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J Virol. Nov 2005; 79(22): 14069–14078.
PMCID: PMC1280183

Induction of Cell Cycle Arrest by Human T-Cell Lymphotropic Virus Type 1 Tax in Hematopoietic Progenitor (CD34+) Cells: Modulation of p21cip1/waf1 and p27kip1 Expression

Abstract

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, an aggressive CD4+ malignancy. Although HTLV-2 is highly homologous to HTLV-1, infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34+ cells with HTLV-1 Tax (Tax1) induced G0/G1 cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21cip1/waf1 (p21) and p27kip1 (p27), and marked suppression of hematopoiesis in immature (CD34+/CD38) hematopoietic progenitor cells in comparison to CD34+/CD38+ cells. HTLV-1 infection of CD34+ cells also induced p21 and p27 expression. Tax1 also protected CD34+ cells from serum withdrawal-mediated apoptosis. In contrast, HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression, failed to induce cell cycle arrest, failed to suppress hematopoiesis in CD34+ cells, and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax221) displayed a phenotype in CD34+ cells similar to that of Tax1, suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34+ hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a fatal CD4+ leukemia (54). Although HTLV-2 was originally isolated from a patient with atypical hairy cell leukemia and shares approximately 70% sequence homology with HTLV-1, infection with HTLV-2 has not been linked to the development of lymphoproliferative disorders (30). The HTLV-1 Tax oncoprotein (Tax1) is a 40-kDa protein that functions as a trans-activator of viral gene expression and is considered to be a key component of the leukemogenic process that results from HTLV-1 infection. Tax1 is capable of trans-activating transcription from numerous cellular promoters by activation of NF-κB and cyclic AMP response element binding protein/activating transcription factor (CREB/ATF). Tax1 also represses the transcription of certain genes, including DNA polymerase β, p53, INK4, lck, c-myc, bax, cyclin A, cyclin D3, and MyoD (6, 33, 36, 41, 55, 60, 64, 69).

Tax1 shares approximately 72 to 74% homology with HTLV-2 Tax (Tax2) at the amino acid level (8, 43). Both Tax1 and Tax2 are capable of immortalizing peripheral blood lymphocytes for growth in culture (56, 57, 65), and in vitro analyses of the transcriptional transactivational capabilities of Tax1 and Tax2 demonstrated remarkably similar transactivation profiles of NF-κB-mediated and CREB-mediated transcription patterns (43).

Distinct phenotypic differences between Tax1 and Tax2 have, however, been documented in cells cultured in vitro. Tax1 and Tax2 display a differential ability to induce cellular gene transcription (17, 38, 51, 65). Tax1 has been shown to induce micronuclei in COS cells, in contrast to Tax2, suggesting that Tax1 displays elevated cytopathicity (60). Tax1 was also demonstrated to inhibit p53 transcriptional activity more potently than Tax2, and Tax2 is less efficient at transforming rat embryo fibroblasts, in comparison to Tax1 (18, 49). Our laboratory recently reported that Tax1 suppressed multilineage hematopoiesis from CD34+ cells in vitro, an activity not displayed by Tax2 (67).

Hematopoiesis is a tightly regulated process involving self-renewal, expansion of lineage-committed progenitors and maturation into terminally differentiated cells. Hematopoietic stem cells generally reside in quiescence, which is critical to prevent exhaustion under conditions of stress. Slow cell cycling and quiescence are necessary for self-renewal of primitive stem cells, while rapid cycling is required for expansion of progenitors and maturation. Hematopoietic cells are subject to strict regulation of cell division and differentiation during normal development (23). Of the two known families of cyclin kinase inhibitors, the Cip/Kip family, consisting of p21cip1/waf1 (p21), p27kip1 (p27), and p57kip2 (p57), plays a critical role in regulation of cell cycle kinetics in the hematopoietic cascade. p21 and p27, in particular, are key molecular mediators of quiescence in hematopoietic stem cells and govern the restriction into cell cycle entry from G0 and progression through G1 (1, 9, 10, 19, 63, 70).

We have previously demonstrated HTLV-1 can infect CD34+ cells and that proviral sequences are maintained during differentiation in vitro and in vivo (21). Lentivirus-mediated transduction of Tax1 markedly suppressed multilineage hematopoiesis in CD34+ cells (67). Here we show that Tax1 upregulates p21 and p27 gene expression in CD34+ cells and induces G0/G1 cell cycle arrest, a function not exhibited by Tax2. Tax1 also protects CD34+ hematopoietic progenitor cells (HPCs) from apoptosis following serum deprivation, in stark contrast to Tax2. Notably, the C terminus of Tax1 encodes domains which account for the unique phenotypic characteristics displayed by Tax1 in HPCs. We postulate that induction of p21 and p27 and induction of cell cycle arrest may facilitate the establishment of HTLV-1 latency in hematopoietic progenitor cells. Tax1-mediated cell cycle arrest and protection from programmed cell death are distinguishing features which differentiates HTLV-1 infection from HTLV-2 infection in humans, and HTLV-1 infection and the establishment of latency in HPCs may be a predisposing event to leukemogenesis.

MATERIALS AND METHODS

Cell lines.

293T cells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (Gemini, Calabasas, CA), 2 mM l-glutamine (Gibco BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gemini) at 37°C in a humidified incubator with 5% CO2. SLB-1 and 729/pH6Neo cells were cultures in Iscove's modified Dulbecco's medium (IMDM) (Gibco-BRL) with 10% fetal bovine serum (Gemini), 2 mM l-glutamine (Gibco-BRL), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gemini).

Generation of vesicular stomatitis virus protein G-pseudotyped lentivirus vectors.

Vesicular stomatitis virus protein G-pseudotyped lentiviral vector virus stocks were generated as previously described (61, 67). Briefly, a three plasmid transfection system was employed to generate vesicular stomatitis virus protein G-pseudotyped virions. Transfer plasmids were cotransfected with the packaging vector, pCMV-R8.2ΔVPR (5), which lacks the human immunodeficiency virus 1 packaging (ψ) site, and a vector encoding the vesicular stomatitis virus protein G envelope protein (pHCMV-G) (7) into 293T cells (2 × 107) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA). Supernatants were harvested at 3 and 5 days posttransfection, filtered, and subjected to ultracentrifugation (50,000 × g for 2 h).

Infection of CD34+ cells.

Human CD34+ cells were prepared from fetal liver by magnetic bead purification (Miltenyi Biotec, Calabasas, CA), as previously described (67). Purified CD34+ cells (3 × 106) were infected with lentivirus vectors (multiplicity of infection, 1) in a final volume of 3.0 ml of Dulbecco's modified Eagle's medium containing 70 μg/ml of Verapimil (13). Cells were resuspended in 3.0 ml of IMDM supplemented with 30 μl of StemSpan cytokine cocktail CC100 (StemCell Technologies, Vancouver, British Columbia, Canada) containing Flt-3 ligand (100 ng/ml), stem cell factor (100 ng/ml), interleukin-3 (IL-3) (20 ng/ml), and IL-6 (20 ng/ml). CD34+ cells were infected with HTLV-1 or HTLV-2 by cocultivation with SLB-1 or 729/pH6Neo cells, as previously described (21). Briefly, purified CD34+ cells (106) were cocultivated either with lethally irradiated (10,000 rad) HTLV-1 transformed SLB-1 cells (5 × 106) or HTLV-2 transformed 729/pH6Neo cells (5 × 106), in 3.0 ml of IMDM supplemented with 30 μl of StemSpan cytokine cocktail CC100. After 3 days, the cell cultures were incubated with either anti-HTLV-1 gp46env mouse monoclonal antibody or anti-HTLV-2 gp46env mouse monoclonal antibody (Zeptometrix, Buffalo, NY), then incubated with a secondary fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G monoclonal antibody (Daco A/S, Glostrup, Denmark). After washing with phosphate-buffered saline, cells were incubated with a phycoerythrin-conjugated anti-CD34 monoclonal antibody and an allophycocyanin-conjugated anti-CD38 monoclonal antibody. gp46env-positive cells were purified by fluorescence-activated cell sorting (FACS) and then further sorted into CD34+/CD38 and CD34+/CD38+ populations.

Cell cycle analysis.

CD34+ cells were stained with Hoechst-33342 (Hoechst 33342) (Molecular Probes, Eugene, OR) and pyronin-Y (Polysciences, Warrington, PA), as previously described (22, 26). Briefly, a cell aliquot (5 × 105) was washed in ice0cold PBS and fixed in ice-cold 70% ethanol overnight. Cells were stained with 1 mg/ml Hoechst 33342 in PBS for 45 min at 37°C, followed by staining with 3.3 mM pyronin-Y in the same buffer for 45 min at 4°C. As a control for G0/G1 cell cycle arrest, CD34+ cells (5 × 105) were cultured in the absence of cytokines in 3 ml of IMDM with 2 mM L-glutamine, and 100 U/ml of penicillin/streptomycin or in the presence of 20 μM roscovitine (Calbiochem, San Diego, CA) (23, 37). RNA staining with pyronin-Y yields a continuous histogram without demarcation between positive and negative cells.

Arbitrary gates were designated for each flow cytometric analysis and gating of cells varied slightly between experiments due to variations in staining intensities displayed by cells following exposure to Hoechst 33342 and pyronin-Y. Cell cycle data were acquired on a LSR II flow cytometer (Becton Dickinson), and analyzed with WinMDI 2.8 software. Statistical significance was analyzed by using the Student t test and single-tail analysis of variance (ANOVA), P < 0.05, using Tax1-transduced cell cultures as a negative control.

Transient transfection assays.

293T cells were cotransfected with survivin promoter-luciferase constructs and Tax1, Tax1(−), Tax2, Tax221, or pUC19 (mock), using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer's protocol. Briefly, cells (105) were transfected with 2 μg of lentivirus constructs and 2 μg of either pLuc-2870, pLuc-cyc1B, pLuc-178, pLuc-268, pLuc-230, pLuc-cyc1.2 (21), or pLuc-cyc1.2 (9, 45). Surviving promoter constructs and the proximal number of nucleotides of upstream sequences and mutation features are described as follows: pLuc-2870, 2.8 kb; pLuc-268, 268 bp; pLuc-178, 178 bp; pLuc-cyc1B, 178 bp of proximal promoter sequences and deletion of two cell cycle-dependent elements (CDE) at −1 to −20; pLuc-230, 230 bp promoter and has deletions of the cell cycle gene homology region and two cell cycle-dependent elements located at −1 to −38; pLuc-cyc1.2 (9), 178 bp promoter with a point mutation in the cell cycle-dependent elements domain; pLuc-cyc1.2 (21), 178-bp promoter and a point mutation in the Sp1 site at ~150 bp. Luciferase assays were normalized to protein concentration levels and performed in triplicate (Luminoskan RS, Labsystems, Needham Heights, MA). Statistical analysis was performed with Tax1(−) as a negative control using the Student t test, P < 0.05.

Clonogenic colony-forming assays.

Clonogenic colony-forming assays were performed by cultivating FACS-purified CD34+/GFP-positive cells in 2 ml of Methocult medium H4433 (StemCell Technologies) at 37°C in a humidified atmosphere with 5% CO2, as previously described (67). Each assay was done in duplicate and all experiments were performed two times. Statistics were performed by single-factor analysis of variance analysis (ANOVA) and Student t test (p< 0.05).

Real-time quantitative RT-PCR of infected CD34+ cells.

Total RNA was extracted from cells using Trizol according to manufacturer's protocol (GIBCO BRL). Quantification of p21, p27, and survivin, and GADPH expression was performed using real-time reverse transcription PCR (real time RT-PCR) using the SYBR-GREEN quantitative real-time PCR kit (QIAGEN). Primers used to detect p21 (63), p27 (66), survivin (31), and GADPH (22) mRNA have previously been described. RT-PCR was performed in a final volume of 20 μl consisting of 10 μl of SYBR-GREEN mix, 2 μl of 10x buffer, 2 μl each of forward and reverse primers at 4 ng/μl, 2 μl of water, and 2 μl of template DNA, according to the manufacturer's instructions. All real-time PCRs were performed in 20 μl in 96 well plates on an iCycler system (Bio-Rad, Hercules, CA). Thermal cycling conditions consisted of 30 min at 50°C, 15 seconds at 95°C, followed by 40 cycles at 94°C for 30 s, 60°C for 1 min, and 74°C for 1 min. The fluorescence signal increase of SYBR-GREEN was automatically detected during the 74°C phase of the PCR.

For each sample, the iCycler system provided an amplification curve constructed by relating the fluorescence signal intensity (ΔRn) to the cycle number. Cycle threshold (Ct) was defined as the cycle number at which the fluorescence signal was stronger than mean background noise collected from the 5th to the 10th cycle and calculated by the Bio-Rad software. The relative differences of p21, p27, and survivin expression were determined using the Ct method as outlined in the Applied Biosystems protocol for RT-PCR. Relative quantitation was calculated using the comparative threshold cycle (Ct) method (described in the User Bulletin 2, ABI PRISM 7700 Sequence Detection System). Ct indicates the fractional cycle number when the amount of amplified target genes reaches a fixed threshold within the linear phase of gene amplification, and is inversely related to the abundance of mRNA transcripts in the initial sample. Mean Ct of duplicate measurements was used to calculate ΔCt as the difference in Ct for target and reference (GADPH) gene. ΔCt for each sample was compared to the corresponding Ct of the control experiment and expressed as ΔΔCt. Relative quantitation was expressed as fold-induction or repression of the gene of interest in comparison to the control condition according to the formula 2−ΔΔCt.

Apoptosis analysis.

Apoptosis analyses were performed as previously described (61). Briefly, CD34+ cells were infected with lentivirus vectors for 4 h. Cells were resuspended in IMDM in the absence of cytokines and serum, and analyzed by flow cytometry after 24 h.

RESULTS

Tax1 induces G0/G1 cell cycle arrest in CD34+ HPCs.

We previously described the design of bicistronic lentivirus vectors which encode HTLV tax genes separated by an internal ribosome entry site sequence (IRES) from the encephalomyocarditis virus (Fig. (Fig.1)1) (67). A Tax2/Tax1 chimera, Tax221, encodes the first 300 amino acids of Tax2B fused in frame to the last C-terminal 53 amino acids of Tax1, encompassing the PDZ binding domain (18).

FIG. 1.
Schematic representation of lentiviral vectors. Bicistronic lentivirus vectors encode an internal ribosomal entry site (IRES) sequence from the encephalomyocarditis virus. The target gene and reporter green fluorescent protein (GFP) gene are driven by ...

To establish whether induction of cell cycle arrest in HPCs accounts for suppression of hematopoiesis as determined by colony-forming assays in Tax1-transduced CD34+ cells, CD34+ HPCs were infected with lentivirus vectors (multiplicity of infection, 1) and GFP-positive cells were analyzed for cell cycle progression by flow cytometry after staining with Hoescht 33342 (Hoechst 33342) and pyronin. Hoechst 33342 and pyronin staining distinguishes cells in distinct phases of cell cycle based on the relative amount of DNA and RNA content, respectively. Quiescent HPCs generally reside in G0, and display a low RNA content (12, 26, 37). As cells transition from G1 to S, RNA levels increase resulting in enhanced pyronin staining, while Hoechst 33342 staining concurrently increases as cells enter S phase.

CD34+ HPCs transduced with Tax1 showed significantly higher levels of cell cycle arrest in G0/G1 in comparison to Tax2, Tax1(−), Vpr, or mock-transduced HPCs at 24 h posttransduction (Fig. (Fig.2A2A and and2B;2B; Table Table1).1). Notably, the percentage of Tax1-transduced CD34+ cells which accumulated in G0/G1 was strikingly similar to levels of cell cycle arrest demonstrated by CD34+ cells cultured in the absence of cytokine growth factors (IL-3, IL-6, stem cell factor, and Flt-3L) or in the presence of roscovitine, a selective cdk inhibitor which induces G1 arrest (2) (Fig. (Fig.2B).2B). Tax1-transduced HPCs and roscovitine-treated cells also display marked levels of G0/G1 cell cycle arrest at 48 h (68% and 83%, respectively), in contrast to Tax2 (35%), Tax1(−) (30%), Vpr (26%), or mock-transduced HPCs (27%) (Fig. (Fig.2C).2C). As expected, transduction of CD34+ cells with HIV-1 Vpr resulted in significant accumulation of cells in G2/M (19.5%), in comparison to mock or Tax1(−)-transduced HPC cultures (6% and 8.5%, respectively) at 24 h. Notably, Tax221 induced G0/G1 arrest with a similar efficiency as Tax1 in CD34+ cells (Table (Table1).1). Although previous investigators have shown that Tax1 mediates cell cycle arrest of immortalized cell lines in G1/S and G2 (15, 28, 47, 48), this is the first demonstration that Tax1 can arrest primary hematopoietic cells in G0/G1.

FIG. 2.
Cell cycle analysis of transduced human hematopoietic progenitor cells (HPC). Human CD34+ cells (106) were infected with lentivirus vectors (multiplicity of infection, 1) in 3 ml of IMDM supplemented with 70 μg/ml of verapimil (Sigma) ...
TABLE 1.
G0/G1 cell cycle analysis of lentivirus vector-transduced human CD34+ HPCsa

Elevated sensitivity of immature CD34+/CD38 cells to Tax1 mediated suppression of hematopoiesis.

CD34+ HPCs represent a heterogeneous cell population which retains the capacity of differentiating into multiple hematopoietic lineages. Hematopoietic stem cells have been shown to reside in the CD34+/CD38 cell subpopulation and are predominantly in quiescence (4, 26, 37, 39). To determine if Tax1 displays an elevated propensity to suppress hematopoiesis in hematopoietic stem cells and establish if these cells are particularly susceptible to the effects of Tax1, purified CD34+ cells were infected with lentivirus vectors (multiplicity of infection, 1), GFP-positive cells were purified into CD34+/CD38 (immature) and CD34+/CD38+ (mature) cells by FACS and tested with clonogenic colony-forming assays (CFA) (67).

Tax1 transduction of CD34+/CD38+ cells resulted in a threefold reduction in the number of clonogenic colonies in comparison to cells transduced with the Tax1(−) vector (Fig. (Fig.3).3). In contrast, Tax1-transduced CD34+/CD38 cells showed a 10-fold reduction in colony-forming assay in comparison to Tax1(−)-transduced cells, demonstrating that immature HPCs have an elevated sensitivity to Tax1-mediated suppression of hematopoiesis. Consistent with our previous results, no effect on the colony-forming assay was detected in Tax2-transduced CD34+/CD38+ cells (67). Tax2 transduction of CD34+/CD38 cells modestly reduced the colony-forming activity by 33% in comparison to Tax1(−). No significant alteration of the ratio of myeloid to erythroid colony types was detected in Tax1-transduced CD34+ cells, as previously reported (data not shown) (67).

FIG. 3.
Clonogenic colony-forming activity of Tax-transduced CD34+ subpopulations. CD34+ HPCs were infected with lentivirus vectors and CD34+/CD38+/GFP-positive cells and CD34+/CD38/GFP-positive cells (103) were ...

Interestingly, transduction of CD34+ cells with Tax221 resulted in a significant reduction in CFA activity in both CD34+/CD38 and CD34+/CD38+ subpopulations (threefold and twofold decrease, respectively), suggesting that the C-terminal domain of Tax1 modulates the suppression of hematopoiesis. Transduction of CD34+ cells with HIV-1 Vpr resulted in the complete elimination of colony-forming activity in vitro in CD34+/CD38+ and CD34+/CD38 HPC subpopulations, consistent with the ability of Vpr to induce cell cycle arrest and apoptosis (3, 72). These results suggest that CD34+/CD38 HPCs are highly susceptible to Tax1-mediated colony-forming activity suppression in comparison to more mature CD34+/CD38+ cells, and that motifs localized in the Tax1 C terminus may play a key role in suppression of hematopoiesis.

Tax1 modulation of p21cip1/waf1 and p27kip1 expression in CD34+ cells.

The cell cycle-dependent kinase (CDK) inhibitors p21cip1/waf1 (p21) and p27kip1 (p27) are regulatory molecules involved in the modulation of cell cycle progression of HPCs and hematopoietic stem cells (9, 10, 66). p21 sustains HPCs in quiescence while p27 expression has been previously implicated in inhibiting expansion of more mature progenitor cells (62, 70). We previously demonstrated that Tax1 robustly activates the p21 promoter in transient transfection assays, in contrast to Tax2 (61). To determine if induction of p21 and p27 expression by Tax1 might correlate with cell cycle arrest and suppression of hematopoiesis, CD34+ HPCs were transduced with lentivirus vectors.

CD34+/CD38+/GFP-positive and CD34+/CD38/GFP-positive cells were isolated by FACS, and RNA was purified and analyzed by real-time reverse-transcription PCR (RT-PCR) (Fig. (Fig.4).4). RT-PCR analysis confirmed that Tax1 significantly upregulated p21 in CD34+/CD38 and CD34+/CD38+ subpopulations (5-fold and 13.5-fold, respectively) (Fig. (Fig.4A).4A). Tax1 modestly upregulated p27 expression (2-fold in both CD34+/CD38 and CD34+/CD38+ cells), in comparison with Tax1(−)-transduced CD34+ cells (Fig. (Fig.4B).4B). Tax2 did not significantly alter expression of p21 or p27 transcription in CD34+/CD38 or CD34+/CD38+ cells. Interestingly, Tax221 demonstrated upregulation of p21 in both CD34+/CD38 and CD34+/CD38+ cell subpopulations (fivefold and sevenfold, respectively). Tax221 showed a twofold activation of p27 expression in CD34+/CD38 and CD34+/CD38+ cell subpopulations, in comparison to Tax1(−)-transduced cells. These data suggest that Tax1- and Tax221-mediated upregulation of p21 and p27 in CD34+/CD38+ and CD34+/CD38 cells is concurrent with induction of cell cycle arrest and multilineage suppression of hematopoiesis. In addition, sequences localized in the C terminus of Tax1 may function in promoting transcriptional transactivation of the p21 and p27 CDK inhibitor genes.

FIG. 4.
Real-time PCR (RT-PCR) analysis of p21cip1/waf1 and p27kip1 expression. CD34+ cells were infected with lentivirus vectors encoding GFP, Tax1, Tax2, Tax1(−), Tax221 or Vpr. CD34+/CD38/GFP-positive and CD34+/CD38 ...

To establish whether HTLV infection alters expression of p21 and p27, CD34+ cells were infected by cocultivation with lethally irradiated HTLV-1-transformed or HTLV-2-transformed cell lines (SLB-1 and 729/pH6Neo cells, respectively). HTLV is poorly infectious in vitro and requires cell-to-cell contact for efficient transmission of viral infection (16, 20, 54). Infected CD34+ HPCs were purified by FACS after 72 h on the basis of cell surface coexpression of CD34 and HTLV envelope protein (gp46env). HTLV-1-infected HPCs showed a sixfold upregulation of p21 and twofold induction of p27 transcription in CD34+/CD38+ cells and a threefold upregulation of both p21 and p27 in CD34+/CD38 cells, in comparison to mock-infected HPCs (Fig. (Fig.4).4). In contrast, HTLV-2-infected HPCs showed no significant upregulation of p21 or p27 expression. These results demonstrate that HTLV-1 infection of CD34+ cells mediates transcriptional upregulation of p21 and p27, and suggests that Tax1 may initiate a cellular environment conducive for establishment of viral latency by induction of cell cycle arrest (Fig. (Fig.77).

FIG. 7.
Model for HTLV-1 infection of hematopoietic progenitor cells and establishment of viral latency.

Tax1 protects CD34+ HPCs from apoptosis.

We previously demonstrated that Tax1 protects transformed T cell lines from apoptosis (61). To define the role of Tax1 and Tax2 in modulating programmed cell death (PCD) in HPCs, CD34+ cells were transduced with lentivirus vectors and cultured in serum-free media and in the absence of cytokines. Tax1 and Tax221-transduced CD34+ cells displayed significant protection from apoptosis 24 h after serum and cytokine deprivation (Fig. (Fig.5).5). In contrast to transduction of CD34+ HPCs with Tax2 failed to protect cells from PCD.

FIG. 5.
Apoptosis analysis of CD34+ cells. Human CD34+ cells (106) were infected with lentivirus vectors encoding Tax1, Tax2, and Tax221 or with the anti-sense vector, Tax1(−) (multiplicity of infection, 1) in 2 ml of IMDM supplemented ...

Tax1 fails to alter endogenous survivin expression.

Survivin, originally identified as an inhibitor of apoptosis (IAP) protein, plays a pivotal role in initiating entry into the cell cycle in human CD34+ HPCs (22, 44, 46). Cotransfection of Tax1 with a constructs containing proximal survivin promoter sequences resulted in a significant reductions in luciferase expression in transient transfection assays, in comparison to cotransfection with Tax2 or Tax1(−) (data not shown) (45). Notably however, survivin expression was not detectably down-regulated in Tax1-transduced CD34+ cells in comparison to HPCs transduced with lentivirus encoding Tax1(−) (Fig. (Fig.6).6). In contrast, transduction of Vpr demonstrably increased survivin expression 4-fold in CD34+/CD38+ and CD34+/CD38 cell subpopulations, confirming a recent report that HIV-1 Vpr induces expression from the survivin promoter (71). Infection of CD34+ cells with HTLV-1 also failed to detectably modulate survivin RNA levels, although infection with HTLV-2 modestly induced mean survivin mRNA levels by twofold. These results show that Tax1 does not detectably alter expression of survivin in HPCs following HTLV-1 infection or after Tax1 transduction.

FIG. 6.
Real-time PCR analysis of survivin expression in CD34+ cells. CD34+ cells were transduced with lentivirus vectors encoding GFP, Tax1, Tax2, Tax1(−), Tax 221 or Vpr. CD34+/CD38/GFP-positive and CD34+/CD38 ...

DISCUSSION

HTLV-1 infection of CD34+ HPCs and Tax1-mediated cell cycle arrest by induction of p21 and p27 identifies pluripotent hematopoietic progenitor and stem cells as a potential viral reservoir for HTLV-1 infection in vivo. p21 and p27 are regulatory molecules involved in the modulation of cell cycle progression of CD34+ HPCs (66). p21 sustains HPCs in quiescence, while p27 expression has been previously implicated in inhibiting expansion of more mature progenitor cells, therefore Tax1-mediated dysregulation of cdk inhibitors would be expected to suppress hematopoiesis in CD34+ cells (62, 70). We previously demonstrated that Tax1 robustly activates the p21 promoter in transient transfection assays, in contrast to Tax2 (61).

Establishment of viral latency in CD34+ cells may account for the relatively long persistence of HTLV-1 infection in adult T-cell leukemia patients, most of whom are infected peri- or postnatally. The sequestration and concomitant differentiation of HTLV-1-infected HPCs may be a mechanistic pathway which allows the virus to evade immune surveillance while also continuously providing a supply of infected cells in vivo (Fig. (Fig.7).7). Indeed, the bone marrow, a site enriched for CD34+ HPCs, has been previously proposed to represent a site for HTLV-1 latency and a reservoir of infected cells (27, 32, 42). We recently showed Tax1 transduction of CD34+ cells resulted in the suppression of multilineage hematopoiesis in vitro, a function not demonstrated by Tax2. Although Tax1 and Tax2 share greater than 72% amino acid homology, the ability of Tax1 to induce cell cycle arrest in CD34+ HPCs with concomitant activation of p21 and p27 transcription suggests that induction of G0/G1 cell cycle arrest may be a feature which distinguishes HTLV-1 from HTLV-2 infection in vivo.

We speculate that infection of CD34+ HPCs by HTLV-1 and the induction of cell cycle arrest may be predisposing factors to the development of adult T-cell leukemia and that these unique functions of Tax1 may potentially account for the distinct pathogenic potential of HTLV-1. It is noteworthy that the degree of G0/G1 cell cycle arrest induced by Tax1 and the p21 and p27 transcription induction profiles were similar to levels of cell cycle arrest following the withdrawal of cytokine growth factors from mock-infected CD34+ cells. Notably, Tax1-arrested CD34+ cells are also highly resistant to apoptosis in contrast to the majority of the cells in the cytokine-deprived sample, which demonstrated significant cell death after 48 h. These results are in concordance with previous reports demonstrating that Tax1 increases cellular resistance to apoptosis and suggests that HTLV-1-infected HPCs have a selective advantage for survival in vivo (15, 52, 59, 61).

Human CD34+ HPCs have previously been demonstrated to be cellular targets for viral infection. Measles virus infection of CD34+ cells results in the disruption and suppression of hematopoiesis in vitro (50). Kaposi's sarcoma-related herpesvirus/human herpesvirus 8 has been detected in CD34+ cells isolated from Kaposi's sarcoma patients (29), and our laboratory has recently demonstrated that Kaposi's sarcoma-related herpesvirus/human herpesvirus 8 can infect CD34+ cells in vitro (Wu and Feuer, unpublished observations). Cytomegalovirus has been shown to establish a productive, latent infection in CD34+/CD38 cells, but not in more mature CD34+/CD38+ cells (24, 25).

Maintaining normal hematopoiesis requires appropriate cell cycle control, and both p21 and p27 are intimately involved in maintaining HPCs in quiescence. p21 is a key molecule which dominates the kinetics of hematopoietic stem cell proliferation and expansion (10, 63). p27 modulates HPC proliferation and pool size, and p27 has been implicated as a negative regulator of cell cycle progression (1, 9, 11, 19, 70). The robust concomitant upregulation of p21 and p27 that occurs following HTLV-1 infection and Tax1 transduction of CD34+ cells indicates that these target cells may be particularly vulnerable to cell cycle arrest as a result of HTLV-1 infection. Although a previous report failed to detect HTLV-1 proviral sequences in CD34+ cells derived from adult T-cell leukemia patients (53), we speculate that HTLV-1 infection and establishment of latency in HPCs is an early event in the infection process and precedes development of adult T-cell leukemia by decades. Evaluation of CD34+ cells directly obtained from HTLV-1 seropositive and HTLV-1-associated myelopathy/tropical spastic paraparesis patients for evidence of HTLV-1 proviral sequences may determine whether infection of HPCs occurs in vivo.

We previously postulated that suppression of multilineage hematopoiesis was due to Tax1 perturbation of differentiation at an early stage of hematopoietic cell development (67). Indeed, the observation that Tax1 suppressed hematopoiesis more robustly in CD34+/CD38 HPCs, in comparison to CD34+/CD38+ cells, suggests that hematopoietic stem cells may be more sensitive to G0/G1 cell cycle arrest following infection with HTLV-1. It is of particular interest to precisely determine if Tax1 arrests cycling CD34+ cells in G1, or whether quiescent CD34+ cells are prevented from progressing from G0 following transduction. Moreover, it remains to be established whether Tax1 retards cell cycle progression or irreversibly arrests CD34+ HPCs in G0/G1.

In vivo modeling of HTLV-1 latency by reconstitution of lymphopoiesis in the SCID-hu chimeric mouse using Tax1-transduced or HTLV-1-infected CD34+ cells will be used to determine if HTLV-1 proviral sequences are preferentially maintained in HPCs and hematopoietic stem cells reservoirs in vivo. Although induction of p21 in HTLV-1-infected T-cell lines has been shown to facilitate cyclin D2/cdk4 complex formation and accelerate G1 progression, primary hematopoietic cells may have a fundamentally different response to Tax-mediated induction of cdk inhibitors, particularly since many of these cells are in quiescence (35). Tax1 has previously been shown to arrest cell lines in G2/M (28, 47, 48). Others have reported that Tax1 inhibits cell cycle progression from G1 to S (14, 15) and from G2 to M (48).

Clearly, demonstration of p21 and p27 CDK inhibitor gene induction following HTLV-1 infection suggests that G0/G1 cell cycle arrest may also occur following HTLV-1 infection. Preliminary evidence in our laboratory demonstrates that HTLV-1 infection of CD34+ cells results in accumulation of cells in G0/G1, a property not displayed following infection with HTLV-2 (data not shown). It remains to be determined whether HTLV-1 infection of CD34+ HPCs results in cell cycle arrest and whether suppression of multilineage hematopoiesis occurs as a result of infection.

The discordance between Tax1-mediated transcriptional repression of the survivin promoter in transient transfection assays and the failure of Tax1 to repress expression of endogenous survivin expression remains to be precisely defined. Consistent with our observations, a recent report failed to detect alterations in endogenous survivin activity following induction of Tax1 expression in the JPX-9 Jurkat cell line (34). It will be important to further define the role of Tax1 in modulating expression from the survivin promoter, particularly since survivin modulates fundamental cellular processes associated with HTLV-1 infection and Tax1 expression: apoptosis and cell cycle progression.

The phenotypic differences displayed by Tax1 and Tax2 in CD34+ HPCs suggest that unique domains encoded by Tax1 may function to modulate cell cycle progression in immature hematopoietic cells. Indeed, the chimeric Tax221 displayed a profile similar to Tax1's with respect to suppression of hematopoiesis and activation of p21 and p27 gene expression. The PDZ and the P/CAF binding domains are localized in the C-terminal region of Tax1, and it remains to be determined which domain distinguishes the functional activities of Tax1 and Tax2 in our model. The PDZ binding domain was found to be involved in binding and inhibition of the tumor suppressor protein hDlg (40, 58) and this domain has recently been shown to mediate T-cell proliferation and transformation (68) (Patrick Green, personal communication). Tax221 was previously shown to mediate transformation of rat embryo fibroblasts at levels indistinguishable from Tax1 (18).

Our data suggest that the PDZ and P/CAF binding domains may function in conferring the unique phenotypes displayed by Tax1 in CD34+ cells. Alternatively, it could be speculated that the C terminus of Tax1 promotes protein stabilization, which may confer these activities on Tax2. Future studies will elucidate the function of the domains located at the C-terminal domain of Tax1 and clarify their role in HTLV-1 pathogenesis.

Acknowledgments

This work was supported by the National Institutes of Health (R29 CA77567) and the Leukemia Research Foundation.

We thank Bert Vogelstein for the pC53-SN3 vector and Masahiro Fugii for the Tax221 construct.

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