PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.


Introduction
Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus that infects an estimated 15-20 million people worldwide [1]. This blood-borne pathogen is the causative infectious agent of adult T-cell leukemia/lymphoma (ATLL), a disease of CD4+ T-cells [2][3][4]. HTLV-1 is also associated with inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis Currently, there have been no studies investigating the role of PRMT5 in T-cell malignancies, including HTLV-1-associated disease. Therefore, we sought to determine if PRMT5 plays a role in HTLV-1 transformation/malignancy. Indeed, we found PRMT5 levels were upregulated during T-cell transformation and in established lymphocytic leukemia/lymphoma cell lines. Our data suggested that PRMT5 negatively regulated HTLV-1 viral gene expression, which indicated that PRMT5 could be an important cellular regulator of the viral transformation process. Furthermore, selective inhibition of PRMT5 by a novel small molecule inhibitor (PRMT5i) in HTLV-1-positive cell lines reduced cell survival; therefore, PRMT5 may represent an important therapeutic target for ATLL.

Plasmids and Cloning
Plasmid DNA was purified on maxi-prep columns according to the manufacturer's protocol (Qiagen, Valencia, CA, USA). The flag-tagged PRMT5 expression vector was described previously [37]. p30 cDNA was cloned into the pcDNA3.1 expression vector (Invitrogen, Grand Island, NY, USA) to create pcDNA3.1-p30. The S-tagged Tax and HBZ expression vectors contained Tax or HBZ cDNA inserted into a pTriEx™-4 Neo vector (Novagen, Madison, WI, USA) for mammalian cell expression of S-tagged Tax and HBZ proteins. The plasmid containing the wild-type HTLV-1 infectious proviral clone, ACHneo, was described previously [43]. The LTR-1-luciferase reporter plasmid and transfection efficiency control plasmid TK-renilla were described previously [41].

Quantitative RT-PCR
Total RNA was isolated from 10 6 cells per condition using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Isolated RNA was quantitated and DNase-treated using recombinant DNase I (Roche). Reverse transcription was performed using the Omniscript RT Kit (Qiagen) according to the manufacturer's instructions. The instrumentation and general principles of the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) are described in detail in the operator's manual. PCR amplification was carried out in 96-well plates with optical caps. The final reaction volume was 20 µL consisting of 10 µL iQ™ SYBR Green Supermix (Bio-Rad), 300 nM of each specific primer, and 2 µL of cDNA template. For each run, sample cDNA and a no-template control were assayed in triplicate. The reaction conditions were 95˝C for 5 min, followed by 40 cycles of 94˝C for 30 s, 56˝C for 30 s, and 72˝C for 45 s. Primer pairs to specifically detect viral mRNA species (tax, hbz), prmt5, st7, and gapdh were described previously [28,44]. Data are presented in histogram form as means with standard deviations from triplicate experiments.

Co-Culture Immortalization Assays
Long-term immortalization assays were performed as detailed previously [45]. Briefly, 2ˆ10 6 freshly isolated human PBMCs were co-cultivated at a 2:1 ratio with lethally irradiated cells (729.B uninfected parental; 729.ACH HTLV-1-producing) in 24-well culture plates (media was supplemented with 10 U/mL rhIL-2). HTLV-1 gene expression was confirmed by the detection of p19 Gag protein in the culture supernatant, and was measured weekly by p19 ELISA (Zeptometrix, Buffalo, NY, USA). Viable cells were counted weekly by Trypan blue dye exclusion.

Packaging and Infection of Lentivirus Vectors
Lentiviral vectors expressing five different PRMT5-directed shRNAs (target set RHS4533-EG10419), and the universal negative control, pLKO.1 (RHS4080) were purchased from Open Biosystems (Dharmacon, Lafayette, CO, USA) and propagated according to the manufacturer's instructions. HEK293T cells were transfected with lentiviral vector(s) expressing shRNAs, plus DNA vectors encoding HIV Gag/Pol and VSV-G in 10-cm dishes with Lipofectamine 2000 according to the manufacturer's instructions. Media containing lentiviral particles was collected 72 h later and filtered through 0.45 µm pore size filters. Lentiviral particles were then concentrated using ultracentrifugation in a Sorvall SW-41 swinging bucket rotor. Lymphoid cell lines were infected with the concentrated lentivirus in 8 µg/mL polybrene by spinoculation at 2000 xg for 2 h at room temperature. HEK293T cells were infected with the concentrated lentivirus in 8 µg/mL polybrene. After 72 h, stable cell lines were selected by treatment with 1-2 µg/mL puromycin for 7 days.

PRMT5i Treatment
A selective PRMT5 inhibitor (PRMT5i) drug was recently described by Alinari et al. [31]. Lymphoid cells were seeded into a 12-well plate at 0.5ˆ10 6 cells/mL. Indicated concentrations of the PRMT5i were added to duplicate wells. Cells were incubated at 37˝C for 48 h. After incubation, cell viability and proliferation were measured. Cell viability was determined using Trypan blue dye exclusion. Cellular proliferation was measured in duplicate in each condition using the CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega). Cells were collected by slow centrifugation (5 min, 800 xg) for downstream qRT-PCR analysis as described above.
2.9. Transient Transfections, Reporter Assays, and p19 Gag ELISA HEK293T cells were transfected using Lipofectamine 2000 Transfection Reagent according to the manufacturer's instructions. Each transfection experiment was performed in triplicate and presented as means plus standard deviations. In general, HEK293T cells in a 6-well dish were transfected with approximately 1-2 µg total DNA consisting of 20 ng TK-renilla (transfection control), 100 ng LTR-1-luciferase, 1 µg ACHneo, 500 ng Flag-PRMT5, 500 ng pcDNA3.1-p30, 100 ng S-tag-Tax, or 500 ng S-tag-HBZ, where indicated. HEK293T cells were harvested 48 h post-transfection in Passive Lysis Buffer (Promega). Relative firefly and Renilla luciferase units were measured in a FilterMax F5 Multi-Mode Microplate Reader using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Each condition was performed in duplicate. Extracts also were subjected to immunoblotting to verify equivalent protein levels. Cell supernatants (48 h) were used for p19 ELISA (Zeptometrix).

Annexin V Staining
Lymphoid cells were seeded into a 6-well plate at 1ˆ10 6 cells/mL. Indicated concentrations of the PRMT5i were added to the appropriate wells. Cells were incubated at 37˝C for 24 h. After incubation, cells were collected by slow centrifugation (5 min, 800 xg) for apoptosis analysis via flow cytometry. Collected cells were stained using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences; San Diego, CA, USA) according to the manufacturer's instructions.

ChIP Assays
pA-18G-BHK-21 cells are a Syrian Hamster kidney cell line stably transfected with a plasmid vector containing the lacZ bacterial gene under the control of a HTLV-1-LTR promoter as previously described [46]. pA-18G-BHK-21 cells were transfected in 10-cm dishes (1 µg ACHneo and 5 µg flag-PRMT5) using Lipofectamine 2000 according to the manufacturer's instructions. Cells were cross-linked in fresh 1% paraformaldehyde for 10 min at room temperature. The cross-linking reaction was quenched using 125 mM glycine. Following cell lysis and DNA fragmentation by sonication, DNA-protein complexes were immunoprecipitated with anti-PRMT5 (Santa Cruz) and control anti-IgG (Santa Cruz) antibodies. Immunoprecipitated DNA-protein complexes were washed using sequential low-salt, high-salt, lithium chloride, and Tris-EDTA (TE) buffers. DNA was purified using the Qiagen Gel Extraction Kit (Qiagen). The presence of specific DNA fragments in each precipitate was detected using PCR. Primers used for amplifying the HTLV-1 LTR were 5'-CCACAGGCGGGAGGCGGCAGAA-3' and 5'-TCATAAGCTCAGACCTCCGGGAAG-3' and LacZ-coding region were 5'-AAAATGGTCTGCTGCTG-3' and 5'-TGGCTTCATCCACCACA-3'. Quantification of each ChIP experiment was performed using ImageJ software.

PRMT5 Was Upregulated in T-Cell Leukemia/Lymphoma Cells
Recently, PRMT5 over-expression was identified to be involved in the pathogenesis of hematologic (lymphoma) and solid tumors (melanoma, astrocytomas) [27][28][29][30][31][32]. To determine whether PRMT5 is important to HTLV-1 biology and pathogenesis, we first examined the levels of PRMT5 protein ( Figure 1A) and RNA ( Figure 1B Interestingly, although protein and RNA levels were upregulated, PRMT5 RNA was not directly correlated to PRMT5 protein levels, which suggested a post-transcriptional method of regulation. PRMT5 protein ( Figure 1C) and RNA ( Figure 1D) were also upregulated in 3 of 4 and 4 of 4 PBMC samples from ATLL patients, respectively, relative to HTLV-1-negative naïve T-cells.   , and naïve T-cells were subjected to immunoblot analysis to compare the levels of endogenous PRMT5 expression. β-Actin expression was used as a loading control. The amount of PRMT5 in each cell line was measured relative to β-actin; the level of PRMT5 expression obtained with naïve T-cells was set at 1; (B) Quantitative RT-PCR for PRMT5 and GAPDH was performed on mRNA isolated from cells in panel A. Total PRMT5 mRNA level was determined using the ΔΔCt method [47] and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments; (C) Lysates of ATLL PBMCs from four independent donors and naïve T-cells were subjected to immunoblot analysis to compare endogenous PRMT5 protein expression levels. β-Actin expression was used as a loading control. The amount of PRMT5 in each condition was measured relative to β-actin and depicted in histogram form; the level of expression obtained with naïve T-cells was set to 1. Each sample was measured in duplicate; (D) Quantitative RT-PCR for PRMT5 and GAPDH was performed on mRNA isolated from cells in panel C. Total PRMT5 mRNA level was determined using the ΔΔCt method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments.

PRMT5 Levels Were Elevated during HTLV-1-Mediated Cellular Transformation
We next determined whether PRMT5 becomes dysregulated and over-expressed during HTLV-1-driven T-cell transformation. Freshly isolated human PBMCs co-cultured with lethally irradiated HTLV-1 producer cells (729.ACHi) in the presence of 10 U/mL of human IL-2 showed progressive growth consistent with HTLV-1 immortalization (Figure 2A, left panel). As a control, PBMCs co-cultured with lethally irradiated 729.B (HTLV-1-negative parental line) were unable to sustain progressive growth. We also detected continuous accumulation of p19 Gag in the culture supernatants of PBMCs co-cultured with 729.ACHi cells, which indicated viral replication and virion production; as expected, irradiated HTLV-1 producer cells alone failed to grow or produce p19 over time ( Figure 2A, right panel). We examined PRMT5 protein ( Figure 2B) and RNA ( Figure  2C) levels throughout the 10-week in vitro transformation assay. Protein and RNA were isolated from two independent wells of cells at weekly time points. Our data revealed that PRMT5 protein and RNA were upregulated at each time point, to varying degrees, throughout the transformation assay. , and naïve T-cells were subjected to immunoblot analysis to compare the levels of endogenous PRMT5 expression. β-Actin expression was used as a loading control. The amount of PRMT5 in each cell line was measured relative to β-actin; the level of PRMT5 expression obtained with naïve T-cells was set at 1; (B) Quantitative RT-PCR for PRMT5 and GAPDH was performed on mRNA isolated from cells in panel A. Total PRMT5 mRNA level was determined using the ∆∆Ct method [47] and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments; (C) Lysates of ATLL PBMCs from four independent donors and naïve T-cells were subjected to immunoblot analysis to compare endogenous PRMT5 protein expression levels. β-Actin expression was used as a loading control. The amount of PRMT5 in each condition was measured relative to β-actin and depicted in histogram form; the level of expression obtained with naïve T-cells was set to 1. Each sample was measured in duplicate; (D) Quantitative RT-PCR for PRMT5 and GAPDH was performed on mRNA isolated from cells in panel C. Total PRMT5 mRNA level was determined using the ∆∆Ct method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments.

PRMT5 Levels Were Elevated during HTLV-1-Mediated Cellular Transformation
We next determined whether PRMT5 becomes dysregulated and over-expressed during HTLV-1-driven T-cell transformation. Freshly isolated human PBMCs co-cultured with lethally irradiated HTLV-1 producer cells (729.ACHi) in the presence of 10 U/mL of human IL-2 showed progressive growth consistent with HTLV-1 immortalization (Figure 2A, left panel). As a control, PBMCs co-cultured with lethally irradiated 729.B (HTLV-1-negative parental line) were unable to sustain progressive growth. We also detected continuous accumulation of p19 Gag in the culture supernatants of PBMCs co-cultured with 729.ACHi cells, which indicated viral replication and virion production; as expected, irradiated HTLV-1 producer cells alone failed to grow or produce p19 over time (Figure 2A, right panel). We examined PRMT5 protein ( Figure 2B) and RNA ( Figure 2C) levels throughout the 10-week in vitro transformation assay. Protein and RNA were isolated from two independent wells of cells at weekly time points. Our data revealed that PRMT5 protein and RNA were upregulated at each time point, to varying degrees, throughout the transformation assay. Gag protein production at weekly intervals are presented. Means and standard deviations of data from each time point were determined from four random independent wells; Cells were also collected at weekly intervals and analyzed by immunoblotting for PRMT5 protein expression (B); and qRT-PCR analysis for PRMT5 RNA levels (C). PRMT5 levels are shown relative to an internal control (β-actin, GAPDH) for each time point. Resting PBMC PRMT5 levels were set to 1. Means and standard deviations of data from each time point were determined from two random independent wells.

Loss of Endogenous PRMT5 Increased HTLV-1 Gene Expression
To determine whether PRMT5 over-expression is a marker for T-cell transformation and/or contributes to the process of HTLV-1 transformation, we utilized shRNA vectors to knockdown PRMT5 expression in three different HTLV-1-transformed cell lines. As shown in Figure 3A, shRNA-mediated knockdown of endogenous PRMT5 expression in the early passage HTLV-1 immortalized T-cell line, PBL-ACH, resulted in a significant increase of viral p19 Gag protein production (left and right panel) and a significant increase in the levels of Tax and HBZ gene expression (middle panel). Knockdown of PRMT5 expression likewise significantly increased Tax and HBZ transcript levels in another HTLV-1-transformed cell line, SLB-1 ( Figure 3B). Finally, knockdown of PRMT5 protein expression in the Tax-negative ATL-derived T-cell line, TL-Om1, significantly increased HBZ transcript levels ( Figure 3C). Gag protein production at weekly intervals are presented. Means and standard deviations of data from each time point were determined from four random independent wells; Cells were also collected at weekly intervals and analyzed by immunoblotting for PRMT5 protein expression (B); and qRT-PCR analysis for PRMT5 RNA levels (C). PRMT5 levels are shown relative to an internal control (β-actin, GAPDH) for each time point. Resting PBMC PRMT5 levels were set to 1. Means and standard deviations of data from each time point were determined from two random independent wells.

Loss of Endogenous PRMT5 Increased HTLV-1 Gene Expression
To determine whether PRMT5 over-expression is a marker for T-cell transformation and/or contributes to the process of HTLV-1 transformation, we utilized shRNA vectors to knockdown PRMT5 expression in three different HTLV-1-transformed cell lines. As shown in Figure 3A, shRNA-mediated knockdown of endogenous PRMT5 expression in the early passage HTLV-1 immortalized T-cell line, PBL-ACH, resulted in a significant increase of viral p19 Gag protein production (left and right panel) and a significant increase in the levels of Tax and HBZ gene expression (middle panel). Knockdown of PRMT5 expression likewise significantly increased Tax and HBZ transcript levels in another HTLV-1-transformed cell line, SLB-1 ( Figure 3B). Finally, knockdown of PRMT5 protein expression in the Tax-negative ATL-derived T-cell line, TL-Om1, significantly increased HBZ transcript levels ( Figure 3C).  (middle panel) for Tax, HBZ, and GAPDH was performed on mRNA isolated from shControl and shPRMT5 cells. Transcript levels were determined using the ΔΔCt method and normalized to relative GAPDH levels. Levels of Tax and HBZ relative to GAPDH in shControl cells were set to 1. Data are presented in histogram form with means and standard deviations from triplicate experiments. HTLV-1 gene expression was quantified by the detection of the p19 Gag protein in the culture supernatant using ELISA (right panel); (B) SLB-1 cells (HTLV-1-transformed) were infected with a pool of five different lentiviral vectors directed against PRMT5 or control shRNAs. The cells were selected for 7 days using puromycin. Immunoblot analysis was performed to compare the levels of PRMT5 and β-actin protein (loading control) in each condition (left panel). Quantitative RT-PCR for Tax, HBZ, and GAPDH was performed on mRNA isolated from shControl and shPRMT5 cells (right panel). Transcript levels were determined using the ΔΔCt method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments. Levels of Tax and HBZ relative to GAPDH in shControl cells were set to 1; (C) TL-Om1 cells (ATL-derived, Tax-negative) were infected with a pool of five different lentiviral vectors directed against PRMT5, or control shRNAs. The cells were selected for 7 days using puromycin. Immunoblot analysis was performed to compare the levels of PRMT5 and β-actin protein (loading control) in each condition (left panel). Quantitative RT-PCR for HBZ and GAPDH was performed on mRNA isolated from shControl and shPRMT5 cells (right panel). HBZ transcript level was determined using the ΔΔCt method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments. Levels of HBZ relative to GAPDH in shControl cells were set to 1. Student's t test was performed to determine significant differences in viral transcript levels between shControl and shPRMT5 cells; p < 0.05 (*). were infected with a pool of five different lentiviral vectors directed against PRMT5 or control shRNAs. The cells were selected for 7 days using puromycin. Immunoblot analysis was performed to compare the levels of PRMT5 and β-actin protein (loading control) in each condition (left panel). Quantitative RT-PCR for Tax, HBZ, and GAPDH was performed on mRNA isolated from shControl and shPRMT5 cells (right panel). Transcript levels were determined using the ∆∆Ct method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments. Levels of Tax and HBZ relative to GAPDH in shControl cells were set to 1; (C) TL-Om1 cells (ATL-derived, Tax-negative) were infected with a pool of five different lentiviral vectors directed against PRMT5, or control shRNAs. The cells were selected for 7 days using puromycin. Immunoblot analysis was performed to compare the levels of PRMT5 and β-actin protein (loading control) in each condition (left panel). Quantitative RT-PCR for HBZ and GAPDH was performed on mRNA isolated from shControl and shPRMT5 cells (right panel). HBZ transcript level was determined using the ∆∆Ct method and normalized to relative GAPDH levels. Data are presented in histogram form with means and standard deviations from triplicate experiments. Levels of HBZ relative to GAPDH in shControl cells were set to 1. Student's t test was performed to determine significant differences in viral transcript levels between shControl and shPRMT5 cells; p < 0.05 (*).   Recently, a first-in-class, small-molecule PRMT5 inhibitor (PRMT5i) was developed [31]. This novel inhibitor selectively blocks S2Me-H4R3 (symmetric di-methylation of H4R3) but is inactive against other type I and type II PRMT enzymes, which highlights its specificity for PRMT5. To evaluate whether PRMT5 over-expression is a marker for T-cell transformation or contributes to the process of HTLV-1 transformation, we treated six different HTLV-1-transformed cell lines with titrating amounts of PRMT5i ranging from 10 µM to 50 µM. As shown in Figure 4A-D, inhibition of PRMT5 resulted in a significant increase in Tax and HBZ viral gene expression in the HTLV-1-transformed T-cell lines PBL-ACH, ACH.2 SLB-1, and Hut-102. ST7 transcript levels were measured as a control to ensure successful PRMT5 inhibition because PRMT5 was reported to repress the tumor suppressor ST7 in MCL [28]. Treatment with PRMT5i also resulted in a significant increase in HBZ transcript levels in the Tax-negative, ATL-derived T-cell lines TL-Om1 and ATL-ED ( Figure 4E,F). HTLV-1-negative cell lines, Jurkat and Hut-78, were also treated with titrating amounts of PRMT5i. ST7 transcript levels were measured to ensure successful PRMT5 inhibition ( Figure 4G). PRMT5 expression levels were examined and found to be relatively unchanged in all PRMT5i treated cells tested (western blot lower panels; Figure 4A-G). ACH-2 cells (HIV-1 LAV ) were also treated with titrating amounts of PRMT5i ( Figure 4H) to examine if PRMT5 might regulate viral gene expression of another human retrovirus. Treatment with PRMT5i did not significantly alter the expression of HIV-1 as measured by RT activity in the cell supernatant.

Selective Inhibition of PRMT5 Decreased Cell Proliferation and Viability
In a recent report, PRMT5 was linked to proliferation in B-cell lines and MCL because knockdown of PRMT5 expression reduced cell proliferation [28]. Treatment of HTLV-1-positive T-cell lines with titrating amounts of PRMT5i or shRNA-mediated knockdown of PRMT5 resulted in a significant decrease in cellular proliferation ( Figure 5A,B) and cell viability ( Figure 5C). Surprisingly, the same dose of PRMT5i had minimal effects on proliferation and viability of Jurkat and Hut-78 cells (HTLV-1-negative). The level of cellular apoptosis ( Figure 5D) and senescence ( Figure 5E-H) was also measured in response to titrating amounts of PRMT5i treatment. The number of apoptotic cells was increased in all cell lines in response to PRMT5i treatment; however, the amount of apoptotic cells in HTLV-1-transformed lines was higher. We found a slight increase in the level of cellular senescence (as measured by increased p21 and p27 levels and decreased cyclin B1 levels) in response to PRMT5i in the HTLV-1-positive cell lines examined. Recently, a first-in-class, small-molecule PRMT5 inhibitor (PRMT5i) was developed [31]. This novel inhibitor selectively blocks S2Me-H4R3 (symmetric di-methylation of H4R3) but is inactive against other type I and type II PRMT enzymes, which highlights its specificity for PRMT5. To evaluate whether PRMT5 over-expression is a marker for T-cell transformation or contributes to the process of HTLV-1 transformation, we treated six different HTLV-1-transformed cell lines with titrating amounts of PRMT5i ranging from 10 μM to 50 μM. As shown in Figure 4A-D, inhibition of PRMT5 resulted in a significant increase in Tax and HBZ viral gene expression in the HTLV-1-transformed T-cell lines PBL-ACH, ACH.2 SLB-1, and Hut-102. ST7 transcript levels were measured as a control to ensure successful PRMT5 inhibition because PRMT5 was reported to repress the tumor suppressor ST7 in MCL [28]. Treatment with PRMT5i also resulted in a significant increase in HBZ transcript levels in the Tax-negative, ATL-derived T-cell lines TL-Om1 and ATL-ED ( Figure 4E,F). HTLV-1-negative cell lines, Jurkat and Hut-78, were also treated with titrating amounts of PRMT5i. ST7 transcript levels were measured to ensure successful PRMT5 inhibition ( Figure 4G). PRMT5 expression levels were examined and found to be relatively unchanged in all PRMT5i treated cells tested (western blot lower panels; Figure 4A-G). ACH-2 cells (HIV-1LAV) were also treated with titrating amounts of PRMT5i ( Figure 4H) to examine if PRMT5 might regulate viral gene expression of another human retrovirus. Treatment with PRMT5i did not significantly alter the expression of HIV-1 as measured by RT activity in the cell supernatant.

PRMT5 Negatively Regulated HTLV-1 Gene Expression
A recent report identified PRMT5 as a binding partner of the HTLV-1 accessory protein p30, a known negative regulator of HTLV-1 gene expression [37]. To investigate the effect(s) of exogenous PRMT5 on p30, HEK293T cells were transfected with a LTR-1-luciferase reporter vector (LTR-1-luc), TK-renilla control, the ACHneo proviral clone, a flag-tagged PRMT5 expression vector, and a p30 expression vector as indicated ( Figure 6A). Luciferase activity was measured after 48 h. The luciferase activity of the empty control reflected the amount of Tax and therefore, was a measure of transcription from the provirus. In the presence of either exogenous p30 or PRMT5, LTR-1-luciferase reporter was significantly repressed (left panel). However, in the presence of both p30 and PRMT5, there was an additive effect on LTR-1-luciferase repression. The amount of viral p19 Gag protein present in the culture supernatant of each condition also was examined using ELISA, which provided another method to quantify HTLV-1 gene expression (middle panel). Similar to the luciferase results, p30 and PRMT5 individually repressed viral p19 Gag production, and the presence of both p30 and PRMT5 repressed viral gene transcription further. We next asked whether PRMT5 was required for p30 function by transducing HEK293T cells with shRNA vectors directed against PRMT5 or a scramble control ( Figure 6B). Scramble and shPRMT5 HEK293T cell lines were selected for 7 days using puromycin to ensure sufficient knockdown of endogenous PRMT5. After selection, each cell line was transfected with LTR-1-luc, TK-renilla control, the ACHneo proviral clone, and a p30 expression vector as indicated. Luciferase activity was measured after 48 h. Knockdown of endogenous PRMT5 significantly enhanced viral transcription, as measured by LTR-1-luciferase activity (left panel) and p19 Gag ELISA (middle panel). In addition, reduced levels of PRMT5 did not significantly affect the ability of p30 to repress viral transcription. selected for 7 days using puromycin to ensure sufficient knockdown of endogenous PRMT5. After selection, each cell line was transfected with LTR-1-luc, TK-renilla control, the ACHneo proviral clone, and a p30 expression vector as indicated. Luciferase activity was measured after 48 h. Knockdown of endogenous PRMT5 significantly enhanced viral transcription, as measured by LTR-1-luciferase activity (left panel) and p19 Gag ELISA (middle panel). In addition, reduced levels of PRMT5 did not significantly affect the ability of p30 to repress viral transcription. The decreases in relative LTR-1-luc activity compared to control were significant (p < 0.05 (*)). HTLV-1 gene expression was quantified by the detection of the p19 Gag protein in the culture supernatant of each condition using ELISA (middle panel). The decreases in p19 Gag levels compared to control were significant (p < 0.05 (*)). Immunoblot analysis was performed to compare the levels of PRMT5 (Flag), p30, and β-actin (loading control) in each condition (right panel); (B) HEK293T cells were infected with a pool of five different lentiviral vectors directed against PRMT5 or control shRNAs. The cells were selected for 7 days using puromycin. HEK293T shControl and shPRMT5 cells were then transfected with 20 ng TK-renilla, 100 ng LTR-1-luciferase reporter, 1 μg ACHneo, and 500 ng p30 expression plasmid as indicated. Forty-eight hours post-transfection, cell lysates were collected, and luciferase levels measured; relative luciferase activity for each condition is shown (left panel). The differences in relative LTR-1-luc activity were significant (p < 0.05 (*)). HTLV-1 gene expression was quantified by the detection of the p19 Gag protein in the culture supernatant of each condition using ELISA (middle panel). The differences in p19 Gag levels were significant (p < 0.05 (*)). Immunoblot analysis was performed to compare the levels of endogenous PRMT5, p30, and β-actin (loading control) in each condition (right panel). The decreases in relative LTR-1-luc activity compared to control were significant (p < 0.05 (*)). HTLV-1 gene expression was quantified by the detection of the p19 Gag protein in the culture supernatant of each condition using ELISA (middle panel). The decreases in p19 Gag levels compared to control were significant (p < 0.05 (*)). Immunoblot analysis was performed to compare the levels of PRMT5 (Flag), p30, and β-actin (loading control) in each condition (right panel); (B) HEK293T cells were infected with a pool of five different lentiviral vectors directed against PRMT5 or control shRNAs. The cells were selected for 7 days using puromycin. HEK293T shControl and shPRMT5 cells were then transfected with 20 ng TK-renilla, 100 ng LTR-1-luciferase reporter, 1 µg ACHneo, and 500 ng p30 expression plasmid as indicated. Forty-eight hours post-transfection, cell lysates were collected, and luciferase levels measured; relative luciferase activity for each condition is shown (left panel). The differences in relative LTR-1-luc activity were significant (p < 0.05 (*)). HTLV-1 gene expression was quantified by the detection of the p19 Gag protein in the culture supernatant of each condition using ELISA (middle panel). The differences in p19 Gag levels were significant (p < 0.05 (*)). Immunoblot analysis was performed to compare the levels of endogenous PRMT5, p30, and β-actin (loading control) in each condition (right panel).   We next asked what effect PRMT5 had on Tax and HBZ transcriptional activity. HEK293T cells were transfected with LTR-1-luc, TK-renilla control, the ACHneo proviral clone, flag-PRMT5 expression vector, S-tag-Tax expression vector, and S-tag-HBZ expression vector as indicated ( Figure 7A). Tax activated transcription while HBZ repressed Tax-mediated transcriptional activation (left and middle panels), as expected [17][18][19]. In the presence of exogenous PRMT5, Tax transcriptional activity and HBZ-mediated repression of Tax transcriptional activity were decreased. To determine if PRMT5 was able to specifically repress Tax in the absence of other viral genes, HEK293T cells were transfected with LTR-1-luc, TK-renilla control, flag-PRMT5 expression vector, and an S-tag-Tax expression vector ( Figure 7B). Tax was able to activate LTR-1-luciferase activity, while PRMT5 had no effect in the presence or absence of Tax on LTR-1 luciferase activity. Our results suggested that PRMT5 requires the HTLV-1 proviral DNA to suppress Tax-induced LTR activation. To examine if viral factors other than Tax are implicated in suppression of viral transcription by PRMT5, we examined the effect of HBZ or p30 with PRMT5 on LTR-1 luciferase activity. HEK293T cells were transfected with LTR-1-luc, TK-renilla control, flag-PRMT5 expression vector, and an S-tag-HBZ or p30 expression vector ( Figure 7C,D). Neither HBZ nor p30 activated or repressed LTR-1-luciferase activity in the absence of Tax, as expected [17][18][19][20][21][22]41,42]. Also, PRMT5 had no effect in the presence or absence of HBZ or p30 on LTR-1-luciferase activity. To determine if PRMT5 associated with the viral LTR promoters in vivo, we performed ChIP assays using pA-18G-BHK-21 cells. The Syrian Hamster kidney cell line was stably transfected with a plasmid vector containing the HTLV-1-LTR promoter that drives expression of lacZ [46]. pA-18G-BHK-21 cells were transfected with ACHneo proviral DNA and flag-PRMT5 expression vector ( Figure 7E). PRMT5 was associated with the viral LTR, but not the downstream LacZ-coding region (negative control).

Discussion
HTLV-1 is a tumorigenic retrovirus and the causative infectious agent of ATLL, an extremely aggressive and fatal disease of CD4+ T-cells [2][3][4]. In culture, HTLV-1 can effectively immortalize and eventually transform primary human T-cells. However, in infected individuals, the incidence of disease is only 2%-6% [7] after an extensive clinical latency period. Evidence suggests both genetic and epigenetic changes in the cellular environment that accumulate over time contribute to the development of ATLL [23]. While many aspects of HTLV-1 biology have been revealed, the detailed mechanisms of ATLL development remain poorly defined. Recently, the cellular protein PRMT5 has been shown to play a critical role in EBV-driven B-cell transformation as well as the pathogenesis of many types of hematologic and solid tumors [27][28][29][30][31][32]. We hypothesized that PRMT5 could be important in HTLV-1-mediated cellular transformation and in regulation of viral replication. Given the development of a novel small molecular inhibitor (PRMT5i) [31], identification of PRMT5 as a factor during HTLV-1 transformation will provide valuable insights into improved strategies to treat patients with ATLL.
To determine the importance of PRMT5 in HTLV-1-infected cells, we first examined the expression level of endogenous PRMT5 in a variety of HTLV-1-transformed, ATLL-derived, and HTLV-1-negative T-cells lines ( Figure 1A). PRMT5 proteins levels were upregulated in HTLV-1-positive cells, but also in all transformed T-cell lines, regardless of origin. This is not surprising given that PRMT5 over-expression has recently been identified in lung carcinoma, glioblastoma, B-cell lymphoma, mantle cell lymphoma, and melanoma, to name just a few [28,31,[48][49][50]. It appears that PRMT5 over-expression is a hallmark of most transformed cells, not specifically HTLV-1-transformed cells. Previous work by Pal et al. found decreased PRMT5 mRNA levels in mantle cell lymphoma cell lines despite abundant PRMT5 protein over-expression [28]. In this instance, the increase in PRMT5 protein was not due to an increase in mRNA levels, but instead was due to a decrease in the inhibitory miRNAs miR-92b and miR-96, which allowed for enhanced PRMT5 translation. Conversely, a recent report by Shilo et al. found both PRMT5 protein and mRNA were upregulated in lung tumors [48]. We examined the level of PRMT5 mRNA in a variety of HTLV-1-transformed, ATLL-derived, and HTLV-1-negative T-cell lines and found that the PRMT5 mRNA level was increased in every transformed cell line relative to naïve T-cells ( Figure 1B). However, the increase in PRMT5 mRNA did not directly correlate with the level of PRMT5 protein expression, which suggested some degree of post-transcriptional regulation. Because these experiments were conducted in cell lines grown in vitro, we also examined the level of PRMT5 protein and RNA in total PBMCs isolated from ATLL patients ( Figure 1C,D). Both PRMT5 protein and RNA were upregulated in a majority of ATLL patient samples. The increased level of PRMT5 RNA and protein expression in patient PBMCs was not as prominent as what was found in transformed cell lines, likely due to the use of total PBMCs, which contain a mixture of normal and leukemic cells.
HTLV-1 infection of CD4+ T-cells does not always lead to transformation. A delicate balance must be achieved between viral gene expression and certain genetic and epigenetic events to result in transformation. Using a long-term immortalization co-culture assay, we found both PRMT5 protein and RNA were upregulated throughout the immortalization process ( Figure 2). It is important to note the producer cells were lethally irradiated, and although there was some residual p19 Gag detected in the supernatant, the producer cells were dead by week 1. Since only a portion of the total co-culture assay was tested, the levels of both protein and RNA fluctuated from week to week; however, the overall trend showed that PRMT5 was upregulated.
Regulation of viral gene expression early after infection is highly relevant for successful transformation; for example, too much Tax expression can cause a phenomenon known as Tax-induced senescence (TIS) [51]. Using shRNA vectors directed against PRMT5, we found that knockdown of PRMT5 enhanced HTLV-1 viral gene expression and decreased cellular proliferation in HTLV-1-infected cell lines (Figures 3 and 5B). Given the importance of PRMT5 in cellular proliferation, long-term stable cell lines were difficult to create. Thus, we transduced cells and selected with drug for less than two weeks, which provided the added benefit of less antigenic drift within the cell population over time. Similar results were obtained using a novel, small molecule inhibitor of PRMT5 ( Figure 4A-F). Because Tax expression is lost in a majority of ATLL-transformed cells and only HBZ is expressed in every cell, we included the Tax-negative ATLL transformed cell lines, TL-Om1 and ATL-ED, in our studies. PRMT5 knockdown and inhibition enhanced HBZ expression in ATLL transformed cell lines. Of interest, PRMT5i did not affect HIV-1 gene expression, which suggested that PRMT5 was not a global repressor of all retrovirus gene expression ( Figure 4G). Because Tax and HBZ are driven from separate viral promoters (5' LTR and 3' LTR opposite strand, respectively), this finding would suggest that PRMT5 is a global repressor of HTLV-1 transcription. In support of this hypothesis, we found PRMT5 associated with the viral LTR using ChIP analysis ( Figure 7E).
Using reporter gene assays, we found PRMT5 inhibited HTLV-1 gene transcription, but not Tax protein specifically (Figures 6 and 7A,B). We also found LTR promoter activation was unaffected by PRMT5 (with or without viral accessory proteins HBZ or p30) in the absence of the proviral genome ( Figure 7B-D). This result was not surprising since one of the functions of HBZ is to repress Tax-mediated transcriptional activation of the viral LTR and the function of p30 is to retain unspliced tax/rex mRNA in the nucleus. Taken together, these results suggested that other viral proteins were required for the repressive effects of PRMT5, and/or PRMT5 affected a cellular transcription factor responsible for activating viral transcription. Although not required for the repressive effects of p30 on viral gene expression, we did find PRMT5 and p30 had additive repressive effects on viral transcription, which adds yet another level of regulation to HTLV-1 gene expression. The roles of additional PRMT5 interacting partners, such as MEP50, in PRMT5-mediated HTLV-1 gene regulation are also a possibility to explore in the future. MEP50 is a WD-40 repeat protein and a common PRMT5 cofactor, likely present in most PRMT5-containing complexes in vivo [52]. Phosphorylation of MEP50 by Cdk4 alters the activity and targeting of the PRMT5 protein in cells [53].
Importantly, we found PRMT5i treatment or shRNA-mediated knockdown of PRMT5 in HTLV-1-positive cell lines caused a decrease in cell proliferation compared to HTLV-1-negative cell lines ( Figure 5A,B). Furthermore, PRMT5i was selectively toxic to HTLV-1-positive cell lines ( Figure 5C). These results suggested that HTLV-1-positive cells rely strongly on PRMT5 for cellular growth and survival. Treatment with PRMT5i induced cellular apoptosis to some degree in all cell lines ( Figure 5D). Interestingly, HTLV-1-transformed cell lines underwent noticeably more apoptosis than either the HTLV-1-negative or the ATL-derived cell lines. Previous reports have found that aberrant expression of Tax protein can lead to TIS in cells [51]. We did observe a slight increase in cellular senescence in response to PRMT5i in the HTLV-1-positive cell lines tested, including Tax-expressing HTLV-1-transformed cells and Tax-negative ATL-derived cells ( Figure 5E-H). We would predict an increase in cellular senescence in the HTLV-1-transformed cell lines, as they are the only Tax-expressing lines. However, these cell lines also express HBZ, which has been reported to repress TIS. Another possibility is the level of Tax expression induced in response to PRMT5i in our cell lines was not substantial enough to elicit a measurable increase in cellular senescence. In summary, our study highlighted the significance of PRMT5 in HTLV-1-mediated cellular transformation and its importance as a target for the newly developed PRMT5i, presenting a viable strategy for treatment of ATLL.