Distinct gene expression signatures induced by viral transactivators of different HTLV 1 subgroups that confer a different risk of HAM/TSP

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1 Retrovirology RESEARCH Open Access Distinct gene expression signtures induced by virl trnsctivtors of different HTLV 1 subgroups tht confer different risk of HAM/TSP Tdsuke Nito 1, Jun ichirou Ysung 2, Yuichi Mitobe 2,8, Kzums Shiri 3, Hiroe Sejim 1, Hiroshi Ushirogw 1, Yuetsu Tnk 4, Ttsufumi Nkmur 5, Kousuke Hnd 3, Mshiro Fujii 6, Mso Mtsuok 2,7 nd Mineki Sito 1* Abstrct Bckground: Among humn T cell leukemi virus type 1 (HTLV-1)-infected individuls, there is n ssocition between HTLV-1 tx subgroups (subgroup-a or subgroup-b) nd the risk of HAM/TSP in the Jpnese popultion. To investigte the role of HTLV-1 subgroups in virl pthogenesis, we studied the functionl difference in the subgroupspecific virl trnscriptionl regultors Tx nd HBZ using microrry nlysis, reporter gene ssys, nd evlution of virl-host protein protein interction. Results: (1) Trnscriptionl chnges in Jurkt Tet-On humn T-cells tht express ech subgroup of Tx or HBZ protein under the control of n inducible promoter reveled different trget gene profiles; (2) the number of differentilly regulted genes induced by HBZ ws 2 3 times higher thn tht induced by Tx; (3) Tx nd HBZ induced the expression of different clsses of non-coding RNAs (ncrnas); (4) the chemokine CXCL10, which hs been proposed s prognostic biomrker for HAM/TSP, ws more efficiently induced by subgroup-a Tx (Tx-A) thn subgroup-b Tx (Tx-B), in vitro s well s in unmnipulted (ex vivo) PBMCs obtined from HAM/TSP ptients; (5) reporter gene ssys indicted tht lthough trnsient Tx expression in n HTLV-1-negtive humn T-cell line ctivted the CXCL10 gene promoter through the NF-κB pthwy, there ws no difference in the bility of ech subgroup of Tx to ctivte the CXCL10 promoter; however, (6) chromtin immunoprecipittion ssys showed tht the ternry complex contining Tx-A is more efficiently recruited onto the promoter region of CXCL10, which contins two NF-κB binding sites, thn tht contining Tx-B. Conclusions: Our results indicte tht different HTLV-1 subgroups re chrcterized by different ptterns of host gene expression. Differentil expression of pthogenesis-relted genes by subgroup-specific Tx or HBZ my be ssocited with the onset of HAM/TSP. Keywords: HTLV-1, Virus subgroup, HAM/TSP, Tx, HBZ, Microrry *Correspondence: mineki@med.kwski m.c.jp 1 Deprtment of Microbiology, Kwski Medicl School, 577 Mtsushim, Kurshiki, Okym , Jpn Full list of uthor informtion is vilble t the end of the rticle The Author(s) This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( iveco mmons.org/licen ses/by/4.0/), which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( iveco mmons.org/ publi cdom in/zero/1.0/) pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Pge 2 of 20 Introduction Humn T-cell leukemi virus type 1 (HTLV-1) ws the first humn oncogenic retrovirus to be identified [1] nd ssocited with distinct humn diseses such s dult T-cell leukemi (ATL) [2, 3] nd HTLV-1-ssocited myelopthy/tropicl spstic prpresis (HAM/TSP) [4, 5]. HTLV-1 cn be divided into 7 subtypes (subtypes 1 1g), including 5 Africn subtypes (subtypes 1b, 1d, 1e, 1f, nd 1g), Melnesin/Austrlin subtype (subtype 1c), nd cosmopolitn subtype (subtype 1) bsed on phylogenetic nlysis of its long terminl repet (LTR) region. The cosmopolitn subtype hs spred worldwide nd is further divided into 5 subgroups (subgroup A E of subtype 1) [6 8]. In the Jpnese popultion, nucleotide sequence vrition in the virl trnsctivtor tx lso determines the HTLV-1 subgroups nmely, tx subgroup-a nd tx subgroup-b correspond to LTRbsed cosmopolitn subtype 1 subgroup A nd cosmopolitn subtype 1 subgroup B, respectively [9]. We therefore refer to tx subgroup-a nd tx subgroup-b s subgroup-a nd subgroup-b herefter. It is well estblished tht both the Tx nd HBZ proteins of HTLV-1 trnsctivte virl nd cellulr genes nd ply key role in HTLV-1 repliction nd pthogenesis [10 16]. A difference of four nucleotides exists in tx nd HBZ coding regions (i.e., nucleotides 7897, 7959, 8208 nd 8344) between subgroup-a Tx (Tx-A) nd subgroup-b Tx (Tx-B), which result in two nd one mino cid coding chnges, respectively, in Tx nd HBZ [9]. The most importnt observtion concerning these virus subgroups is tht the incidence of HAM/TSP in symptomtic helthy crriers (HCs) infected with subgroup-a is 2.5 times higher thn tht in individuls infected with subgroup-b in southern Jpn, where both subgroups co-exist [9]. Recently, we reported tht this is lso the cse for inhbitnts of Okinw Prefecture, Jpn, which consists of 160 islnds nd is locted in the subtropicl southernmost point of Jpn [17]. We hve lso reported tht lthough different HTLV-1 subgroups re chrcterized by different ptterns of HBZ nd FoxP3 gene expression in HAM/TSP ptients vi independent mechnisms of direct trnscriptionl regultion, these differences do not significntly ffect the clinicl nd lbortory chrcteristics of HAM/TSP ptients [18]. Thus, the mechnism by which HTLV-1 subgroups differ in the risk for HAM/TSP is still lrgely unknown. The rtionle of this study is tht microrry-bsed study of subgroup-specific Tx- or HBZ-induced chnges of cellulr genes would revel the downstrem trgets nd effectors of these virl trnscriptionl fctors nd identify which trgets differ between the virl strins. The results will cst light on the cuses of HAM/TSP nd identify ttrctive trgets for novel therpeutics. Methods Ptients nd preprtion of clinicl smples This study ws pproved by the Reserch Ethics Committee of Kwski Medicl School (pprovl number: ). Written informed consent ws obtined from ll individuls. Clinicl smples from 37 ptients with HAM/TSP (19 subgroup-a nd 18 subgroup-b infected ptients), 20 HCs, nd 20 HTLV-1-uninfected norml control subjects (NCs) were nlyzed. The dignosis of HAM/TSP ws mde ccording to the World Helth Orgniztion dignostic criteri [19]. The detil informtion of the ptients chrcteristics including provirl lod (PVL) ws presented in Tble 1. Fresh peripherl blood mononucler cells (PBMCs) were isolted using Histopque-1077 (Sigm, St. Louis, MO, USA) density grdient centrifugtion, wshed twice in RPMI medium, nd stored in liquid nitrogen s stocked lymphocytes until use. Cells Twelve HTLV-1-infected humn T-cell lines (MT2, MT4, C5MJ, MT1, ATL43Tb, ATL55T, ED, TLOm1, ILT-M1, HCT1, HCT4, nd HCT5) nd three HTLV-1-uninfected T-cell lines (Jurkt, CEM, nd Molt4) were used in this study. MT2, MT4, nd C5MJ re chroniclly HTLV- 1-infected cell lines derived from cord blood mononucler cells exposed to HTLV-1 from ptients with ATL, i.e., HTLV-1-trnsformed T-cell lines. MT1, ATL43Tb, ATL55T, ED, nd TLOm1 re HTLV-1-infected cell lines derived from ptients with ATL. Among these ATL cell lines, only ATL55T is IL-2-dependent. ILT-M1, HCT1, HCT4, nd HCT5 re IL-2-dependent HTLV-1-infected T-cell lines derived from ptients with HAM/TSP. The Tx-inducible JPX-9 cell line ws derived from the Jurkt HTLV-1-negtive humn T-cell leukemi cell line nd expresses biologiclly ctive Tx protein under the Tble 1 Clinicl profiles of HTLV-1-ssocited myelopthy/ tropicl spstic prpresis (HAM/TSP) ptients Tx A (n = 19) Tx B (n = 18) p vlue Age t onset 47.0 ± ± Sex (Mle: Femle) 9: 19 14: Serum nti-htlv-1 ntibody ± ± titer HTLV-1 provirus lod in ± ± PBMCs b OMDS c 5.9 ± ± Durtion of illness 15.1 ± ± The results represent the men ± SD Anti-HTLV-1 ntibodies were titrted using the prticle gglutintion method b HTLV-1 Tx copy number per 10 4 PBMCs c OMDS: Osme Motor Disbility Score (see Additionl file 1: Tble S1)

3 Pge 3 of 20 control of the metllothionein promoter [20]. These cells were cultured in RPMI 1640 medium supplemented with 10% het-inctivted fetl clf serum (FCS), 50 U/ml penicillin, nd 50 µg/ml streptomycin (Wko Pure Chemicl, Osk, Jpn) t 37 C nd 5% CO 2. For IL-2-dependent cell lines, 10 U/ml (for ATL55T, HCT1, HCT4, nd HCT5) or 30 U/ml (for ILT-M1) of recombinnt humn IL-2 (Wko) were dded to the culture. Jurkt Tet-On 3G (Tkr Bio USA, Mountin View, CA) is Jurkt-derived T-cell line mde for use in the Tet-On 3G Inducible Gene Expression System (Tkr Bio USA). Jurkt Tet-On 3G cells were cultured in RPMI1640 supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin (Wko), nd 200 µg/ml G418 (SIGMA-ALDRICH Jpn, Tokyo, Jpn) t 37 C in 5% CO 2. Humn embryonic kidney 293T cells were grown in Dulbecco s modified Egle s medium (DMEM) supplemented with 4 mm glutmine, 10% FCS, 100 units/ml penicillin, nd 100 µg/ml streptomycin t 37 C nd 5% CO 2. Plsmid construction All primers used for the plsmid construction re listed in Additionl file 2: Tble S2. The following ptre3gbsed plsmids were constructed for inducible expression in the presence of doxycycline (Dox) (Tkr Bio USA) in Jurkt Tet-On 3G cells. To construct ptre3g- Tx-A or ptre3g-tx-b, DNA frgments corresponding to the Tx coding sequence were mplified vi PCR, using Tx-FOR nd Tx-REV s primers nd pcg-tx- A or pcg-tx-b [18] s templte. PCR products were digested with SlI nd BglII, then cloned into SlI- nd BmHI-digested ptre3g-ires (Tkr Bio USA). To construct ptre3g-hbz-a or ptre3g-hbz-b, DNA frgments corresponding to the HBZ coding sequence were mplified vi PCR, using HBZ-FOR nd HBZ-REV s primers nd pcmv-ha-hbz-a or pcmv-ha-hbz- B [18] s the templte. PCR products were digested with SlI nd BglII, then cloned into SlI- nd BglII-digested ptre3g-ires. The following pcaggs-p7- nd pgl3-bsed plsmids were constructed for the immunoprecipittion ssy nd luciferse ssy. To construct pcaggs-p7-tx-a nd pcaggs-p7-tx-b, DNA frgments corresponding to the Tx coding sequence were mplified by PCR using Tx-FOR nd Tx-Stop-REV s primers nd pcg- Tx-A or pcg-tx-b [18] s templte. PCR products were phosphorylted with T4 polynucleotide kinse nd digested with SlI nd cloned into SlI- nd EcoRVdigested pcaggs-p7. To construct pcaggs-p7-tx-a-flag, pcaggs- P7-Tx-B-FLAG, nd pcaggs-p7-tx-1 ( )- FLAG, DNA frgments corresponding to the Tx coding sequence were mplified vi 1st-PCR using Tx-FOR nd Tx-FLAG-REV s primers nd pcaggs-p7-tx- A, pcaggs-p7-tx-b, or CSII-EF-Tx-1( )- FLAG [21] s the templtes. To dd tndem FLAG-tg sequence to the C-terminus of Tx proteins, DNA frgments were mplified vi 2nd-PCR using Tx-FOR nd FLAG-Tndem-REV s primers nd the 1st-PCR products s templtes. Next, to fuse the NotI-recognized DNA sequence t the 3 site of the FLAG-tg, DNA frgments were mplified vi 3rd-PCR using Tx-FOR nd FLAG-Tndem-NotI-REV s primers nd the 2nd- PCR products s templtes. The 3rd-PCR products were digested with SlI nd NotI, nd cloned into SlI- nd NotI-digested pcaggs-p7. To construct pcaggs-p7- Tx-2B-FLAG, DNA frgments were mplified vi PCR using Tx-2-FOR nd Tx-2-FLAG-Tndem-REV s primers nd CSII-EF-Tx-2-FLAG [21] s the templte. To dd tndem FLAG-tg sequence to the C-terminus of Tx proteins, DNA frgments were mplified vi 2nd- PCR using Tx-2-FOR nd FLAG-Tndem-REV s primers nd the 1st-PCR products s templtes. Next, to fuse the NotI-recognized DNA sequence to the 3 site of the FLAG-tg, DNA frgments were mplified vi 3rd-PCR using Tx-2-FOR nd FLAG-Tndem-NotI-REV s primers nd the 2nd-PCR products s templtes. The 3rd-PCR products were digested with SlI nd NotI, nd cloned into SlI- nd NotI-digested pcaggs-p7. To mplify the humn C-X-C motif chemokine 10 (CXCL10) promoter region, hrboring sequence spnning nucleotide positions 875 to + 97 (where the trnscription strt site is set to be + 1) or 279 to + 97 [22], PCR ws crried out using CXCL FOR or CXCL FOR nd CXCL10-REV s primer sets nd genomic DNA derived from Jurkt cells s templtes. PCR products were digested with KpnI nd XhoI nd cloned into KpnI- nd XhoI-digested pgl3-bsic (Promeg Corportion, Mdison, WI). The resultnt plsmid ws designted pgl3-cxcl10-long-luc or pgl3-cxcl10-short-luc. Muttions in the NF-κB1 (5 - GGG ACT TCCC-3 ), NF-κB2 (5 -GGG AAA TTCC-3 ),

4 Pge 4 of 20 nd AP-1 (5 -TAA GTC A-3 ) binding sites in the CXCL10 promoter were generted using overlpping-pcr using the primers listed in Additionl file 2: Tble S2. To construct pgl3-cxcl10 (κb1-mut)-luc (κb1 mutnt sequence: 5 -GTG ACT TCAC-3 ), two DNA frgments corresponding to the CXCL10 promoter sequence were mplified vi PCR using CXCL FOR nd κb1- mut-rev or CXCL10-REV nd κb1-mut-for s primers nd pgl3-cxcl10-short-luc s the PCR templte. Fulllength CXCL10 (κb1-mut) DNA frgment ws mplified vi PCR using CXCL FOR nd CXCL10-REV s primers. PCR products were digested with KpnI nd XhoI nd cloned into the KpnI- nd XhoI-digested pgl3-bsic. Likewise, pgl3-cxcl10 (κb2-mut)-luc (κb2 mutnt sequence: 5 -GTG AAA TTAC-3 ), pgl3- CXCL10 (κb1 + κb2-mut)-luc (muttion of both the κb1 nd κb2), nd pgl3-cxcl10 (AP-1-mut)-Luc (AP-1 mutnt sequence: 5 -TAA GAG A-3 ) were constructed using primers listed in Additionl file 2: Tble S2. The sequences of ll recombinnt plsmids were confirmed using Snger sequencing. Trnsfection by electroportion DNA electroportion ws performed with the Neon Trnsfection System (Invitrogen, Crlsbd, CA). For the electroportion, Jurkt nd Jurkt Tet-On 3G cells were grown in non-treted cell culture dishes nd trnsferred into 15 ml polypropylene tube. Cells were centrifuged t 700 g for 3 min. The pellet ws re-suspended in 10 ml of PBS, nd cells were counted. Cells were pelleted gin nd re-suspended in Buffer R (included with Neon Kits) to finl concentrtion of /ml. 100 µl or 10 µl of cell suspension contining cells or cells, respectively, nd 10 µg or 3 µg of DNA plsmid were trnsferred into the Neon tip (100 µl tip or 10 µl tip). The electroportion ws crried out under pproprite conditions s per the mnufcturer s instructions. After 24 h trnsfection, Tx-A, Tx-B, HBZ-A, or HBZ-B proteins were induced in Jurkt Tet-On 3G cells by dding Dox (finl concentrtion: 3 ng/ml) for 24 h. Uninduced Jurkt Tet-On 3G cells were used s bseline reference. Western blotting Whole-cell lystes were subjected to SDS polycrylmide gel electrophoresis (SDS-PAGE) nd trnsferred to polyvinylidene difluoride (PVDF) membrnes (pore size 0.45 µm, Merck Millipore, MA, USA). PVDF membrnes were blocked with 5% skim milk or Blocking One (Ncli tesque, Kyoto Jpn) in Tris-buffered sline contining 0.1% Tween 20 (TBS-T) nd probed with nti-tx mouse monoclonl (Lt-4), nti-hbz mouse monoclonl (#7-1) [23], nti-hbz rt monoclonl (#4B12), ntiβ-ctin rbbit polyclonl (PM053, MEDICAL & BIOLOGICAL LABORATORIES, Ngoy, Jpn), or nti-α-tubulin rbbit polyclonl (PM054, MEDICAL & BIOLOGICAL LABORATORIES) ntibodies. PVDF membrnes were wshed with TBS-T nd incubted with IRDye 680RD Got nti-mouse IgG, IRDye 800CW got nti-rt IgG, or IRDye 800CW got nti-rbbit IgG (LI-COR Biosciences, Lincoln, NE). After wshing with TBS-T, the proteins were detected using Odyssey CLx Infrred Imging System (LI-COR Biosciences). Microrry nlysis The microrry experiments were performed in triplicte, nd dt re shown s men vlues. RNA processing nd lbeling After 24 h of cultivtion, the trnsfected cells were hrvested nd processed for subsequent nlysis. RNA ws prepred using the RNAesy Mini Kit column purifiction (QIAGEN, Tokyo, Jpn) following the mnufcturer s instructions. RNA ws quntified using NnoVue spectrophotometer (GE Helthcre Jpn, Tokyo, Jpn) nd qulity ws monitored using 1% Agrose/Formldehyde gel electrophoresis. Cynine-3 (Cy3) lbeled crna ws prepred from 0.1 µg RNA using the One-Color Low Input Quick Amp Lbeling Kit (Agilent Technologies, Tokyo, Jpn) ccording to the mnufcturer s instructions, followed by RNAesy Mini Kit column purifiction (QIAGEN). Dye incorportion nd crna yield were ssessed using the NnoDrop spectrophotometer (Thermo Fisher Scientific) nd the Agilent 2100 Bionlyzer (Agilent). crna hybridiztion nd scnning 600 ng of Cy3-lbelled crna (specific ctivity > 6.0 pmol Cy3/µg crna) ws frgmented t 60 C for 30 min in rection volume of 25 µl contining 1 Agilent frgmenttion buffer nd 2 Agilent blocking gent following the mnufcturer s instructions. On completion of the frgmenttion rection, 25 µl of 2 GE Agilent hybridiztion buffer, HI-RPM ws dded to the frgmenttion mixture nd hybridized to Agilent SurePrint G3 Humn GE 8 60 K V2 Microrrys (Ctlog Code: G4851B) nd V3 Microrrys (G4851C) (Agilent) for 17 h t 65 C in rotting Agilent hybridiztion oven. After hybridiztion, microrrys were wshed for 1 min t room temperture with GE Wsh Buffer 1 (Agilent) nd 1 min with 37 C

5 Pge 5 of 20 GE Wsh buffer 2 (Agilent), then dried immeditely by brief centrifugtion. Slides were scnned immeditely fter wshing on the Agilent DNA Microrry Scnner (G2505C) (Agilent) using one color scn setting for 8 60 k rry slides (Scn Are mm, Scn resolution 3 µm, dye chnnel set to Green). The scnned imges were nlyzed using Feture Extrction Softwre (Agilent) t defult prmeters (protocol GE1-1100_Jul11 nd Grid: _D_F_ ) to obtin bckground subtrcted nd sptilly detrended Processed Signl intensities. Fetures flgged in Feture Extrction s Feture Non-uniform outliers were excluded. Dt were nlyzed using GeneSpring GX (Agilent). Microrry dt hve been deposited in the NCBI Gene Expression Omnibus ( (GEO ID: GSE nd GSE121201). To detect differentilly expressed genes, we pplied Significnce Anlysis of Microrrys (SAM) with R pckge smr. In the SAM nlysis, we used two-clss unpired comprison with threshold of q-vlue < 0.05 between control nd Tx-A, control nd Tx-B, control nd HBZ-A, control nd HBZ-B. When gene hs more thn two probes, fold-chnge in the gene is defined to be the verge fold-chnges in multiple probes ssigned to be the gene. Genomic DNA nd RNA extrction nd cdna synthesis Genomic DNA ws extrcted from PBMCs using the QIAmp Blood Kit (Qigen, Tokyo, Jpn). RNA ws extrcted from PBMCs using the RNesy Mini Kit with on-column DNse digestion (Qigen). cdna ws synthesized using the PrimeScript RT Regent Kit (Tkr, Kyoto, Jpn). All rection procedures were performed s suggested by the mnufcturer. Quntifiction of HTLV 1 provirl lod nd nti HTLV 1 ntibody titers To exmine the HTLV-1 provirl lod (PVL), quntittive PCR ws performed using 100 ng of genomic DNA (roughly equivlent to 10 4 cells) from PBMCs s previously reported [24]. The mount of HTLV-1 provirl DNA ws determined using the following formul: copy number of HTLV-1 tx per PBMCs = [(copy number of tx)/(copy number of β ctin/2)] All smples were exmined in triplicte. Antibody titers to HTLV-1 were determined using prticle gglutintion method (Serodi-HTLV-1 ; Fujirebio, Tokyo, Jpn), ccording to the mnufcturer s instructions. Rel time quntittive reverse trnscription PCR nlysis To estimte tx nd HBZ mrna expression levels, reltime quntittive reverse trnscription PCR (rel-time qrt-pcr) ws performed s previously reported [14]. Humn CXCL10 (Applied Biosystems Hs _m1) gene specific ssys were used for CXCL10 quntifictions. The expression levels of these genes were normlized to the expression levels of humn hypoxnthine phosphoribosyltrnsferse 1 (HPRT1) (Humn HPRT1 Endogenous Control ; Applied Biosystems, Foster City, CA). All ssys were performed in triplicte. ELISA Humn CXCL10 levels were determined using ELISA, employing the Quntikine ELISA Humn CXCL10/ IP-10 Immunossy kit (R&D Systems, Minnepolis, MN) ccording to the mnufcturer s instruction. Luciferse ssy To compre CXCL10 promoter ctivity, Jurkt cells ( cells) were trnsfected with 1.5 µg of firefly luciferse reporter plsmid, i.e., pgl3-cxcl10- Long-Luc, pgl3-cxcl10-short-luc, pgl3-cxcl10 (κb1-mut)-luc, pgl3-cxcl10 (κb2-mut)-luc, pgl3- CXCL10 (κb1 + κb2-mut)-luc, or pgl3-cxcl10 (AP- 1-mut)-Luc, 1.5 µg of Tx protein expression plsmid pcaggs-p7-tx-a-flag or pcaggs-p7-tx-b- FLAG), nd 250 ng of Renill reporter plsmid (prl- RSV) per well by electroportion. The reporter construct contining luciferse gene fused to five repets of the NF-κB site of the IL-2R gene [25] ws tested with sme procedure. We lso tested CXCL10 promoter ctivity using 293T cells. The reporter plsmid pgl3-cxcl10- Long-Luc or pgl3-cxcl10-short-luc, 1.5 µg of Tx protein expression plsmid (pcg-tx-a or pcg-tx- B), nd 250 ng of Renill reporter plsmid (prl-rsv) were trnsfected into 293T cells ( cells) per well by using Lipofectmine LTX with PLUS regent (Invitrogen, Crlsbd, CA, USA) ccording to the mnufcturer s instructions. After 24 h trnsfection, cells were hrvested nd lysed, nd reporter ctivities were mesured using the Dul-Luciferse Reporter Assy System (Promeg, Tokyo, Jpn). Ech experiment ws performed in triplicte, nd the dt represent the men ± SD of three independent experiments. Firefly Luciferse vlues were normlized to vlues of the Renill reporter.

6 Pge 6 of 20 Immunoprecipittion ssy Jurkt cells ( cells) were trnsfected with 10 µg of ech indicted plsmid, i.e., pcaggs-p7-empty, pcaggs-p7-tx-a-flag, pcaggs-p7-tx-b-flag, pcaggs-p7-tx-1( )-flag, nd pcaggs-p7- Tx-2B-FLAG, by electroportion nd then cultured for 24 h t 37 C. The hrvested cells were solubilized using lysis buffer (20 mm Tris HCl [ph 7.9], 100 mm NCl, 0.1% Triton X-100). After soniction, homogentes were centrifuged t 14,000 g t 4 C for 5 min, nd the superntnt frction ws used s extrcts for immunoprecipittion. Then, Tx protein ws immunoprecipitted with nti-flag gels. The cell extrcts were incubted with ANTI-FLAG M2 Affinity Gel (SIGMA-ALDRICH Jpn, Tokyo, Jpn) t 4 C for 1 h. After incubtion, the resins were collected vi brief centrifugtion nd wshed two times with the lysis buffer. The resin-bound proteins were eluted by boiling in the SDS-PAGE Smple Loding Buffer (Tkr Bio USA) nd subjected to 10% SDS-PAGE, followed by western blotting using nti-flag M2 (SIGMA- ALDRICH), nti-p105/p50 (#3035: Cell Signling Technology Jpn, Tokyo, Jpn), nti-p100/p52 (#4882: Cell Signling Technology), nti-p65 (#4764: Cell Signling Technology), nti-relb (#4922: Cell Signling Technology), nd nti-c-rel (#4727: Cell Signling Technology) ntibodies. An liquot of the cell lystes removed before immunoprecipittion ws lso chrcterized s n input (Input). The mounts of p100, p52, RelB, p65, p105, p50, nd c-rel proteins in immunoprecipitted smples were quntified by the densitometry scnning of corresponding bnds of the western blot using Imge J softwre. Chromtin immunoprecipittion (ChIP) ssy Jurkt cells ( cells) were co-trnsfected with 9 µg of ech indicted plsmid, i.e., pcaggs-p7-empty, pcaggs-p7-tx-a-flag, nd pcaggs-p7-tx-b- FLAG, nd 1 µg of pef-321t (plsmid encoding SV40 T ntigen for mplifiction of trnsfected pcaggs-p7) by electroportion nd then cultured for 24 h t 37 C. Trnsfected cells were treted with 1% formldehyde t room temperture for 15 min. Fixtion ws quenched by the ddition of glycine t the finl concentrtion of 125 mm, then cells were wshed twice with PBS nd collected by centrifugtion. Cell pellets were resuspended in ChIP lysis buffer (50 mm Tris HCl [ph 7.9], 1% Triton X-100, 0.1% SDS, nd 10 mm EDTA). Cell lystes were sonicted to sher the chromtin DNA to ~ 500 bsepirs in size nd then diluted with four volumes of ChIP dilution buffer (12.5 mm Tris HCl [ph 7.9], mm NCl, nd 1% Triton X-100). Lystes clrified by centrifugtion were incubted with nti-lt-4 ntibody t 4 C overnight. Antibody-protein-DNA complexes were incubted with Slmon Sperm DNA/Protein A Agrose (MILLIPORE, Temecul CA), nd immunoprecipittes collected onto Protein A Agrose were wshed with high slt wsh buffer (20 mm Tris HCl [ph 7.9], 500 mm NCl, 2 mm EDTA, 0.1% SDS, nd 1% Triton X-100), low slt wsh buffer (20 mm Tris HCl[pH 7.9], 100 mm NCl, 2 mm EDTA, 0.1% SDS, nd 1% Triton X-100), LiCl buffer (10 mm Tris HCl[pH 7.9], 250 mm LiCl, 1% NP-40, nd 1 mm EDTA), nd twice with TE (10 mm Tris HCl[pH 7.9] nd 1 mm EDTA) successively. Then, bound proteins were eluted from the grose beds in elution buffer (1% SDS nd 100 mm NHCO 3 ) by incubtion t room temperture for 15 min. Crosslinking ws reversed by incubtion t 65 C overnight. All smples were treted with 45 μg/ml of proteinse K (Ncli tesque, Kyoto, Jpn) t 50 C for 2 h, then 40 µg of glycogen (Ncli tesque) ws dded, followed by extrction with phenol:chloroform:isomyl lcohol nd precipittion with ethnol. DNA frgments were subjected to PCR or quntittive rel-time PCR using EmerldAmp Mster Mix (TKR, Shig, Jpn) or SYBR Premix EX Tq II (TKR, Shig, Jpn) with specific primer sets: 5 -GAA AGT GAA ACC TAA TTC ACT ATT ACC AA-3 nd 5 -ACT TAG CAA AAC CTG CTG GCT GTT-3 for genomic DNA frgment contining both NF-κB nd AP-1 binding sites. Three independent experiments were performed, nd the dt represent the men ± SD. Sttisticl nlysis To test for significnt differences mong four different groups of subjects (HAM/TSP ptients with subgroup- A, HAM/TSP ptients with subgroup-b, HCs nd NCs), the dt were sttisticlly nlyzed using one-wy nlysis of vrince (ANOVA). Inter-group comprisons were done by Scheffe s post hoc multiple comprisons test. The Mnn Whitney U test ws used to compre dt between two groups (i.e., difference between Tx-A nd Tx-B with respect to trnscriptionl ctivity for both the short nd long CXCL10 promoters, see Fig. 6b). Correltions between vribles were exmined using Spermn rnk correltion nlyses. The results shown re the men ± SD where pplicble. Results were considered sttisticlly significnt t p vlues < 0.05.

7 Pge 7 of 20 Tx-A Tx-B HBZ-A HBZ-B b Dox HBZ Tx β-ctin 13 P S empty HBZ-A HBZ-B N Dox induc on: 24 hr Fig. 1 Expression of ech subgroup Tx or HBZ protein under trnscriptionl control of doxycycline (Dox)-inducible promoter. Schemtic representtion of the subgroup-specific Tx nd HBZ proteins. Proteins re shown s grey brs, nd differences in mino cid sequences between subgroup-specific Tx, i.e., Tx-A nd Tx-B, or HBZ, i.e., HBZ-A nd HBZ-B, proteins re indicted by single letter mino cid bbrevition. b The Dox-induced expression of ech Tx or HBZ protein is shown. Western blot nlysis confirmed tht ech subgroup-specific Tx nd HBZ protein ws ppropritely induced following Dox tretment Results Expression of ech subgroup Tx or HBZ protein under the control of n inducible promoter reveled different trget gene profiles The differences in mino cid sequences between subgroup-specific Tx, i.e., Tx-A nd Tx-B, or HBZ, i.e., HBZ-A nd HBZ-B, proteins re depicted in Fig. 1. Protein expression in Jurkt Tet-On 3G cells ws induced by dding Dox to culture medi t finl concentrtion of 3 ng/ml for 24 h t 37 C. Western blot nlysis confirmed tht ech subgroup-specific Tx nd HBZ protein ws ppropritely induced following Dox tretment (Fig. 1b). Microrry ws performed on RNAs derived from these smples. The results of microrry nlysis re shown in Fig. 2. In our microrry nlysis, genes were considered V A Tx-A Tx-B S differentilly expressed on the significnt level using SAM nlysis (q-vlue < 0.05). The numbers of genes tht occurred in ech cluster re shown in Fig. 2; we identified 231, 676, nd 1712 genes specificlly regulted by Tx-A, by both Tx-A nd Tx-B, nd by Tx-B, respectively. In HBZ, 500, 731, nd 6843 genes were specificlly regulted by HBZ-A, by both HBZ-A nd HBZ-B, nd by HBZ-B respectively. These dt indicted tht the number of genes regulted by HBZ ws 2 3 times higher thn tht regulted by Tx. Next, to define the gene expression profiles of ech subgroup-specific Tx or HBZ, we mnully creted list of the top 50 genes up-regulted by ech subgroup of Tx, i.e., Tx-A nd -B) (Tble 2), or HBZ, i.e., HBZ-A nd -B (Tble 3). The dt show the verge fold-chnge expression in ech subgroup-specific Tx (Tble 2) or HBZ (Tble 3) in Dox-treted Jurkt Tet-On 3G cells, compred to untreted cells. Gene expression levels were represented with red (high expression) or blue (low expression) in ech block. The genes re rnked in order of their verge fold-chnge expression of Tx-A, nd the next column represents the verge fold-chnge expression of Tx-B. Interestingly, s shown in Tble 2, ll of the top 50 genes regulted by Tx were more strongly upregulted by Tx-A thn Tx-B (Tble 2). In contrst, s shown in Tble 3, some trget genes were preferentilly induced by HBZ-A, rther thn by HBZ-B, wheres other trget genes were preferentilly induced by HBZ-B, rther thn by HBZ-A. There were lso some trget genes regulted by both subtypes HBZ t similr efficiency. These results indicte tht the Tx nd HBZ molecule of ech respective subgroup hs different trget gene profile. Importntly, some of the genes listed in Tble 2 (shown in red letters), such s EBI3 (= Epstein- Brr virus-induced gene 3, component of IL-27) [26], VCAM-1 [27], IL-13 [28], nd CCL1 [29] hve lredy been identified s Tx trget genes in previous studies, vlidting our gene induction system. It is lso noteworthy tht mong the Tx- or HBZ-regulted genes, there were set of ncrnas, including mirnas nd long noncoding RNAs (lncrnas) (Fig. 2b nd Tbles 2, 3). Vlidtion of differentilly expressed genes identified by microrry using qrt PCR in PBMCs of HAM/TSP ptients with HTLV 1 subgroup A or B To confirm whether the obtined in vitro gene expression profile hd clinicl significnce, we performed reltime qrt-pcr on the PBMCs of 37 ptients with HAM/ TSP (19 subgroup-a nd 18 subgroup-b), 20 HCs, nd 20 NCs (Fig. 3). Among the identified Tx-responsive genes,

8 Pge 8 of 20 Tx subtype HBZ subtype Tx-A Tx-B HBZ-A HBZ-B Regulted genes b ncrna ncrna Tx-A HBZ-A Tx-B HBZ-B Fig. 2 Results of cdna microrrys to exmine subgroup-specific Tx or HBZ-medited trnscriptionl chnges in Jurkt Tet-On humn T-cells. After trnsfection, Tx-A, Tx-B, HBZ-A or HBZ-B proteins were induced in Jurkt Tet-On 3G cells by dding Dox (finl concentrtion: 3 ng/ml) for 24 h. Uninduced Jurkt Tet-On 3G cells were used s bseline reference. Microrry ws performed on RNAs derived from these smples. The numbers of genes tht occurred in ech cluster re shown. We identified 231, 676, nd 1712 genes tht were specificlly up- or down-regulted by Tx-A, by both Tx-A nd Tx-B, nd by Tx-B, respectively. In HBZ, 500, 731, nd 6843 genes were specificlly up- or down-regulted by HBZ-A, by both HBZ-A nd HBZ-B, nd by HBZ-B respectively. b Among 231, 676, nd 1712 genes were specificlly upregulted by HBZ-A, by both HBZ-A nd HBZ-B, nd by HBZ-B, respectively, 47, 62, nd 410 genes were ncrnas. Similrly, mong 500, 731, nd 6843 genes tht were specificlly upregulted by Tx-A, by both Tx-A nd Tx-B, nd by Tx-B, respectively, 167, 241, nd 1245 genes were ncrnas we mesured the expression levels of CXCL10 mrna, since CXCL10 ws preferentilly induced by Tx-A rther thn by Tx-B (Tble 2). In ccordnce with the microrry dt, the results showed tht the expression levels of CXCL10 in HAM/TSP ptients with subgroup-a were significntly higher thn in HAM/TSP ptients with subgroup-b (p = by Scheffe s post hoc multiple comprisons test.) (Fig. 3). Menwhile, there ws no significnt difference in HTLV-1 PVL between HAM/TSP ptients with subgroup-a or -B (Fig. 3b). More efficient induction of chemokine CXCL10 by Tx A thn by Tx B Among the genes differentilly induced by Tx-A nd Tx-B (Tble 2), we focused our ttention especilly on the chemokine genes (Tble 4), prticulrly becuse chemokines hve been considered to ply relevnt roles in the pthogenesis of HAM/TSP [30, 31], nd both CXCL9 [32] nd CXCL10 [33] (listed in Tble 2) hve lredy been identified s Tx trget genes in previous studies, providing further vlidtion of our gene induction system. In prticulr, CXCL10 hs been proposed s the most vible, prognostic biomrker for HAM/TSP, s the cerebrospinl

9 Pge 9 of 20 Tble 2 Top 50 up-regulted genes in Tx-induced Jurkt Tet-On 3G cells Gene symbol Tx-A b Tx-B c Gene nme KIF kinesin fmily member 25 EBI Epstein-Brr virus induced 3 EGR erly growth response 2 UNQ unchrcterized LOC FAM129A fmily with sequence similrity 129 member A GEM GTP binding protein overexpressed in skeletl muscle WNT10A Wnt fmily member 10A CDH cdherin 16 CXCL C-X-C motif chemokine lignd 10 ANXA8L nnexin A8 like 1 EGR erly growth response 1 LYZL lysozyme like 4 FRG FSHD region gene 2 ENTPD ectonucleoside triphosphte diphosphohydrolse 2 IL interleukin 3 GADD45G growth rrest nd DNA dmge inducible gmm CRTAM cytotoxic nd regultory T cell molecule SDC syndecn 4 FSCN fscin ctin-bundling protein 1 VCAM vsculr cell dhesion molecule 1 FRG2C FSHD region gene 2 fmily member C LRG leucine rich lph-2-glycoprotein 1 BCL B cell CLL/lymphom 3 BCL2A BCL2 relted protein A1 JPH junctophilin 4 SGPP sphingosine-1-phosphte phosphtse 2 RGS regultor of G protein signling 1 IKZF IKAROS fmily zinc finger 4 IL interleukin 13 TSPO trnsloctor protein PTGIR prostglndin I2 receptor IL36G interleukin 36 gmm KCNJ potssium voltge-gted chnnel subfmily J member 18 BDKRB brdykinin receptor B2 DEPP DEPP1, utophgy regultor CSF colony stimulting fctor 2 UBD ubiquitin D TNFRSF TNF receptor superfmily member 4 LINC long intergenic non-protein coding RNA 1589 HGC unchrcterized LOC TNFAIP TNF lph induced protein 2 LTB lymphotoxin bet LAIR leukocyte ssocited immunoglobulin like receptor 2 CXCL C-X-C motif chemokine lignd 9 GPR G protein-coupled receptor 183 CCL C-C motif chemokine lignd 1 KIF25-AS KIF25 ntisense RNA 1 AHSP lph hemoglobin stbilizing protein LOXL lysyl oxidse like 4 LHX LIM homeobox 2 Genes were rnked in order of fold chnge by Tx-A induction b,c In order to identify up-regulted genes in Tx-A or Tx-B induction, cells trnsfected with ptre3g-empty were used s normliztion smples. The microrry experiments were performed in triplicte, nd dt were shown s men vlues fluid (CSF) levels of CXCL10 were well correlted with disese progression of HAM/TSP, better even thn HTLV-1 PVL in PBMCs, i.e., the number of HTLV-1-infected cells [31, 32]. Our dt demonstrted tht CXCL10 ws more efficiently upregulted by Tx-A, which is ssocited with n incresed risk of developing HAM/TSP, thn by Tx-B (Tble 2), suggesting tht the HTLV-1 subgroups re ssocited with chnges in host gene expression closely ssocited with HAM/TSP pthogenesis. To further vlidte the results using different inducible protein expression

10 Pge 10 of 20 Tble 3 Top 50 up-regulted genes in HBZ-induced Jurkt Tet-On 3G cells Gene symbol HBZ-A b HBZ-B c Gene nme lnc-gadd45g lnc-gadd45g-5:2 RNU6ATAC RNA, U6tc smll nucler (U12-dependent splicing) NRARP NOTCH regulted nkyrin repet protein OR52E olfctory receptor fmily 52 subfmily E member 8 CCL C-C motif chemokine lignd 21 LINC long intergenic non-protein coding RNA 353 RPPH ribonuclese P RNA component H1 LINC long intergenic non-protein coding RNA 399 CLDN cludin 6 CPXM crboxypeptidse X, M14 fmily member 2 STARD StAR relted lipid trnsfer domin contining 13 LOC unchrcterized LOC lnc-c17orf lnc-c17orf48-4:1 lnc-ndufv lnc-ndufv2-1:1 TRHDE-AS TRHDE ntisense RNA 1 LOC unchrcterized LOC OR10J olfctory receptor fmily 10 subfmily J member 1 LOC unchrcterized LOC TM4SF trnsmembrne 4 L six fmily member 5 LOC unchrcterized LOC THBS thrombospondin 4 lnc-ttll lnc-ttll2-1:1 RALYL RALY RNA binding protein like GCSAML-AS GCSAML ntisense RNA 1 lnc-map2k lnc-map2k6-2:1 LINC long intergenic non-protein coding RNA 2218 LOC unchrcterized LOC LOC unchrcterized LOC LOC unchrcterized LOC CNTNAP3B contctin ssocited protein like 3B LOC unchrcterized LOC lnc-rad23b lnc-rad23b-1:1 XAGE XAGE-4 protein lnc-tnp lnc-tnp1-2:1 LINC long intergenic non-protein coding RNA 944 lnc-cdh lnc-cdh3-2:1 LINC long intergenic non-protein coding RNA 2219 CDC14C cell division cycle 14C lnc-reep lnc-reep5-1:1 KRT kertin 79 TMEM238L trnsmembrne protein 238 like LOC unchrcterized LOC RTL retrotrnsposon Gg like 1 lnc-stxbp lnc-stxbp6-1:2 POLR2M RNA polymerse II subunit M FAM216B fmily with sequence similrity 216 member B PPP1R1A protein phosphtse 1 regultory inhibitor subunit 1A LOC unchrcterized LOC CA crbonic nhydrse 6 LOC unchrcterized LOC Genes were rnked in order of fold chnge by HBZ-A induction b,c In order to identify up-regulted genes in HBZ-A or HBZ-B induction, cells trnsfected with ptre3g-empty were used s normliztion smples. The microrry experiments were performed in triplicte, nd dt were shown s men vlues

11 Pge 11 of 20 b Fig. 3 Vlidtion of differentilly expressed genes identified by microrry using qrt-pcr in PBMCs of HAM/TSP ptients with HTLV-1 subgroup () or (b). To confirm whether the obtined in vitro gene expression profiles hve clinicl significnce, we performed rel-time qrt-pcr on PBMCs of 37 ptients with HAM/TSP (19 subgroup-a nd 18 subgroup-b), 20 HCs, nd 20 NCs. Among identified Tx-responsive genes, we mesured the expression levels of CXCL10 mrna levels. The expression levels of CXCL10 in HAM/TSP ptients with subgroup-a ws significntly higher thn in HAM/TSP ptients with subgroup-b (p = by Scheffe s post hoc multiple comprisons test). b There ws no significnt difference in HTLV-1 PVL between HAM/TSP ptients with subgroup-a or -B system, we used JPX-9 cells [20], subclone of Jurkt generted by stble trnsfection of functionl Tx expression-plsmid vector, nd induced Tx expression by dding cdmium chloride (CdCl 2 ) to the culture medium (10 µm finl concentrtion). Tx protein ws lmost undetectble in JPX-9 before the induction but becme detectble 6 h fter the ddition of CdCl 2 to the culture medium (Fig. 4). Quntittive rel-time PCR results indicte tht CXCL10 mrna expression ws induced long with Tx protein expression in JPX-9 cells (Fig. 4b, white brs). ELISA dt indicted tht CXCL10 protein becme detectble 12 h fter the ddition of CdCl 2 to the culture medium, i.e. 6 h fter detection of Tx protein nd 3 h fter detection of CXCL10 mrna (Fig. 4b, gry brs), nd the CXCL10 protein levels were still incresing even 120 h fter the ddition of CdCl 2. Preferentil expression of CXCL10 in HTLV 1 infected T cell lines derived from ptients with ATL nd HAM/TSP We mesured the levels of CXCL10 mrna nd protein expression in HTLV-1-infected nd -uninfected humn T-cell lines. As shown in Fig. 5, CXCL10 mrna ws preferentilly expressed in HTLV-1-infected humn T-cell lines derived from ptients with ATL (3 out of 5 tested) nd HAM/TSP (4 out of 4 tested), compred with HTLV-1-trnsformed T-cell lines (0 out of 3) nd HTLV-1 negtive humn T-cell lines (0 out of 3). Quntittive rel-time PCR nd ELISA nlysis reveled tht, lthough the expression levels of the virl RNAs tx nd HBZ in ATL-derived cell lines were low (Fig. 5b), high levels of CXCL10 mrna (Fig. 5) nd protein expression (Fig. 5c) were observed in ATL cell lines, suggesting tht over-expression of CXCL10 in ATL-derived cell lines ws independent of tx gene expression. Western blot lso reveled tht over-expression of CXCL10 in ATL-derived cell lines ws independent of Tx protein expression (Fig. 5d). Importntly, consistent with the cell line dt, the expression levels of CXCL10 mrna in PBMCs of HAM/TSP ptients were lso not significntly correlted with the tx expression (p = , r = by Spermn rnk correltion nlysis), indicting tht incresed CXCL10 expression is independent of tx expression. Thus, our dt indicted tht incresed expression of CXCL10 mrna is ssocited with hving HTLV-1 subgroup-a or subgroup-b, not tx expression in infected cells both in vitro nd ex vivo (i.e., in unmnipulted PBMCs obtined from HAM/TSP ptients). No difference in subgroup specific Tx molecules with respect to trnscriptionl ctivtion of the CXCL10 promoter vi NF κb We further exmined whether there were ny differences in the bility of subgroup-specific Tx to ctivte

12 Pge 12 of 20 Tble 4 Chemokine genes differentilly induced by Tx-A nd Tx-B Fold chnge Tx-A Tx-B Tx A/B rtio Gene nme Prtner (receptor or lignd) References Chemokine CXCL10 CXCR3 [32, 45, 46] CXCL9 CXCR3 [32, 46] N.D. 2.4 CCL22 CCR4 [47] CCL1 CCR8 [29, 48] N.D. 2.9 CCL5 CCR1, CCR3, CCR5 [49, 50] CCL20 CCR6 [51] XCL1 XCR1 [52] Chemokine receptor CXCR6 CXCL16 None reported CCR7 CCL19, CCL21 [53] 1.5 N.D. CCRL2/CCR11 CCL2, CCL5, CCL7, CCL8 None reported Not detected b Fig. 4 CXCL10 expression is induced long with Tx in JPX-9 cells. Western blot nlysis of Tx expression in JPX-9 cells treted with 10 μm CdCl 2. Cell lystes were prepred from CdCl 2 -treted JPX-9 cells t the indicted time points, nd Tx expression ws confirmed by western blotting with Lt-4 nti-tx monoclonl ntibody. Equl smple loding ws verified with nti-α-tubulin (bottom). Tx protein ws lmost undetectble in JPX-9 before the induction, but becme detectble 6 h fter the ddition of CdCl 2 to the culture medium. b Induction of CXCL10 mrna nd protein expression in JPX-9 cells treted with CdCl 2. CXCL10 mrna nd protein levels fter Tx induction were detected by quntittive rel-time PCR nd ELISA, respectively. The copy number of CXCL10 mrna ws normlized by the copy number of hypoxnthine gunine phosphoribosyl trnsferse 1 (HPRT1) mrna. Quntittive rel-time PCR results indicte tht CXCL10 mrna expression ws induced long with Tx protein expression in JPX-9 cells (white brs). ELISA dt indicted tht CXCL10 protein becme detectble 12 h fter the ddition of CdCl 2 to the culture medium, i.e. 6 h fter detection of Tx protein nd 3 h fter detection of CXCL10 mrna, nd the CXCL10 protein levels were still incresing even t 120 h fter the ddition of CdCl 2 (gry brs). The dt re representtive of 3 independent experiments. Dt shown s men ± SD, n = 3

13 Pge 13 of 20 Fig. 5 Preferentil expression of CXCL10 in Humn T-cell leukemi virus type-1 (HTLV-1)-infected T-cell lines derived from ptients with dult T-cell leukemi (ATL) nd HTLV-1-ssocited myelopthy/ tropicl spstic prpresis (HAM/TSP). Expression of CXCL10 mrna ws exmined by rel-time PCR in HTLV-1-infected nd -uninfected T-cell lines. CXCL10 mrna ws preferentilly expressed in HTLV-1-infected humn T-cell lines derived from ptients with ATL (3 out of 5 tested) nd HAM/TSP (4 out of 4 tested), compred with HTLV-1-trnsformed T-cell lines (0 out of 3) nd HTLV-1 negtive humn T-cell lines (0 out of 3). b The expressions of tx nd HBZ mrna were exmined by quntittive rel-time PCR in HTLV-1-infected T-cell lines. The expression levels of the virl RNAs tx nd HBZ were reltively high in T-cell lines derived from ptients with HAM/TSP nd HTLV-1-trnsformed T-cell lines when compred with those in ATL cell lines. c CXCL10 levels in culture superntnts from HTLV-1-infected nd -uninfected humn T-cell lines were ssessed by ELISA. Significnt levels of CXCL10 ws observed in the cultured superntnts from HTLV-1-infected humn T-cell lines derived from ptients with ATL nd HAM/TSP, wheres it ws not detectble in ny of the other cell lines tested. d Expression of Tx nd HBZ ws exmined by Western blot in HTLV-1-infected nd -uninfected T-cell lines b the humn CXCL10 promoter [22]. Jurkt cells were cotrnsfected with Tx effector plsmids nd reporter gene construct contining the region between 875 nd +97 nucleotides (pgl3-cxcl10-long-luc) nd 279 nd + 97 nucleotides (pgl3-cxcl10-short-luc) of the CXCL10 upstrem regultory sequences (Fig. 6). After 24 h trnsfection, Tx-A or Tx-B protein ws induced by dding Dox for 24 h. Then, the reporter gene ssy ws crried out for Tx-medited trnscriptionl ctivtion. As shown in Fig. 6b, there ws no difference between Tx-A nd Tx-B with respect to trnscriptionl ctivity for both the short nd long CXCL10 promoters in the epithelil cell line 293T nd the T cell line, Jurkt. The dt lso showed tht the Tx mutnt M22 [34], which is defective in NF-κB ctivtion, filed to ctivte the CXCL10 promoters (both short nd long) (Fig. 6b). In contrst, the Tx 703 mutnt [35], which cn ctivte NF-κB but not CREB, efficiently ctivted the CXCL10 promoters (both short nd long) (Fig. 6b). To further determine whether either the NF-κB or the AP-1 sequence ws required for Tx-medited ctivtion of the CXCL10 promoter, we constructed reporter plsmids by site-directed mutgenesis. Then mutnt reporter constructs were co-trnsfected long with the Tx expression plsmid, nd luciferse ctivity ws determined for ech of the four mutnts (Fig. 6c). As result, Tx-induced luciferse ctivity ws significntly reduced by muttion in one of the two NF-κB binding site sequence, but not reduced by muttion in the AP-1 site, indicting tht Tx trnsctivtion of the CXCL10 involves both κb binding sites. Nmely, Tx-induced ctivtion of NF-κB pthwy is responsible for the upregultion of CXCL10 expression, c d but subgroup-specific Tx molecules hve similr effects on the trnscriptionl ctivity of the CXCL10 promoter. This ws lso the cse for nother reporter construct contining NF-κB binding site sequence. As shown in Additionl file 3: Fig. S1, subgroup-specific Tx molecules do not differ in the trnscriptionl ctivtion of the reporter construct contining luciferse gene under control of the NF-κB binding sequence of the IL-2R gene [25].

14 Pge 14 of 20 (See figure on next pge.) Fig. 6 Subgroup-specific Tx molecules do not differ in the trnscriptionl ctivtion of the CXCL10 promoter vi NF-κB. Schemtic representtion of the CXCL10 promoter sequence cloned into pgl3-bsic. The firefly luciferse gene ws used to monitor the ctivity of the CXCL10 gene promoter. b These reporter constructs were independently trnsfected into Jurkt humn T-cells nd 293T cells with or without the Tx expression plsmid. Luciferse ssys were performed 24 h fter trnsfection. There ws no difference between Tx-A nd Tx-B with respect to trnscriptionl ctivity for both the short nd long CXCL10 promoters. The dt lso showed tht the Tx mutnt M22, which is defective in NF-κB ctivtion, filed to ctivte the CXCL10 promoters (both short nd long). In contrst, the Tx 703 mutnt, which cn ctivte NF-κB but not CREB, efficiently ctivted the CXCL10 promoters (both short nd long). c To determine whether either the NF-κB or the AP-1 sequence ws required for Tx-medited ctivtion of the CXCL10 promoter, mutnt reporter constructs were co-trnsfected long with the Tx expression plsmid, nd luciferse ctivity ws determined for ech of the four mutnts. Tx-induced luciferse ctivity ws significntly reduced by muttion in one of the two NF-κB binding site sequence, but not reduced by muttion in the AP-1 site, indicting tht Tx trnsctivtion of the CXCL10 involves both κb binding sites. Three independent experiments were performed. Dt shown s men ± SD, n = 3 No difference in Tx A nd Tx B with respect to binding to NF κb proteins While HTLV-1 is the custive gent of ATL nd HAM/ TSP, the closely relted virus HTLV-2 is not clerly ssocited with known clinicl disese [36]. It hs been reported tht HTLV-1 Tx (i.e., Tx1) but not HTLV-2 Tx (Tx2) intercts with NF-кB2/p100/p52 nd RelB, nd this interction ws medited by the Tx1 mino cid region [37]. As one of the two sites of mino cid differences between Tx-A nd Tx-B is locted just beside this region (i.e., t position 221), we tested whether this mino cid difference ffects binding ffinity to NF-кB protein, thereby ltering the bility to ctivte the NF-кB pthwy nd its downstrem trget genes. Jurkt cells were trnsfected with ech Tx expression plsmid nd cells were hrvested fter 24 h, then western blot nd immunoprecipittion nlyses were performed. The mounts of p100, p52, RelB, p65, p105, p50, nd c-rel proteins in immunoprecipitted smples were quntified by densitometric scnning of corresponding bnds of the western blot. As shown in Fig. 7, b, western blot nlysis performed on immunoprecipitted Tx protein reveled tht ech subgroup of Tx (i.e., Tx-A nd -B) intercted with ech NF-κB component (i.e., c-rel, RelA, RelB, p50/ p105, nd p52/p100) with similr efficiency. Higher bundnce of the DNA frgment bound by ternry complex including Tx A thn tht including Tx B in the CXCL10 promoter To investigte whether HTLV-1 subgroups ffect the binding of Tx to the endogenous CXCL10 promoter, we performed ChIP ssys (Fig. 8). Chromtin frgments were prepred from Jurkt T-cells trnsfected with plsmid expressing Tx nd immunoprecipitted with specific nti-tx monoclonl ntibodies (Lt-4). The immunoprecipitted DNA ws then mplified vi PCR using specific primer pirs tht mplify the CXCL10 proximl promoter sequence including the two NF-κB sites nd n AP-1 site (Fig. 8, Trget region ). As shown in Fig. 8, the ternry protein complex including Tx ws found to be ssocited with the CXCL10 proximl promoter sequence including the two NF-κB sites nd n AP-1 site (Fig. 8). Moreover, the bundnce of specific DNA frgment bound by ternry complex including Tx is higher for Tx-A thn for Tx-B (Fig. 8b, c). Discussion In this study, to investigte the role of HTLV-1 subgroups in virl pthogenesis, we first performed microrrybsed study to define the gene expression profiles of ech subgroup-specific Tx or HBZ. Our results showed tht some Tx-responsive genes identified by microrry, such s EBI3 [26], VCAM-1 [27], IL-13 [28], nd CCL1 [29] hve lredy been identified s Tx trget genes in previous studies, vlidting our gene induction system. Furthermore, Tx-induced expression of CXCL10 gene identified by microrry ws confirmed by qrt-pcr. Our microrry nlysis showed tht out of totl of 50,599 genes including Entrez genes nd lincrna genes screened, pproximtely 2619 nd 8074 were differentilly expressed upon Tx or HBZ induction, respectively. The list of the top 50 genes up-regulted by ech subgroup of Tx (i.e., Tx-A nd -B) or HBZ (i.e., HBZ-A nd -B) showed tht the genes tht were differentilly expressed in the presence of Tx included those relted to poptosis (BCL2-relted protein A1: BCL2A1), the cell cycle nd DNA repir (growth rrest nd DNA-dmgeinducible, gmm: GADD45G), cytokines (IL3, IL13, IL36G), chemokines (CXCL9, CXCL10), nd dhesion molecules (vsculr cell dhesion molecule 1: VCAM1). In contrst, the genes tht were differentilly expressed in the presence of HBZ include those relted to RNA-splicing (RNA, U6tc smll nucler: RNU6ATAC), the Notch signling pthwy (NOTCH-regulted nkyrin repet protein: NRARP) nd chemokine (CCL21). These results suggest tht Tx minly induces expression of genes relted to the ctivtion nd trnsformtion of CD4+ T cells, wheres HBZ modultes vriety of cellulr signling pthwys which re relted to the immune response, differentition, nd growth of T-cells.

15 Pge 15 of 20 b c

16 Pge 16 of 20 (See figure on next pge.) Fig. 7 Tx-A nd Tx-B do not differ in their binding to NF-κB proteins. To test whether one of the two mino cid differences between Tx-A nd Tx-B, i.e., t position 221, which re locted just beside the Tx1 ( ) motif is involved in the p100 processing, we tested whether this mino cid difference ffects the binding ffinity to the NF-кB protein, thereby ltering the bility to ctivte the NF-кB pthwy nd their downstrem trget genes. Jurkt cells were trnsfected with ech expression plsmid nd cells were hrvested fter 24 h, then immunoprecipittion nd western blot nlyses were performed. Western blot nlysis performed on immunoprecipitted Tx protein reveled tht ech subgroup of Tx, i.e., Tx-A nd -B, intercted with ech NF-κB components, i.e., c-rel, RelA, RelB, p50/p105, nd p52/p100, with similr efficiencies. b The mounts of p100, p52, RelB, p65, p105, p50, nd c-rel proteins in immunoprecipitted smples were quntified by densitometric scnning of corresponding bnds of the western blot using Imge J softwre. Verticl brs indicte men ± SD of the densitometric nlysis from four independent experiments Our microrry results lso showed tht vrious kinds of ncrnas frequently ppered mong both Tx- nd HBZ-regulted genes. It is now well estblished tht the mjority of trnscribed genomic sequences re ssocited with ncrnas rther thn protein-coding RNAs, nd such ncrnas, including regultory mirnas nd lncr- NAs tht re functionlly involved in vriety of host immune responses nd in the pthogenesis of humn diseses [38]. It is, therefore, plusible to propose tht deregultion of the ncrna signture cused by virus infection will strongly ffect the phenotype nd function of the infected cell. Indeed, in HTLV-1 infection, it hs been reported tht the expression of mir-31, which is negtive regultor of the noncnonicl NF-κB pthwy, ws geneticlly nd epigeneticlly silenced in ATL cells, resulting in constitutive NF-κB ctivtion [39]. In ddition, lncrnas, heterogeneous clss of RNAs defined s non-protein-coding trnscripts longer thn 200 nucleotides, re thought to ply role in protesoml nd ubiquitintion pthwys, poptosis, DNA dmge responses, nd cell cycle regultion [40]. lncrnas hve been directly linked to humn diseses such s certin cncers nd utoimmune nd neurodegenertive diseses [40]. However, to dte, the role of lncrnas in virl infections remins lrgely unknown. Our results provide new insights into the hitherto unknown functions of lncrnas in infection by viruses including HTLV-1. A previous report suggested tht serum CXCL10 is significntly higher in HAM/TSP ptients thn in HCs, nd the CSF level of CXCL10 ws strongly correlted with disese severity [32]. Thus, CXCL10 concentrtion ws proposed s potentil prognostic biomrker for HAM/ TSP. Our dt showed tht mong the Tx-regulted trget genes, CXCL10 ws pproximtely 1.5 times more strongly induced by Tx-A thn Tx-B. More importntly, rel-time qrt-pcr on PBMCs obtined from HAM/TSP ptients indicted tht the expression levels of CXCL10 in HAM/TSP ptients with subgroup-a were significntly higher thn those in HAM/TSP ptients with subgroup-b. The difference in trnscription is likely to be due to difference in the ction of the NF-κB/Rel fmily of trnscription fctors. In HTLV-1 infection, Txmedited NF-κB ctivtion is recognized s crucil fctor ssocited with the development of HTLV-1-ssocited diseses [41], since NF-κB, which consists of five molecules (RelA (p65), RelB, c-rel, p50, nd p52) tht form trnscriptionlly ctive complexes in vrious combintions, hs n essentil role in inflmmtion, innte immunity, nd mny steps of cncer initition nd progression [42]. Indeed, ATL cells nd their derivtive cell lines crry constitutively ctive NF-κB regrdless of their Tx expression, nd NF-κB is required for immortliztion nd lso the survivl of HTLV-1 trnsformed cells [43]. We therefore determined whether the HTLV-1 virl protein Tx ctivtes the expression of CXCL10 t the trnscriptionl level nd whether there were ny differences in the bility of subgroup-specific Tx molecules to ctivte the CXCL10 promoter. However, contrry to our expecttions, there ws no difference in the bility of ech subgroup Tx to ctivte the CXCL10 promoter, lthough trnsient Tx expression in n HTLV-1-negtive humn T-cell line ctivted the CXCL10 gene promoter through the NF-κB pthwy. In cler contrst to HTLV-1, HTLV-2 hs not been ssocited with ATL or other types of mlignncies [36]. As HTLV-1 Tx (Tx1) nd HTLV-2 Tx (Tx2) hve mny shred ctivities but lso certin significntly distinct ctivities [36], the difference between those two Tx proteins my revel the key roles in HTLV-1-induced mlignnt trnsformtion. Most importntly, lthough Tx2 ctivtes the clssicl pthwy of NF-κB, similr to Tx1, mlignnt trnsformtion by Tx2 hs rrely been reported [44]. Thus, one significnt difference between Tx1 nd Tx2 is the ctivtion of trnscription fctor NF-κB2/p100/p52, which is key plyer in the lterntive, non-clssicl NF-κB pthwy [44]. Interestingly, Tx1 but not Tx2 ws reported to interct with NF-κB2/ p100/p52 nd RelB, nd the distinct interction ctivity ws medited by the Tx1 mino cid region , nd one of the two sites of mino cid differences between Tx-A nd Tx-B is locted just beside this region (i.e., t position 221). We therefore tested whether this mino cid difference ffected the binding ffinity to NF-кB protein, thereby ltering the bility to ctivte the NF-кB pthwy nd their downstrem trget genes. The results showed tht both Tx-A nd Tx-B showed

17 Pge 17 of 20 b