Feedback Loop Regulation between Pim Kinases and Tax Keeps Human T-Cell Leukemia Virus Type 1 Viral Replication in Check

Abstract

The Pim family of serine/threonine kinases promote tumorigenesis by enhancing cell survival and inhibiting apoptosis. Three isoforms exist, Pim-1, -2, and -3, that are highly expressed in hematological cancers, including Pim-1 in adult T-cell leukemia (ATL). Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of ATL, a dismal lymphoproliferative disease known as adult T-cell leukemia. The HTLV-1 virally encoded oncogene Tax promotes CD4⁺ T-cell transformation through disruption of DNA repair pathways and activation of survival and cellular proliferation pathways. In this study, we found Tax increases the expression of Pim-1 and Pim-3, while decreasing Pim-2 expression. Furthermore, we discovered that Pim-1, -2, and -3 bind Tax protein to reduce its expression thereby creating a feedback regulatory loop between these two oncogenes. The loss of Tax expression triggered by Pim kinases led to loss in Tax-mediated transactivation of the HTLV-1 long terminal repeat (LTR) and reductions in HTLV-1 virus replication. Because Tax is also the immunodominant cytotoxic T cell lymphocytes (CTL) target, our data suggest that Pim kinases may play an important role in immune escape of HTLV-1-infected cells. IMPORTANCE The Pim family of protein kinases have established pro-oncogenic functions. They are often upregulated in cancer; especially leukemias and lymphomas. In addition, a role for Pim kinases in control of virus expression and viral latency is important for Kaposi sarcoma-associated herpesvirus (KSHV) and human immunodeficiency virus type 1 (HIV-1). Our data demonstrate that HTLV-1 encodes viral genes that promote and maintain Pim kinase activation, which in turn may stimulate T-cell transformation and maintain ATL leukemic cell growth. HTLV-1 Tax increases expression of Pim-1 and Pim-3, while decreasing expression of Pim-2. In ATL cells, Pim expression is maintained through extended protein half-life and heat shock protection. In addition, we found that Pim kinases have a new role during HTLV-1 infection. Pim-1, -2, and -3 can subvert Tax expression and HTLV-1 virus production. This may lead to partial suppression of the host immunogenic responses to Tax and favor immune escape of HTLV-1-infected cells. Therefore, Pim kinases have not only pro-oncogenic roles but also favor persistence of the virus-infected cell.

Keywords: ATL; HBZ; Pim kinase; STAT signaling; Tax; human T-cell leukemia virus; leukemia; replication; transformation; virus.

FIG 1

ATL-derived cell lines express high levels of Pim kinases. (A) Real-time PCR expression of Pim-1, Pim-2, and Pim-3 from whole cell extracts of ATL-derived (Gray graphs: Tl-Om1, ED-40515(-), MT1, ATLT, ATL25, ATL43T, ATL55T, KK1, LM-Y1, and KOB) and HTLV-1 (Black graphs: MT2, MT4, C91PL, 1186.94, C8166, 1185, LAF, MUO4, and SP) cell lines. Resting PBMCs (RPBMC) and HL-60 were used as controls. Gene expression was normalized to GAPDH expression and performed at least twice. (B) Protein expression data for Pim kinases in ATL-derived and HTLV-1-derived cell lines. Actin served as a loading control. A nonspecific band was detected for Pim-1. Fisher EZRun protein ladder is marked, when needed. (C) Tax expression in ATL-derived (left blot) and HTLV-1 (right blot) cell lines. Actin served as loading control. (D) Correlation between Tax/Rex gene expression and Pim kinases in ATL-derived and HTLV-1 cell lines. Gene expression was normalized to GAPDH expression; and correlations between genes were determined using Pearson’s correlation coefficient (r).

FIG 2

Tax increases Pim-1 and Pim-3 kinase expression, while inhibiting Pim-2. (A) 293T cells were transfected for 48 h with Tax or HBZ expression vectors and then probed for Pim kinases expression. Actin served as a loading control. (B) Comparison of Tax expression between 293T-Tax expressing cells and Tax-positive, HTLV-1 cell lines, MT4 and C91PL. Actin served as a loading control. (C) Western blot analysis of whole cell lysate of Jurkat and K562 cells for Pim expression. Actin served as a loading control. (D) Jurkat TET-Tax lines were induced with or without doxycycline for 6 days. Representation of at least two independent experiments is shown. Gene expression was normalized to GAPDH expression and performed at least twice. (E) Western blots for Pim kinase expression following induction of Jurkat TET-Tax lines. Cells were induced with or without doxycycline for 6 days. Actin served as a control. (F) Knock-down of Tax expression in ATLT cells. Cells were transduced with a Tax shRNA and 72 h later, cells were analyzed for Tax and Pim kinase expression. Experiments were performed at least twice. Gene expression was normalized to GAPDH.

FIG 3

Tax transduction of normal PBMCs induces Pim kinases early after infection. (A–B) Four Tax transduced cultures of PBMCs and two cycling PBMCs (cPBMCs) were compared for Pim kinase expression (A) or Tax expression (B). Gene expression was normalized to GAPDH expression and performed at least twice. For Tax expression, Tax transduced cultures were compared with the MT4 cell line. (C–D) Tax transduced PBMCs express Pim kinases in as little as 4- or 7-weeks post-transduction. Two separate, healthy donor PBMCs were transduced with Tax and grown for 4 or 7 weeks. Gene expression of Pim kinases (C) and Tax (D) were examined at both time points and compared with non-transduced PBMCs at 4 weeks. Gene expression was normalized to GAPDH.

FIG 4

Pim kinases inhibit Tax expression. (A) 293T cells were co-transfected with pcTax and pCDNA-Pim kinases, -1, -2, or -3 for 48 h and probed for Tax or Pim kinase expression. Actin served as an internal control. (B) 293T cells were co-transfected with HBZ-HA and pCDNA-Pim kinases, -1, -2, or -3 for 48 h and probed for HA (HBZ) or Pim kinase expression. Actin served as an internal control. (C–D) 293T cells were transfected for 72 h with three individual shRNA to Pim-1 to determine the level of individual Pim-1 shRNA. Pim kinase gene expression was normalized to GAPDH expression from at least two independent experiments. Western blots for Pim-1 following Pim-1 knock-down are shown. A nonspecific band for Pim-1 is present (D). (E–F) 293T cells were transfected with pcTax and two individual shRNA to Pim-1. Pim kinase gene expression was normalized to GAPDH expression from at least two independent experiment (D). Elevation of Tax expression following knock-down of Pim-1 was also seen at the protein level (F). Actin served as a loading control.

FIG 5

Pim kinases bind Tax. (A) 293T cells were co-transfected with pcTax and PMH-Pim kinases for 48 h. Lysates were subject to co-immunoprecipitation with HA antibody and probed with anti-Tax and anti-HA antibodies. Whole cell lysates were probed for Tax and HA-Pim expression to verify expression. Actin demonstrates the amount of protein in each lysate. (B) 293T cells were transfected with pcTax and HA-tagged Pim-1 expression plasmids for 48 h. Cells were treated with or without proteosome inhibitor, MG132 (10 μM) for the last 8 h. (C) 293T cells were transfected with pcTax and HA-tagged Pim-1 expression plasmids for 48 h. Cells were treated with or without proteosome inhibitors, MG132 (10 μM) and lactacystin (10 μM) for the last 8 h. (D–F) 293T cells were transfected with pcTax and HA-tagged Pim-1, -2, or-3 expression plasmids for 48 h. Cells were treated with or without proteosome inhibitor, MG132 (10 μM), or lysosomal inhibitors, chloroquine (100 μM) or leupeptin (100 μM) for the last 8 h. For (B–F), whole cell lysates were probed with Tax and anti-HA (Pim) antibodies. Actin served as a loading control.

FIG 6

(A–F) 293T cells were transfected for 48 h with pcTax and HA-Pim kinases. (A) Cell lysates were fractionated into nuclear and cytoplasmic lysates and probed with anti-Tax, anti-HA, and anti-Cyclin B1 and α-Tubulin, control antibodies. (B) Cell lysates were lysed directly in urea buffer and whole cell lysates were probed with anti-Tax and anti-HA antibodies. Actin served as a loading control. (C–D) Cell lysates were fractionated into soluble and insoluble nuclear and cytoplasmic lysates and probed with anti-Tax, anti-HA, anti-Cyclin B1, and anti-α-Tubulin antibodies. In (D) cell lysates were treated for 7 h with MG132 (10 μM) or leupeptin (100 μM). Long and short exposures of tax are presented. (E–F) 293T cells were transfected for 48 h with pcTax, pcDNA-Pim-1, Pim-2, or Pim-3, and/or HA-tagged K48 (E) or K63 (F). All cells were treated with MG132 (10 μM) for 6 h, prior to collection. Cell lysates were immunoprecipitated overnight with anti-Tax antibody. Due to Pim repression of Tax, prior to Western blot analysis, additional lysate was run for extracts containing Pim kinases. Western blots were performed on immunoprecipitates and lysates with anti-Tax, anti-HA (Ub), and Pim kinase antibodies. Actin served as a loading control. (G) Quantification of K48 and K63 immunoprecipitation. Bands were quantified from several HA-immunoprecipitates and normalized to lysates.

FIG 7

Pim kinases inhibit Tax transactivation activity. (A) 293T cells were transfected with the HTLV-1 LTR luciferase construct along with pcTax and increasing amounts of pcDNA-Pim-1, -2, and -3 kinases. Fold change was compared with 293T-pCDNA transfected controls. Luciferase activity was normalized to RL-TK renilla expression. Results represent the average of at least two experiments. (B) Verified expression of Pim kinases, -1, -2, and -3 in 293T transfected cells. Actin served as a loading control. (C) 293T cells were transfected with pcTax along with individual Pim kinases. 48-h post-transfection, gene expression was performed for Tax expression. Experiments were performed at least twice, and GAPDH served as a control. (D) 293T cells were transfected with HTLV-1 LTR luciferase construct along with pcTax and/or Pim-1 shRNA A and B. Fold change was compared with 293T-pCDNA transfected controls. Luciferase activity was normalized to RL-TK renilla expression. Results represent the average of at least two results. Western blots confirming Tax over-expression are provided. (E) 293T cells were transfected for 48 h with Tax. The pan-Pim kinase inhibitor, AZD1208 (10 μM), was added during the last 30 h. Actin served as a loading control.

FIG 8

Inhibition of Pim kinases leads to elevation of Tax expression in HTLV-1 lines. (A–C) Treatment of MT1, MT2, ATL25 (A), and ATLT (B–C) with 5, 10, or 20 μM AZD1208, or 10 μM M-100 or PimIV inhibitor. Treatments were performed for 48 h or 72 h. Representation of at least two independent experiments is shown. Gene expression was normalized to GAPDH expression and performed at least twice. (D–E) Jurkat TET-Tax cells were induced with doxycycline for 72 h. 10 μM AZD1208 was added for the final 18 h for gene expression (D) or 72 h for protein expression (E). Actin served as a loading control. (F) ATLT and MT4 cells were treated with 10 μM AZD1208 and/or 10 μM MG132. Total cell lysate was probed for Tax and actin served as loading control. (G) ATLT and MT4 were treated with 10 μM AZD1208. Cytoplasmic, soluble nuclear, and insoluble nuclear fractions were probed with Tax antibody. Purity of fractions was confirmed with Cyclin B1 and α-Tubulin antibodies.

FIG 9

Pim kinases inhibit HTLV-1 replication and virus production. (A–B) 293T cells were transfected with the HTLV-1 LTR luciferase construct along with the HTLV-1 molecular clones PBST (A) or ACH (B), along with increasing amounts of Pim-1, -2, and -3 kinases. Fold change was compared with 293T-pCDNA transfected controls. Luciferase activity was normalized to RL-TK renilla expression. Results represent the average of at least two experiments. (C–D) 293T cells were transfected with or without PBST along with Pim-1, -2, or -3. cDNA was analyzed by (C) PCR or (D) real-time PCR for HTLV-1 and Pim gene expression. Fold change is compared with PBST transfected cells, with GAPDH serving as an internal control. Results represent the average of two experiments. (E–F) 293T cells were transfected with PBST with or without Pim-1, -2, or -3. Whole cell lysates were probed with anti-HTLV p24 antibody (E). Actin expression served as a loading control. Supernatant was analyzed for HTLV p19 antigen by ELISA (F). Results were performed at least twice. (G–H) 293T cells were transfected with PBST with or without Pim-1 shRNA A or B. In (G) HTLV-1 luciferase plasmid was transfected, and Luciferase activity was normalized to RL-TK renilla expression. Results were performed at least twice. For (H) whole cell lysates were probed with anti-HTLV p24 antibody and demonstrate the precursor (Pr55) and p24. Actin expression served as a loading control.

FIG 10

Pim kinase expression is stabilized in ATL cells through Hsp90. (A) Nuclear and cytoplasmic extracts of ATL lines, ED-40515(-), MT1, ATL25, and KOB. Cyclin B1 served as control for subcellular fractionation. (B–D) Analysis of Pim kinase protein stabilization following treatments with (B) cycloheximide (100 μg/mL CHX for 0, 2, 4, 6, or 8 h) in ED-40515(-) and MT1 ATL-derived cells, with (C) proteases (10 μM MG132 and 1 μM Lactacystin for 6 h) in ED-40515(-) and MT1 ATL-derived cells, and with (D) protease (10 μM MG132 for 6 h) in ATLT, C8166, C91PL, KK1, and ATL43T cells. For (B–D) actin expression served as a loading control. For (B) Cyclin B1 degradation confirmed that CHX treatment was working. Half-life of each protein is labeled next to the gel. (E–F) Pim kinases are stabilized by heat shock proteins in ATL-derived lines. ED-40515(-), MT1, KK1, and ATL43T cells were treated with the heat shock inhibitor, geldanamycin (1, 2, 4, or 6 μg/mL GA for 24 h). Whole cell lysates were analyzed for Pim protein expression (E). Actin served as a loading control. Proliferation, after GA treatment, was measured with XTT assays (F). Results were carried out in duplicate and calculated as a fold change compared with cells treated without GA. (G) Illustration of feedback loops between Tax/HBZ proteins and Pim kinases. Tax can drive the expression of Pim-1 and Pim-3, possibly contributing to the cellular transformation of T-cells. However, Pim-1, -2, and -3 and HBZ can also inhibit Tax expression, leading to loss of HTLV-1 transactivation and viral replication. This may allow for immune escape and viral latency. Pim kinase levels are stabilized in ATL cells through interaction with heat shock protein, Hsp90, which protects Pim kinases from degradation.