencies reported previously (17
) for patients on suppressive HAART regimens (0.1 to 1 infectious unit per 106
Having established the proviral burden in resting CD4+
T lymphocytes, we analyzed HIV-1 transcription. HIV-1 mRNAs are transcribed by RNA polymerase II. Alternative utilization of any one of five alternate splice donors and more than 10 alternate splice acceptors gives rise to 1.7- to 2.0-kb MS mRNAs, 4.3- to 5.5-kb singly spliced mRNAs, and 9.2-kb US mRNAs (Fig. ) (42
). We measured MS and US mRNA species (Fig. ). Detection of only MS mRNAs would suggest posttranscriptional mechanisms for latency (40
), while the absence of HIV-1 mRNAs would suggest that latency operates at the level of transcription.
FIG. 2. Detection of MS and US HIV-1 mRNAs associated with resting CD4+ T lymphocytes. (A) Schematic structures of MS and US HIV-1 RNAs. The HIV-1 genome and viral proteins encoded by it are shown at the top. The proteins that are translated from MS mRNA (more ...)
For detection of HIV-1 mRNAs, we used sensitive, semiquantitative, single-round amplifications to approximate the number of RNA molecules in each sample. To confirm the presence or absence of very low levels of HIV-1 mRNA, nested versions of these assays that can detect a single molecule were used in parallel. To validate these assays, in vitro-transcribed RNA standards were generated from plasmids carrying a spliced sequence representing exons 1, 5, and 7 for MS RNA and a sequence spanning the major splice donor site for US RNA (Fig. ). RNA synthesis was performed in the presence of [3H]UTP, allowing precise radiochemical quantification of synthetic RNA standards. Both MS and US synthetic mRNAs were simultaneously serially diluted in known copy numbers in lysates of 106 resting CD4+ T lymphocytes from healthy donors. After serial dilutions, these mRNA standards were carried through the entire procedure, including mRNA isolation, DNase treatment, cDNA synthesis, and single-round or nested PCR performed in parallel with patients' samples. Representative experiments are shown in Fig. . The single-round assays were sensitive to 2.5 copies of RNA standard per 5 × 104 cell equivalents of mRNA, resulting in a dynamic range of 50 to 5,000 copies of RNA per 106 resting CD4+ T lymphocytes (Fig. , top panels). The nested RT-PCR assays readily detected as few as 2.5 molecules of MS and US HIV-1 RNA standards in a background of mRNA isolated from 5 × 104 resting CD4+ T lymphocytes (Fig. ). Consistent with radiochemical quantification, the nested assay signal was lost as standards were diluted from 2.5 to 0.25 copy per tube.
Using these sensitive and carefully validated assays, we consistently failed to detect MS mRNA species in resting CD4+
T lymphocytes from patients on long-term HAART (Fig. and Table ). The results of these assays indicated that the number of MS mRNA molecules associated with 106
purified resting CD4+
T lymphocytes was less than 50. In contrast, we detected MS RNA in activated CD4+
T lymphocytes infected in vitro with R5 HIV-1. We also detected MS RNA in unfractionated PBMCs isolated from viremic patients because of the presence of productively infected, activated CD4+
T lymphocytes (not shown). These results suggest that the PCR primers and conditions used can readily detect HIV-1 mRNAs in infected cells and that sequence variation in the relatively conserved regions chosen as primer binding sites in HIV-1 isolates does not prevent amplification. In addition, MS RNAs were readily detected in as few as five uninduced cells from the chronically infected ACH-2 cell line serially diluted in 106
T cells from healthy individuals. The final reaction mixture contained RNA from as few as one ACH-2 cell in a background of RNA from 5 × 104
uninfected resting cells. The ACH-2 cell line has been used as a model for latent virus (13
). Although HIV-1 gene expression can be up-regulated in ACH-2 cells by activating stimuli, the basal level of transcription is much higher than the level we observed in latently infected resting lymphocytes in vivo. Thus, unlike some models of latency based on continuously proliferating cell lines, the analysis of highly purified resting G0
T lymphocytes from patients on HAART suggests that MS HIV-1 RNA species are not produced at high levels in latently infected cells.
Levels of MS and US HIV-1 mRNAs associated with highly purified resting CD4+ T lymphocytes and ratios of HIV-1 RNA copies per HIV-1 DNA-positive resting CD4+ T cella
The level of US HIV-1 RNA was also low. In four of nine patients, no US mRNAs were detected using either semiquantitative or nested RT-PCR assays (Table ). In the remaining patients, US RNAs were detected right at the limit of detection of the assays used (50 copies/106 cells). For example, US RNA was detected in one of two semiquantitative PCRs at a level below 2.5 copies/tube for patient 4 and in one of two nested reactions for patient 9 (Fig. ). Therefore, the levels of both MS and US RNA species are low in purified resting CD4+ T lymphocytes from patients on long-term HAART.
Next, we determined whether this apparent lack of transcription of HIV-1 DNA in resting CD4+
T lymphocytes could be reversed by cellular activation. Since the antigen specificity of latently infected resting CD4+
T lymphocytes is heterogeneous (12
), we used the lectin phytohemagglutinin (PHA) to mimic reactivation of HIV-1 latency in vivo. Purified resting CD4+
T lymphocytes from an intensively studied subset of patients (patients 4, 5, 6, 7 and 9) were activated with PHA in the presence of irradiated allogeneic PBMCs in culture medium supplemented with interleukin-2, a procedure previously shown to induce uniform proliferation of resting CD4+
T lymphocytes (18
). To demonstrate uniform cellular activation, we examined carboxy fluorescein diacetate succinimidyl ester (CFSE) dilution upon activation of CFSE-labeled resting CD4+
T lymphocytes. In all experiments, more than 95% of CFSE-labeled lymphocytes underwent at least one cell division by day 3 postactivation (Fig. ). To demonstrate further the uniform activation of resting CD4+
T lymphocytes in this system, we studied the surface expression of early, intermediate, and late markers of T-cell activation: CD69, CD25, and HLA-DR, respectively. As expected, these cellular markers were up-regulated with different kinetics on large proportions of the lymphocytes, with the entire population eventually giving evidence of being activated (Fig. ).
FIG. 3. Reversal of transcriptional silencing by T-cell activation. (A) Activation of purified resting CD4+ T lymphocytes. Prior to activation, resting CD4+ T lymphocytes were labeled with CFSE. Proliferation was measured by a twofold dilution (more ...)
To analyze HIV-1 transcription after activation, we measured MS mRNA species. We chose MS mRNAs because these RNAs encode the Tat and Rev proteins that are indispensable for HIV-1 replication (11
). Moreover, MS RNAs were not detected in latently infected resting CD4+
T lymphocytes in patients on HAART, and MS RNA production thus would represent a molecular exit from HIV-1 latency. Finally, unlike US RNA, MS RNA signals cannot be due to bound extracellular virions.
RT-PCR assays were developed to detect rare mRNA species in the larger pool of cellular RNA present in activated CD4+ T lymphocytes. MS HIV-1 RNA standards were serially diluted in lysates obtained at 0, 3, and 6 days after activation of 100,000 resting CD4+ T lymphocytes. As seen in Fig. , MS HIV-1 RNA standards were readily detected at the single-molecule level in the final nested PCR.
T lymphocytes from patients on HAART were plated at cell concentrations giving approximately 10 HIV-1 DNA-positive lymphocytes per well, based on the measurements described above (Table ). Resting CD4+
T lymphocytes were then subjected to activation under the same conditions, and individual wells were analyzed for HIV-1 transcription on days 2 to 5 postactivation. MS mRNA was detected, but only in a fraction of the wells (Table ). Representative experiments are shown in Fig. . In some HIV-1 DNA-positive wells from later time points, a wide range of spliced HIV-1 mRNAs were seen, consistent with the large number of known splice variants (Fig. ). In a typical experiment, only 1% of HIV-1 DNA-positive lymphocytes transcribed HIV-1 RNA after in vitro activation, even though all lymphocytes in the culture became activated. This finding, when considered together with the measured frequency of resting CD4+
T lymphocytes harboring HIV-1 DNA (Fig. ), suggests that in patients on long-term HAART, only about 1 in 106
T lymphocytes contains a provirus capable of high-level HIV-1 gene expression after cellular activation (Table ). As expected, this frequency is consistent with and somewhat higher than the frequency of resting CD4+
T lymphocytes from which replication-competent virus can be cultured (typically 0.1 to 1 per 106
T lymphocytes) (17
Frequency of HIV-1 DNA-positive resting CD4+ T lymphocytes that can be induced to transcribe HIV-1 mRNA after activation