To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT4b receptor, a GPCR with high constitutive Gs signaling and strong ligand-induced G-protein activation of the Gs and Gs/q pathways. The first receptor in this series, 5-HT4-D100A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced Gs signaling, but only a few  also induced signaling via the Gq pathway. Zacopride-induced Gq signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT2C receptor. Additional point mutations (D66A and D66N) blocked constitutive Gs signaling and lowered ligand-induced Gq signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT1A conferred ligand-mediated Gi signaling. This Gi-coupled RASSL, Rs1.3, exhibited no measurable signaling to the Gs or Gq pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.G protein–coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their  These signals mediate a wide variety of physiological responses, including heart rate, chemotaxis, cell proliferation, neurotransmission, and hormonal responses. Owing to their physiological importance, GPCRs are of great medical interest. Indeed, they are targets for at least 40% of modern pharmaceuticals Heptahelical G protein–coupled receptors (GPCRs) are the largest family of human cell-surface receptors, encompassing more than 340 hormone receptors and 350–460 olfactory receptors in vivo have been hampered by the lack of specific agonists and antagonists for many of the receptors. GPCRs display molecular and functional diversity, such as the type of G-protein signaling pathway associations, different levels of constitutive activity, and different signaling responses due to different ligand-selective conformations Although many drugs target GPCRs, studies of GPCR signaling To better study the diversity of GPCRs, we developed receptors activated solely by synthetic ligands (RASSLs) by modifying their structures to render them unresponsive to endogenous hormones. Instead, RASSLs are activated by small-molecule drugs s-, Gi-, and Gq-coupled GPCRs, which stimulate adenylyl cyclase, inhibit adenylyl cyclase, and stimulate phospholipase-C, respectively. Ro1 (RASSL opioid 1), the prototype RASSL based on a Gi-coupled κ-opioid receptor i response to natural ligands is 0.001% of that of the wildtype receptor, but it is potently activated by the synthetic agonist spiradoline in vivo, RASSLs can yield insights into fundamental aspects of receptor diversity Since it is impractical to convert all GPCRs into RASSLs, we and others have focused on representative Gs-coupled RASSL based on the melanocortin-4 receptor q-coupled RASSL based on the histamine 1 receptor Multiple RASSLs have since been made, including a G4 receptor (100A) in the mouse 5-HT4 receptor dramatically reduced its affinity for serotonin, its endogenous ligand. This mutation also allows synthetic agonists and antagonists for the wildtype receptor to activate the mutant, turning 5-HT4-D100A into a RASSL To better study GPCRs, we built a series of RASSLs based on the human 5-HTreceptor , which hreceptor  than oth4 receptor with other family members. Altering the G-protein selectivity of GPCRs is often difficult because it is based on receptor conformation determined by multiple regions of the receptor 4 receptor belongs to a family of at least 15 receptors, each with different subfamilies that engage different G-protein signaling pathways. The 5-HT4, 5-HT6, and 5-HT7 subfamilies are Gs coupled, the 5-HT1 subfamily is Gi coupled, and the 5-HT2 subfamily is Gq coupled s-, Gi-, and Gq-coupled RASSLs.Finally, we reasoned that novel receptors coupling to other signaling pathways could be created by making chimeras of the 5-HT4 receptor. These modified GPCRs will help us better study the effect of constitutive Gs signaling and ligand-induced Gs, Gs/Gq, and Gi signaling in vivo.Here, we describe a new series of RASSLs developed to modify the ligand-induced and constitutive signaling of the human 5-HT100A mutation in the mouse 5-HT4 receptor converts it into a RASSL 4 receptor have not been tested. We now extend these findings to the human 5-HT4 receptor  and >10-fold higher than that of the control receptors (the β2-adrenergic and parathyroid hormone receptors), which have low levels of constitutive signaling  accumulation by Rs1 via constitutive or ligand-induced signaling. Rs1 showed no measurable difference in constitutive Gq signaling   . Surpris.005)  showed enhanced Gq signaling in response to cisapride, zacopride, RS23597, RS39604, and RS67333  or third intracellular loop (i3) of Rs1 eliminated both Gignaling . Only th RS67333 , showingreserved .s-coupling of the 5-HT4 receptor. They also suggested that the C-terminus of 5-HT2C contains a G-protein coupling domain for Gq signaling or that the C-terminus of 5-HT4 receptor promotes Gq signaling. However, we were unable to completely alter the G-protein preference of this receptor for Gq signaling. We amplified the Gq signaling of Rs1 by domain swapping the C-terminus with 5-HT2C. None of the other 12 chimeras showed enhanced Gq signaling, even when multiple internal segments were combined. We did not proceed further with these experiments because substitutions of multiple internal domains also decreased cell-surface expression of the receptors (data not shown). Since Gq signaling of Rs1-C-5-HT2C was activated by cisapride, zacopride, and RS23597 but not by serotonin, we named it Rs1.1 . Signaling increased 1.3-fold in response to GR125487 (p<0.05), 1.7-fold in response to GR113808 (p<0.05), and 2.2-fold in response to RO110-0235 (p<0.005). This suggests that the carboxylic tail may play a role in functional selectivity in 5-HT4 or 5-HT2C receptors   in HEK293 cells co-transfected with 1.5 µg of Rs1 receptor and 0.5 µg of dopamine 1 receptor  . Interesignaling . Unfortu reduced . While tit Rs1.3 , Table 1s/Gq, Gs, Gi), with different constitutive signaling using a single receptor backbone and different synthetic agonists. These new RASSLs will also allow a systematic examination of the different functional domains of the 5-HT4B receptor.We report here a new series of RASSLs to study multiple G-protein signaling pathways. Many GPCRs activate multiple G-protein signaling pathways and exhibit a wide range of constitutive signaling activities. Our new RASSLs will help us better study the effect of stimulating canonical signaling pathways  system, we have already made an Rs1 transgenic mouse in which Rs1 expression is driven by the osteoblast-specific Col1α-1 2.3-kb promoter fragment. These mice exhibited dramatically increased bone formation in vivo. We found only constitutive Gs signaling in Rs1 but did not observe any constitutive Gq signaling. It would be interesting to generate new or modify existing RASSLs with increased Gi or Gq constitutive signaling in the future to examine the effect of both agonist-mediated and constitutive signaling in various physiological processes. Since signaling would only be dependent on expression rather than on circulating hormones, these RASSLs have an intrinsic advantage for studying constitutive signaling when combined with a Tet system.These new RASSLs could help us better dissect the physiological significance of constitutive signaling 4 RASSLs have a large set of agonists with different activities that could be useful in future studies. Many of the Rs1 agonists used in this study activated Rs1 with an EC50 in the nanomolar range, allowing to us activate Rs1 effectively. Having an abundant selection of ligands for Rs1 helped us find strong evidence of ligand-induced functional selectivity on Rs1. Having a greater variety of drugs to choose from could be valuable for in vivo studies. For instance, we could use GR125487 to activate Rs1 while suppressing the basal activity of wildtype 5-HT4. Alternatively, we could potentially study Rs1 constitutive signaling by using drugs such as RO116-0086 or RO116-1148 4 receptor but not of Rs1.In addition, the 5-HTin vivo studies in which Rs1 is activated with minimal side effects. Since knockout of the 5-HT4 receptor does not cause overt side effects 4 receptors for most in vivo experiments s, Gs/q, or Gi pathway.Finally, this new series of RASSLs may make it possible to perform in vivo, they have several limitations. Despite significant efforts to screen all available antagonists, we could not identify inverse agonists that would lower constitutive signaling by Rs1. Fortunately, we can use conditional expression systems such as the tetracycline transactivator system to control Rs1 expression and constitutive activation. Although we were successful in finding mutations that reduced Rs1 constitutive signaling these same mutations adversely affected the agonist mediated signaling (Rs1.2), suggesting that the D66N and W272A mutations are critical for all aspects of Gs signaling. Other mutations could be explored that may provide a more optimal reduction in Gs basal activity without affecting ligand-induced receptor activation. In addition, the relatively small Gi signaling of Rs1.3 can only be activated by a relatively high concentration of zacopride, thus limiting its usefulness in vivo.While these new RASSLs may be useful for studying G protein signaling 2C chimeras that are expressed on the cell surface, none of the i2 or i3 chimeras showed any Gs or Gq signaling. Evidently, these intracellular loops of Rs1 are crucial for signaling via those pathways. The importance of i2 and i3 for Gs signaling of Rs1 is further supported by the lack of Gs signaling by the Rs1-i2-5-HT1A and Rs1-i3-5-HT1A chimeras. This is the first study showing the importance of i2 and i3 in both Gs and Gq signaling of the human 5-HT4-D100A receptor.Our study yielded insights into the G-protein selectivity and functional selectivity  of Rs1. Of the 12 Rs1-5-HTs signaling and enabled Rs1 to stimulate Gi signaling of 5-HT1A. The role of i2 and i3 in the Gi signaling of 5-HT1A receptor has been extensively reported. The entire N-terminus 1A are thought to be sufficient to support G-protein coupling, but not signaling. On the other hand, the N-terminus 1Ai signaling of 5-HT1A. In fact, replacing the N-terminus of the i3 of the α2-adrenergic receptor with that of 5-HT1A resulted in a chimera that signals like a 5-HT1A receptor when stimulated by a α2-adrenergic receptor agonist 1A chimeras with multiple internal domains replaced are not significantly expressed on the cell surface (data not shown), it may be difficult to further improve the potency of the Rs1.3 using our current approach. We hypothesize that replacing the N- and C-terminal portions of i2 and i3 instead of the whole i2 and i3 loops may increase the potency of Rs1.We also found that i3 domain swapping abolished all G1 receptor 2-adrenergic 22AWe also showed that various drugs can differentially activate G-protein signaling of Rs1. Functional selectivity has been reported for many receptors. It led to divergent fates of internalization for the dopamine Dq signaling of Rs1, Rs1.1, or the 5-HT4 receptor. On the other hand, the benzamide derivatives cisapride, zacopride, RS23597, RS39604, and RS67333 activated the Gq signaling of Rs1. These findings may reflect distinct conformational changes caused by indoleamine and benzamide derivatives.The indoleamine derivatives GR113808, GR125487, and RO110-0235 did not fully activate G2c and Rs1 point mutants (Rs1-D66A and Rs1-D66N). The D100A mutation and replacement of the C-terminus amplified Gq signaling by the 5-HT4 receptor. The addition of D66A and D66N abolished Gq signaling. Since D100A is located in the binding pocket of the 5-HT4 receptor, this mutation in Rs1 may have changed the configuration of the binding pocket, making the receptor more susceptible to Gq activation by zacopride and RS23597. This response was even more pronounced when the D100A mutation was combined with domain swapping of the C-terminus with that of 5-HT2C. Thus, it is reasonable to hypothesize that these changes modified the ligand-selective receptor conformation The possibility of functional selectivity is further supported by the results obtained with Rs1-C-5HTet al. made a series of RASSLs based on the muscarinic M3 and M4 receptors, which have low constitutive activity. These RASSLs each couple different G-protein signaling pathways and can be activated by clozapine-N-oxide, an inert ligand with high bioavailability s, Gi, or Gq) will have different in vivo phenotypes due to noncanonical signaling. This growing collection of RASSLs will greatly facilitate our efforts to understand the physiological significance of the inherent signaling diversity of GPCRs.Our studies with Rs1 provide a proof-of-concept for making a series of RASSLs with different signaling properties. Recently, Armbruster An ideal series of RASSLs would have receptors with different combinations of low and high basal signaling, with robust ligand-induced effects for each major pathway, and potent inverse agonists. Although we have not achieved this goal with the Rs1 series, we are hopeful that it can be achieved with other receptors in the future. Indeed, RASSLs based on the muscarinic receptors 4 receptor cDNA  was used in all experiments. To improve expression and allow detection of the receptor, we added a signal peptide from influenza hemagglutinin 4 was then subcloned by PCR; the primers, ATCGATCGgcggccgcGTGAGCAAGGGCGAGGAGCTGTTC and ATCGATCG gcggccgcCTAAGTGTCACTGGGCTGAGCAGCC, were inserted into the Not1 restriction site (gcggccgc) of the pUNIV-5-HT2C-INI plasmid to replace the 5-HT2C-INI (a gift from Dr. Bryan Roth) in frame with the signal peptide and the FLAG epitope. The receptor was then mutated (D100A) with a Quick-Change site-directed mutagenesis kit  with primer GTCTTGTTCGGACATCTCTGgccGT CCTGCTCACAACGGCAT-CG  HEK293 cells were maintained in high-glucose DMEM  supplemented with sodium pyruvate (Invitrogen) and 10% Fetalplex . Receptors were electroporated into HEK293 cells as described 5-HT, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich . Cisapride, zacopride, GR113808, GR1254875, RS23597-190 HCl, RS39604 HCl, and RS67333 HCl were from Tocris . Human parathyroid hormone peptide (amino acids 1–34) was from Bachem Biosciences . RO110-0235 was generously donated by Renee Martin .2) containing anti-FLAG M1 antibody  for 1 h at 25°C. The samples were washed three times with wash buffer (PBS and 1 mM CaCl2) and stained with 100 µl of staining buffer with rat anti-mouse IgG antibody conjugated with horseradish peroxidase . After 30 min, the samples were washed with wash buffer, placed on a rocker for 10 min, and washed again. This process was repeated two more times. Then, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) liquid substrate  was added to the samples. After rocking for 30 min, 200 µl of the substrate was transferred to new 96-well plates, and optical density was measured with Victor 3  at 405 nm. All samples contain three replicates, and all experiments were repeated at least three times.Cell-surface receptor expression was measured with a FLAG ELISA as described To improve assay consistency and minimize pipetting error in the 384-well plates, we modified the high-range HTRF assay  by seeding, stimulating, and lysing the cells in 96-well plates and using the lysate instead of live cells to determine cAMP production. The remainder of the analysis was performed according to the manufacturer's instructionsi signaling was examined in cells transfected with 1.5 µg of receptor cDNA, 0.6 µg of human dopamine 1 receptor cDNA, and pcDNA3 (up to 6 µg). The co-transfectants were stimulated first with 100 µl of KRBG buffer containing IBMX for 10 min at room temperature and then with 50 µl of PBS containing 10 µM apomorphine (agonist for the dopamine 1 receptor) and 10 µM zacopride for 10 min at 37°C. The cells were lysed in 50 µl of lysis buffer, and 5 µl of lysate was used in the HiRange HTRF assay.G6 HEK293 cells as above To measure calcium mobilization, 4.8 µg of receptor cDNA, 0.6 µg of DsRed plasmid, and 0.6 µg of human bombesin receptor cDNA were electroporated into 5×106) were electroporated as described above. Then, 105 cells were placed in DMEM supplemented with 10% decomplemented, dialyzed against FBS, and seeded onto 96-well plates coated with poly-D-lysine. The next day, the cells were stimulated with agonists in 50 µl of 1× stimulation buffer for 30 min at 37°C and lysed for 10 min with 9 µl of lysis/detection buffer. Then, 14 µl of lysate was added to 384-well plates and subjected to High-Range HTRF assay as described above, except that 3 µl of cAMP-d2 and anti-cAMP-cryptate solution were added to each well.A modified version of the IP1 protocol was used . HEK293 cells were washed once with calcium-free PBS and dissociated from flasks with cell dissociation buffer (Invitrogen). Cells . Calcium mobilization results were analyzed with SoftMax Pro v5 (Molecular Devices). Statistical significance was determined with paired Student's Figure S1Chemical structures of the compounds used in the study. All the chemicals used in the experimental are shown.(1.92 MB EPS)Click here for additional data file.Figure S2Rs1-5-HT2C chimeras. All modifications made to Rs1 . Red lin(12.17 MB EPS)Click here for additional data file.Figure S3The second and third intracellular loops (i2 and i3) of Rs1 are crucial for Gs and Gq signaling.  Rs1-5-HT2C chimeras with swaps of i2 and i3 could no longer process Gs signals, at either 25 ng or 4.8 µg of receptor cDNA per 5×106 HEK293 cells. (C) Gq signaling of Rs1 was abolished when i2 and i3 of Rs1 were replaced with those of 5-HT2C. The Gq signaling was measured by calcium mobilization assay. (D) Only chimeras with a single domain swap were expressed on the cell surface. The results represent three independent experiments. All figures were representative of three independent experiments.(2.03 MB EPS)Click here for additional data file.Figure S4Rs1-5-HT1A chimeras. All modifications were made on Rs1 . Red lin(3.82 MB EPS)Click here for additional data file.Figure S5Replacing the second or third intracellular loop (i2 or i3) of Rs1 with 5-HT1A alters G protein signaling.  Rs1-5-HT1A chimeras with swaps of the second (i2) and third intracellular (i3) loops no longer signal via the Gs pathway, regardless of whether cells were transfected with 25 ng or 4.8 µg of receptor cDNA per 5×106 HEK293 cells. This suggests that the second and third intracellular loops are crucial for acute Gs signaling of Rs1. ***p<0.001 vs. mock transfected (t test). (C) Replacing i3 of Rs1 resulted in a Gi signaling receptor. All HEK293 transfectants were electroporated with 0.6 µg of the human dopamine 1 receptor and 1.5 µg of Rs1, Rs1-5HT1A chimeras, or the mu-opioid receptor. Rs1 and Rs1-5HT1a chimeras were treated with 10 µM apomorphine (an agonist for dopamine 1 receptor) or with 10 µM apomorphine and 10 µM zacopride. Transfectants with 0.6 µg of the human dopamine 1 receptor and 1.5 µg of the mu-opioid receptor served as positive controls. 10 µM DADLE was used in place of zacopride to stimulate mu-opioid receptors. ***p<0.005, *p<0.05 vs. apomorphine (t test). (D) Rs1, .(1.67 MB EPS)Click here for additional data file.Figure S6High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.98 MB EPS)Click here for additional data file.Figure S7High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.96 MB EPS)Click here for additional data file.Figure S8High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.00 MB EPS)Click here for additional data file.Figure S9High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.99 MB EPS)Click here for additional data file.Figure S10High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.99 MB EPS)Click here for additional data file.Figure S11High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.99 MB EPS)Click here for additional data file.Figure S12High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.00 MB EPS)Click here for additional data file.Figure S13High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.99 MB EPS)Click here for additional data file.Figure S14High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.00 MB EPS)Click here for additional data file.Figure S15High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.99 MB EPS)Click here for additional data file.Figure S16High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.00 MB EPS)Click here for additional data file.Figure S17High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.96 MB EPS)Click here for additional data file.Figure S18High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.00 MB EPS)Click here for additional data file.Figure S19High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(2.96 MB EPS)Click here for additional data file.Figure S20High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.40 MB EPS)Click here for additional data file.Figure S21High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 . Red lin(3.03 MB EPS)Click here for additional data file.


Recently, human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), encoded from a minus strand mRNA was discovered and was suggested to play an important role in adult T cell leukemia (ATL) development. However, there have been no reports on the role of HBZ in patients with HTLV-1 associated inflammatory diseases.We quantified the HBZ and tax mRNA expression levels in peripheral blood from 56 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 10 ATL patients, 38 healthy asymptomatic carriers (HCs) and 20 normal uninfected controls, as well as human leukemic T-cell lines and HTLV-1-infected T-cell lines, and the data were correlated with clinical parameters. The spliced HBZ gene was transcribed in all HTLV-1-infected individuals examined, whereas tax mRNA was not transcribed in significant numbers of subjects in the same groups. Although the amount of HBZ mRNA expression was highest in ATL, medium in HAM/TSP, and lowest in HCs, with statistical significance, neither tax nor the HBZ mRNA expression per HTLV-1-infected cell differed significantly between each clinical group. The HTLV-1 HBZ, but not tax mRNA load, positively correlated with disease severity and with neopterin concentration in the cerebrospinal fluid of HAM/TSP patients. Furthermore, HBZ mRNA expression per HTLV-1-infected cell was decreased after successful immunomodulatory treatment for HAM/TSP.in vivo expression of HBZ plays a role in HAM/TSP pathogenesis.These findings suggest that  Human T-cell lymphotropic virus type 1 (HTLV-1) is a replication-competent human retrovirus  which isAmong several HTLV-1 genes, a transcriptional activator Tax encoded in the pX region is thought to play a central role in immortalization, oncogenesis and inflammation through its pleiotropic activity . In HAM/in vivo. Recently, a novel basic leucine zipper protein encoded by the complementary strand of the HTLV-1 genome, named HTLV-1 basic leucine zipper factor (HBZ), was characterized [It has been reported that among fresh leukemic cells isolated from ATL patients, about 60% of cases do not express the tax transcript . In tax cterized . HBZ is cterized , promotecterized , supprescterized ,33, promcterized , and enhcterized .In this study, we investigated whether HTLV-1 HBZ mRNA expression is associated with clinical and laboratory markers reported in HAM/TSP patients, including HTLV-1 proviral load, neopterin concentration in cerebrospinal fluid (CSF), and motor disability score. In addition, to confirm the previous observations ,18, we hHuman leukemic T-cell lines  and HTLV-1-infected T-cell lines  were cultured in RPMI 1640 medium supplemented with 10% FCS. The diagnosis of HAM/TSP was done in accordance with World Health Organization criteria . The dia2 (25 mM), 20 μl dNTPs mixture , 5 μl random hexamers (50 μM), 2 μl RNase inhibitor (20 U/μl), and 2.5 μl (50 U/μl) Moloney murine leukemia virus reverse transcriptase were added to a total volume of 100 μl. Samples were incubated at 25°C for 10 minutes and 48°C for 30 minutes, and reactions were stopped by heating to 95°C for 5 minutes. Genomic DNA was extracted from the frozen PBMCs by QIAamp blood kit . We, then, carried out a real time quantitative PCR using ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems) to examine the HTLV-1 proviral load [6 PBMCs. Neopterin levels were evaluated by HPLC with fluorometric detection methods as described previously [®, Fujirebio, Japan).RNA was extracted from PBMCs using RNeasy Mini Kit with on-column DNase digestion  according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using TaqMan Gold RT-PCR Kit . For cDNA synthesis from extracted mRNA, 2 μg total RNA, 10 μl 10×TaqMan RT buffer, 22 μl MgClral load  and tax ral load  in PBMCsral load . Namely,ral load -39, but eviously . Serum HMotor dysfunction seen in HAM/TSP patients was evaluated by clinical neurologists according to the Osame Motor Disability Score (OMDS) , which gThe Mann-Whitney U test was used to compare data between two groups. Correlations between variables were examined by Spearman rank correlation analysis. Values of p < 0.05 were considered statistically significant.EcoRI digestion [EcoRI digestion in samples from two ATL patients, indicating at least the biclonal integration of HTLV-1 proviral DNA. The incidence of multibands in our cases (two out of ten: 20%) was comparable with the data by Kamihira et al. (17.8%). The number of HTLV-1 proviral load in MT-2 cells measured by our quantitative PCR method (16.2 copies/cell) was also comparable with the previous report (12.6 copies/cell) [A total of 56 HAM/TSP patients, 10 ATL patients and 38 HCs completed the evaluation. Twenty normal uninfected healthy controls (NCs) were used as negative controls. The HTLV-1 proviral load in this study represents the copy number of HTLV-1 tax per cell (for HTLV-1 infected cell lines) or PBMC   .The HTLV-1 proviral load was significantly greater in HAM/TSP patients  than HCs   by the HTLV-1 proviral load (i.e. HTLV-1 tax copy number per cell). As a result, neither tax nor the HBZ mRNA/DNA ratio differed significantly between each clinical group  figure . InteresTo investigate the mutual expression status of HBZ and tax mRNA in different clinical status, we calculated the ratio of HBZ mRNA/tax mRNA in 22 HAM/TSP patients, 11 HCs and 7 ATL patients, who express both tax and HBZ mRNA in PBMCs. HTLV-1 tax mRNA was not expressed in 60.7% (34 out of 56) of HAM/TSP patients, 71.1% (27 out of 38) of HCs and 30.0% (3 out of 10) of ATL patients, whereas HTLV-1 HBZ mRNA was expressed in all the infected individuals tested. As shown in figure To investigate the relationship between HTLV-1 mRNA load and various laboratory markers, HTLV-1 proviral load, CSF neopterin concentration and anti-HTLV-1 antibody titers were quantified and compared with motor dysfunction of HAM/TSP patients. Since neopterin is a low molecular weight pteridine compound released from macrophages upon stimulation with γ-interferon secreted by activated T cells, the measurement of neopterin concentrations in body fluids like blood serum, CSF or urine provides information about cellular immune activation in humans under the control of type 1 T helper cells . As show® by Sumitomo Pharmaceutical Co., Osaka, Japan) were administrated per intramuscular injection. Two patients (HAM1 and 2) showed marked clinical improvement with the changes of the OMDS, whereas two patients (HAM3 and 4) did not show clinical improvement (without the changes of the OMDS)  mRNA load and mRNA/DNA ratio before, during, and after interferon-alpha (IFN-α) treatment in four HAM/TSP patients who received 4 weeks of daily administration. Three million international units (IU) of IFN-α , whereas HTLV-1 HBZ mRNA was expressed in all the infected individuals tested. There was also a statistically significant correlation between HTLV-1 HBZ mRNA load and HTLV-1 proviral load both in HAM/TSP patients and HCs, whereas tax mRNA load correlated with the HTLV-1 proviral load only in HCs but not in HAM/TSP patients. Recently, Usui et al. reported a similar observation . Namely,When HTLV-1 tax or HBZ mRNA load was adjusted with HTLV-1 proviral DNA load , the amount of tax and HBZ mRNA expressed per provirus was not significantly different between HAM/TSP patients and HCs, suggesting that the higher HTLV-1 proviral load seen in HAM/TSP patients caused higher HTLV-1 HBZ mRNA expression. This is consistent with our previous study using different methods for mRNA and DNA quantification , but difAnother important observation is that the amount of HTLV-1 HBZ mRNA expression per provirus was more than a thousand times higher than tax mRNA expression both in HAM/TSP patients and HCs. Surprisingly, the amount of HTLV-1 HBZ mRNA expression per provirus was even higher in HTLV-1-infected PBMCs than in infected cell lines, whereas tax mRNA expression was significantly higher in cell lines than infected PBMCs. Since HBZ suppresses Tax-mediated viral transcription , the abuWe also found that the HTLV-1 HBZ mRNA load significantly correlated with the neopterin concentrations in CSF of HAM/TSP patients. Since neopterin levels in CSF have been used as an immunologic marker for monitoring disease activity and treatment efficacy of HAM/TSP ,42,56, tIn summary, we showed that spliced HBZ gene was transcribed in all the HTLV-1 infected individuals examined, whereas tax mRNA was not transcribed in more than half in the same groups. Moreover, our data demonstrated a significant correlation between HTLV-1 HBZ mRNA load and HTLV-1 proviral load, neopterin concentrations in CSF and motor disability seen in HAM/TSP patients, indicating that HTLV-1 HBZ mRNA load may be a valid predictor of disease progression. Our present findings suggest that HTLV-1 HBZ mRNA expression plays a role not only in ATL, but also in the pathogenesis of the HTLV-1-associated inflammatory disease HAM/TSP.The authors declare that they have no competing interests.MS designed and performed the experiments, analyzed the data, and wrote the paper; TM and KA provided clinical samples and assembled clinical database. YS and JY provided clinical samples and performed experiments. KS performed experiments, analyzed and interpreted data. MM made contribution to the conception and design of the study. YO contributed to obtaining funding and gave advice.Changes in HBZ mRNA load and HBZ mRNA/DNA ratio in PBMCs of HAM/TSP patients after IFN-α treatment.Click here for file


This Primer focuses on detection of the small interaural time differences that underlie sound localization. Animals constantly detect and encode the location of sound sources in the environment. In order to determine the location of sounds in the horizontal plane, or azimuth, the auditory system employs the interaural time differences (ITDs) that arise when sound reaches one ear before the other. Given the tiny time differences involved, animals are remarkably accurate at localizing sounds. The range of time differences that are useful to the animal depends on the head size—for humans, it's about 600 µs, for gerbils about 150 µs. Humans and barn owls are both localization champions, with an ability to resolve sounds about 2° apart ITDs are detected by specialized neurons that act as coincidence detectors in an area of the brainstem called the medial superior olive. The name reflects how these neurons work—they respond most reliably when they receive precisely synchronized, essentially simultaneous inputs from each ear. For coincidence detection to be useful in detecting very small time differences, the incoming sound information must first be encoded very precisely in the periphery, such that neurons fire action potentials in phase with the sound on a cycle-by-cycle basis. The phase-locked activity of the auditory nerve fibers in the periphery are relayed by the cochlear nucleus specialized for timing. The nervous system throws everything it's got at both making and keeping a temporally precise signal, from fast synapses to short sharp responses In 1948, Jeffress proposed a circuit for detection of interaural time differences Jeffress envisaged arrays of ITD detectors for each frequency band, each tuned to a different preferred ITD, so that the whole array could form a place map, also called a “labeled line” population code A landmark paper from McAlpine, Jiang, and Palmer (2001) focused on the relevance of the slope of the ITD curve to localization, and led to reexamination of ITD coding, especially a reevaluation of the Jeffress model's utility as a description of ITD coding It's not often that neurobiologists are able to generate such explicit hypotheses about neural coding, and there has been a great deal of excitement and discussion about how ITDs are detected and which of the various coding strategies are used. The slope and peak solutions for encoding ITDs are not inconsistent, since both depend upon coincidence detection and convey ITDs to the midbrain through the distribution of firing rates across the population of neurons. In the best studied examples, barn owls appear to use the information in both peaks and slopes of the tuning curves The original Jeffress model showed equal path length delay lines converging on an array of coincidence detectors . But stuPLoS Biology, Jercog et al. present a novel mechanism to compensate for the robust delay caused by the longer contralateral path length New computer-aided reconstructions of axonal connections suggest that the observed path lengths cannot account for the distribution of best delays in the cat in vivo, this difference would not be so large. With a brain temperature near 38°C and a Q10 of 1.8 in vivo conduction velocity closer to 7 m/s and a shorter delay between ipsilateral and contralateral inputs.The Jercog et al. data showed that there are asymmetries in the synaptic inputs to the coincident detector neurons such that the contralateral compound postsynaptic potential had a slower rise time than the ipsilateral postsynaptic potential. Why? Either there is an intrinsic biophysical difference between the two sets of dendrites , or there is more variation in the arrival times of the contralateral inputs. The second is more plausible, principally because Jercog et al. stimulate the ipsilateral inputs relatively close to the recording site, while the contralateral stimulating electrode is further away. Thus, any intrinsic variability in conduction velocity in the contralateral input bundle would be magnified. The authors performed their experiments in gerbil brain slices that preserved connectivity between the coincidence detector  and its inputs from the two cochlear nuclei . They obThese measurements made by Jercog and colleagues go a long way toward explaining the measured distributions of best ITDs in gerbils less than the monaural firing rates. Thus coincidence detectors behave more like biophysical AND-gates than simple summation devices. Several mechanisms contribute to this effect, including the segregation of the ipsilateral and contralateral inputs onto different dendrites and the shunting of the postsynaptic current via a critically important potassium channel conductance with a low activation threshold (GKLT) KLT activates with only small depolarization KLT, coincidence detectors typically have very low input resistances and thus very rapid responses to changes in voltage (τm of 0.3–1.5 ms). Spike triggering is very sensitive to the rate of rise of the voltage in these and other coincidence detector neurons Most neurons respond best when their inputs arrive simultaneously, because of spatial and temporal summation. What sets ITD coincidence detection apart is the narrowness of the summation window and the exclusion of nonsynchronous inputs. True coincidence detectors also require more than simple summation—they should fire when inputs from two ears coincide, but not when two inputs from the same ear coincide. A neuron that sums its inputs linearly would not be able to distinguish between these two scenarios. The neurons in the ITD circuit meet this criterion: the minimal firing rate (in the trough of the tuning curve) that occurs during out of phase binaural stimulation is actually KLT, on its amplitude and activation dynamics. The longer the initial rise of the compound synaptic potential, the more time GKLT has to activate, the larger the conductance will be at the time of the peak in the EPSP, suppressing the voltage response, and reducing the likelihood of firing an action potential.Together, synaptic and intrinsic mechanisms determine the time window for coincidence detection. Direct electrical stimulation of the synaptic inputs in bird slices have shown this window is quite narrow, with a symmetry around zero delay, and with excitatory postsynaptic potentials (EPSPs) with similar kinetics for contralateral and ipsilateral synaptic inputs KLT in opposite dendrites. Certainly, the interplay of excitation and inhibition can shift ITD tuning curves. In the final analysis, biophysical examination of coincidence detection offers an outstanding opportunity to ask precise questions about neural coding.Thus, although the source(s) of the asymmetry in medial superior olive inputs remains open to debate, one major point emerges from the Jercog et al. study, which is that asymmetry in bilateral EPSP shapes could greatly influence coincidence detection and neural codes for ITD. In vivo, this asymmetry could come from almost anywhere—variation in the phase locking of the inputs, variation in the best frequency of the inputs, variation in axonal properties of the inputs, changes in inhibitory synaptic inputs, and differential expression of I


In these experiments, we did not detect HTLV-I or HFV DNA sequences in any thymoma or control tissues, despite adequate sensitivity of our assays . Additionally, none of 14 thymoma patients evaluated serologically for HTLV I/II infection was positive by enzyme-linked immunoassay (ELISA), while five (36%) had indeterminate Western blot reactivity. In comparison, one of 20 US blood donors was HTLV-I/II ELISA positive, and nine (45%) donors, including the ELISA-positive donor, had indeterminate Western blot reactivity. Western blot patterns varied across individuals and consisted mostly of weak reactivity. In conclusion, we did not find evidence for the presence of HTLV-I or HFV in US thymoma patients.The cause of thymoma, a rare malignancy of thymic epithelial cells, is unknown. Recent studies have reported the detection of DNA from human T-cell lymphotropic virus type I (HTLV-I) and human foamy virus (HFV) in small numbers of thymoma tumours, suggesting an aetiologic role for these retroviruses. In the present study, we evaluated 21 US thymoma patients and 20 patients with other cancers for evidence of infection with these viruses. We used the polymerase chain reaction to attempt to amplify viral DNA from tumour tissues, using primers from the  Thymoma is a rare malignancy of thymic epithelial cells of unknown aetiology. Thymoma usually arises in middle-aged or elderly adults . The tumOf interest, recent reports have suggested that two retroviruses could play a causal role in thymoma. DNA sequences from human T-cell lymphotropic virus type I (HTLV-I), the viral cause of adult T-cell leukaemia/lymphoma, were detected in tumour tissue obtained from 11 Italian thymoma patients . NonetheIn the present US-based investigation, we sought to confirm the previously reported detection of these two retroviruses, HTLV-I and HFV, in thymoma tumours. We also evaluated patient sera for HTLV-I and HTLV-II antibodies.n=1), B1 (n=9), B2 (n=6), B3 (n=3), and C (n=1). By Masaoka stage (n=5), II (n=8), III (n=2), IVa (n=4), and IVb (n=1). Four patients (20%) had myasthenia gravis, while five patients (25%) had a history of or developed additional cancers during clinical follow-up . Serum samples were available for 14 of these 21 patients.The study included archived tumour samples (stored at or below −70°C) from 21 thymoma patients treated at the Indiana University Cancer Center. Clinical data were unavailable for one patient. For the remainder, 10 were female, and the median age at thymoma diagnosis was 48 years (range 22–76). A total of 18 (90%) were white, two were black (10%), and all were born in and resided in the US. By World Health Organization histologic classification  from US patients with colon cancer (five cases), breast cancer (seven), or lung cancer (eight). For antibody studies, we used sera from 20 US blood donors as controls. All specimens were anonymised and tested only after delinking from clinical information.μl proteinase K solution (20 mg ml−1) at 55°C. Subsequently, 4 μl of RNase A solution (100 mg ml−1) was added and incubated at 37°C for 10 min. The mixture was then placed on a QIAamp spin column. DNA was eluted after two wash steps, and spectrophotometry was used to quantify DNA and assess its purity. DNA yield was similar for thymoma tissues and control specimens . Extracted DNA was of high purity .DNA extraction and polymerase chain reaction (PCR) experiments were performed simultaneously for thymoma cases and controls. DNA was extracted from tissues using the DNeasy Tissue Kit  according to the manufacturer's instructions. Briefly, for each specimen, 15–20 mg of minced tissue was lysed overnight in Tissue Lysis Buffer and 20 pol and tax (HTLV-I) and gag and bel1 (HFV) regions. Specifically, HTLV-I sequences were amplified using SG231/SG238 for pol     , 1.5 mM MgCl2, 250 μM of each dNTP , 2.5 U of AmpliTaq Gold polymerase , and 25 pmol of each primer. Polymerase chain reaction amplification was performed as follows: preheat at 95°C for 10 min, 40 cycles of amplification , followed by a final elongation step at 72°C for 10 min. Amplified products were analysed by 2% agarose gel electrophoresis with ethidium bromide staining. Positive controls were serial 10-fold dilutions of DNA from the HTLV-I-infected T-cell line MT1 or HFV plasmid pcHSRV2/2 , calculated to contain 3 × 104–3 × 10−1 copies of HTLV-I DNA or 106–101 copies of HFV DNA per reaction, respectively.For PCR detection of HTLV-I and HFV, we used primers from the Protective clothing, dedicated equipment, newly prepared reagents and primers, and UV irradiation were used to prevent PCR contamination. Sample preparation, PCR amplification, and postamplification processing were performed in separate rooms. Additionally, to prevent contamination by laboratory controls, all PCR experiments were performed twice: the first PCR experiments were performed in the absence of positive controls and titration series (‘suicide’ PCR) (env proteins) and p19 and p24 (gag proteins). For HTLV-II, bands of 2+ intensity for recombinant gp46II (env) and p24 (gag) were considered diagnostic. Weak reactivity  on these bands or reactivity only for other viral proteins was considered indeterminate for HTLV-I/II infection.Sera were tested for HTLV-I and HTLV-II antibodies by enzyme-linked immunoassay  and Western blot  according to the manufacturer's protocol. Western blot bands were scored on a grey scale from 0 (absent) to 10 (maximum intensity). For HTLV-I, Western blot reactivity was defined as the presence of bands (2+ intensity) for recombinant gp46I and gp21 (pol and tax) and HFV (gag and bel1) were all negative. These experiments were performed twice for each specimen , with complete concordance. Figure 1pol (panel A) and HFV bel1 (panel B). In these experiments, serial dilutions of the positive control DNA demonstrated that PCR could identify three HTLV-I copies and 10 HFV copies per reaction  had reactivity to both gag and env (but this reactivity did not meet our criteria for Western blot positivity), and one donor had reactivity only to Western blot proteins other than gag and env.By ELISA, 14 of 14 thymoma patients and 19 of 20 blood donor controls were HTLV-I/II seronegative, while one blood donor (BD15) was HTLV-I/II seropositive. By Western blot, none of the 35 evaluated subjects was HTLV-I or HTLV-II seropositive. Indeterminate Western blots were observed in five thymoma patients (36%) and nine blood donors (45%); in most of these subjects, reactivity was weak . A DNA sequence corresponding to the HTLV-I regulatory gene tax was amplified from most cases , whereas DNA corresponding to the structural gene pol was found in fewer tissues . Additionally, sera from 83 other myasthenia gravis patients were studied by the same group (gag protein p19).The single prior report of HTLV-I detection in thymoma was from Italy  was the same in both studies. In our experiments, we would have detected as few as three HTLV-I pol or tax copies in 500 ng genomic DNA, equivalent to one copy per 25 000 cells, had the virus been present. Thus, our assays were sufficiently sensitive to rule out the presence of HTLV-I in these specimens.The reasons why our findings regarding HTLV-I, which were convincingly negative, differ from those of Manca et al) and four blood donors (20%). The limited-spectrum and mostly weak-intensity Western blot reactivity, coupled with the negative ELISA results, indicates that thymoma patients were infected with neither HTLV-I nor HTLV-II.Similarly, we did not find evidence for a specific HTLV-I or HTLV-II antibody pattern in thymoma patients. One blood donor control had a positive ELISA result and indeterminate Western blot, suggesting that he might have been exposed to or infected with HTLV-I or HTLV-II. All other subjects were ELISA negative, and overall, equivalent proportions of thymoma patients and blood donor controls manifested indeterminate Western blots. In our study, p21 seroreactivity was weak and observed in only one thymoma patient . All four cases also had low-titer neutralising antibody against HFV. Attempts in both studies to isolate HFV were unsuccessful  perfectly matches the published SFVcpz gag sequence (Using primers designed to detect HFV sequences, we could not confirm these prior reports for US thymoma patients. With the same In conclusion, we did not find evidence for HTLV-I or HFV infection in US thymoma patients. It would be of further interest to study the relationship between HTLV-I, thymoma, and myasthenia gravis in geographic areas where HTLV-I is endemic. Since the cause of thymoma is unknown, further searches for a viral aetiology may be warranted.


CD209) gene promoter region  in DNA samples from four Brazilian ethnic groups  to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that −336A and −139A SNPs were quite common in Asians and that the −201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the −336A variant was more frequent in HTLV-1-infected patients  than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218–5.179). In addition, the −139A allele was found to be associated with protection against HTLV-1 infection  when the HTLV-1-infected patients as a whole were compared with the healthy-control group. These observations suggest that the −139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs −336 and −139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections.This study evaluated four polymorphisms located in the DC-SIGN ( CD209 gene family, is a receptor on DCs that binds to ICAM-3, which is expressed on T cells to facilitate the initial interaction between DCs and T cells (CD209 gene promoter region (position −336) affect its activity by modifying gene transcription significantly. Therefore, variations in this region may have an influence on the pathogenesis of human infectious diseases.Dendritic cells (DCs) are professional antigen-processing cells that act against invading pathogens , being iHuman T-cell lymphotropic virus type 1 (HTLV-1) was the first retrovirus isolated from humans . We also compared SNP distribution between HTLV-1-infected individuals and a control group to determine whether the frequencies of CD209 polymorphisms influence the susceptibility of HTLV-1-infected individuals to developing HAM/TSP.On this basis, the objective of the present study was to investigate the distribution of four single-nucleotide polymorphisms (SNPs) located in the Blood samples were collected from four ethnically distinct Brazilian populations: 45 from Caucasian individuals , 25 from Afro-Brazilians , 28 from Asians  and 25 from Amerindians . The Caucasian, Afro-Brazilian and Asian groups were selected at random from blood donors of the Regional Blood Center of Ribeirão Preto and Núcleo de Haemoterapia of Franca, Brazil. Only individuals who reported the absence of racial admixture in their four grandparents were included in the study. The Amerindian samples were collected from Xavante Amerindians who live in Campinápolis city, Mato Grosso state, Brazil.tax and LTR regions). The total HTLV-1 group consisted of 66 patients , most of whom were Caucasian (77.3 %). The HTLV-1 group was classified into healthy asymptomatic HTLV-1 carriers (HAC) and patients with HAM/TSP. The HAC group consisted of 33 individuals  and the HAM/TSP group consisted of 33 patients . Both groups were mainly of Caucasian origin . The admixed control group consisted of 32 randomly selected blood donors from the same geographical region as the HTLV-1-positive individuals described above, with a mean age of 34.8 years ; the ethnic background was evaluated based on skin colour and individuals were mainly Caucasians (62.5 %) and African descendants (34.4 %). The study was approved by the Institutional Ethics Committee of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo (process number 7639/2005) and all subjects gave written, informed consent to participate.The HTLV-1-seropositive patients studied here were detected among blood-donor candidates of the Regional Blood Center of Ribeirão Preto and among patients of the Neurology Department of the University Hospital, São Paulo, Brazil. Diagnosis of HTLV-1/2 infection was established by antibody screening of serum/plasma samples using an enzyme immunoassay , followed by confirmation by PCR  and diluted in 200 μl ultrapure water. All samples were quantified and diluted to 0.1 μg DNA μl−1.A 5 ml blood sample was collected into EDTA-containing tubes and plasma was separated under aseptic conditions by centrifugation at 900 CD209 gene promoter was amplified in all samples under study by using the primers 5′-CAAAAATGAGGACAGCAGCA-3′ and 5′-CTCCAAGGAACCAAGACTGC-3′, which delimit the promoter region between nt −423 and 28 (defined from GenBank accession no. NC_000019) and amplify a fragment of 452 bp. Reactions were performed in 25 μl mixtures containing 100 ng DNA, 1.0 U Taq DNA polymerase (Invitrogen Life Technologies), 50 mM KCl, 20 mM Tris/HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM each dNTP and 0.3 pmol each primer. Thermocycling was performed in a GeneAmp PCR system 9700 (Applied Biosystems) under the following conditions: an initial cycle at 95 °C for 5 min and 35 cycles at 95 °C for 40 s, 62 °C for 40 s and 72 °C for 60 s, with a final extension step of 72 °C for 10 min. The amplified products were analysed by 1 % agarose gel electrophoresis followed by ethidium bromide staining.For genotyping, the Sequencing reactions were performed using PCR primers and a DYEnamic ET dye terminator kit  according to the manufacturer's instructions. Electrophoresis was carried out using an automated apparatus MegaBace DNA sequencing system 1000 (Amersham Biosciences). The electropherograms were analysed by using Sequence Analyser software version 1 (Amersham Biosciences). Consensus sequences were obtained and compared by multiple alignments, using GenBank accession no. NC_000019 as the prototype sequence.http://www.phrap.org). Multiple alignments were produced by using the polybayes multiple-alignment algorithms . This same method was used to compare the frequencies of SNPs among the ethnic groups. Allelic frequencies and observed heterozygosity were computed by a direct counting method. Adherences of genotypic proportions to expectations under Hardy–Weinberg equilibrium were tested by the exact test of genepop version 3.4 software  and its 95 % confidence interval (CI).CD209 promoter region at positions −336, −332, −201 and −139 that are known to be associated with susceptibility/resistance to pathogens. The allelic frequencies of these SNPs for Brazilian ethnic groups and other populations previously described are presented in Table 1CD209 promoter region fitted the Hardy–Weinberg equilibrium expectations for all groups.We investigated four polymorphisms located in the The −336G allele showed higher frequencies in Afro-Brazilians (42.0 %) and Caucasians (26.7 %) than in Asians (5.4 %). In addition, this allele was not observed in the Amerindian group, and the admixed control group presented frequencies similar to those of the Caucasian group  and was present in the control group at a frequency of 1.6 %. Analysis of the −139A/G position showed that the allelic frequency of SNP −139A was higher in Asians (62.5 %) than in Afro-Brazilians (26.0 %), Caucasians (35.6 %) and Amerindians (30.0 %). The Brazilian control group showed frequencies similar to those of the Caucasian group (Table 1P=0.2835± 0.0060), between Afro-Brazilians and Zimbabweans (P=0.5503±0.0080) or sub-Saharan Africans (P=0.5018±0.0071), and between Brazilian Asians and Asians (P=1.0000±0.0000).An exact test of allelic frequency differentiation, performed based on the frequencies of SNPs −336 and −139, did not reveal any difference between Brazilian Caucasians and European Caucasians (P=0.0138±0.0005) and –139 (P=0.0000±0.0000), and between SNP −332 and SNP −139 (P=0.0028±0.0002). Given the positive association but unknown gametic phase, the phase and em algorithms were used to reconstruct CD209 haplotypes  or HTLV-2. The sex, age and ethnic background distribution of the blood donors are shown in Table 3P=0.1125±0.0079, P=0.2812±0.0089 and P=0.0916± 0.0062, respectively).Table 4P=0.0035) and genotype (P=0.0055) frequencies compared with the controls. However, no differences were found between the HAC and HAM/TSP groups. In order to detect a possible influence of specific alleles or genotypes, 2×2 contingency tables were created to compare the frequencies using Fisher's exact test.An exact test of population differentiation based on the allele and genotype frequencies revealed that the total HTLV-1 group showed differences in the allele . Likewise, the −336GG genotype was found to be associated with protection against HTLV-1 infection when the HTLV-1 group as a whole was compared with the healthy-control group . On this basis, it is plausible to assume that the −336G allele is the variant conferring resistance to HTLV-1 infection. When the asymptomatic HTLV-1-infected patients were compared with the HAM/TSP group, no difference was found regarding this polymorphism (P=0.1353)   and the second one a lower frequency  in patients   when the HTLV-1-infected patients as a whole were compared with the healthy-control group. Likewise, the −139AA genotype was associated with protection against HTLV-1 infection . No differences were found between the asymptomatic group and the HAM/TSP group regarding this polymorphism (P=0.6666). However, the frequency of the −139A allele was considerably lower in the asymptomatic and HAM/TSP groups compared with the healthy-control group   , which may indicate a possible additive or co-dependent effect for both polymorphisms in the resistance/susceptibility to HTLV-1. In order to determine such interactions, haplotypes for each sample were estimated by two distinct methods without taking into account any prior information. Table 5P=0.0000±0.0000). As observed for the allele and genotype frequencies, there was no difference between the HAM/TSP and the asymptomatic group (P=0.3328±0.0206), but both HTLV-1 groups did differ from the healthy-control group .An exact linkage-disequilibrium test taking into account all samples revealed a high linkage disequilibrium between SNPs −336 and −139 , whilst haplotype −336A/−332G/−201G/−139G was associated with susceptibility to HTLV-1 infection . It is noteworthy that both haplotypes conferring protection carried a single dose of a protective allele (−336G or −139A), as well as a single dose of a susceptibility allele (−336A and −139G), whilst the susceptibility haplotype carried a double dose of the susceptibility alleles (−336A and −139G)  in distinct Brazilian ethnic groups and also their association with HTLV-1 infection.We have described here for the first time the distribution of genetic polymorphisms in the In the Brazilian population studied here, the −336G allele was more frequent in the Afro-Brazilian group than in the Caucasian and Asian groups. However, the GG genotype was not detected in the Asian group and most subjects (86 %) were AA homozygotes, with only 13 % being heterozygotes. These data agree with previous studies that reported a higher frequency of the −336G allele in populations of African descent . This variant has recently been described by The −201T allele was exclusively detected in the Afro-Brazilian population and has been reported only in African ethnic groups  than HAC (9 %) individuals compared with controls (30 %). The absence of the GG genotype in the HTLV-1-infected group suggests that this −336G allele is a variant that confers protection against HTLV-1 infection. Thus, the presence of the −336G allele would result in lower DC susceptibility to HTLV-1 in the initial stages of infection, possibly protecting against HTLV-1 infectivity.CD209 gene has been studied extensively. In agreement with the present results, the −336G allele has been associated with reduced DC-SIGN expression on the DC surface and in macrophages, as position −336 is located close to the major transcription site, possibly affecting binding of the transcription factor Sp1 and other factors that modulate transcriptional activity (CD209 were investigated and no significant association was found when the genotypes and alleles of infected individuals were compared with those of controls or when the HAC and HAM/TSP groups were compared.Similarly, in the present study, polymorphisms at positions −332 and −201 of the promoter region of For the SNP at position −139, allele A was found to be associated with protection against HTLV-1 infection when the infected and control groups were compared. Thus, these results suggest that this SNP may be associated with HTLV-1 transmission and not with development of the associated clinical manifestations. Previous studies have already demonstrated a higher frequency of allele −139A in individuals not infected with HIV compared with infected patients (Comparison of the HTLV-1 group and the healthy-control group showed that haplotypes −336G/−332G/−201G/−139G and −336A/−332G/−201G/−139A were associated with protection against HTLV-1 infection, whilst haplotype −336A/−332G/−201G/−139G was associated with susceptibility to HTLV-1 infection. These associations suggest that these polymorphisms may influence only the acquisition of HTLV-1 and not the associated diseases. Comparison of the HAM/TSP and asymptomatic groups with healthy controls revealed the presence of the same haplotypes conferring protection and susceptibility against HTLV-1, suggesting that these polymorphisms also have a hitchhiking effect on HTLV-1 acquisition.CD209 gene may not be involved directly in triggering the development of HAM/TSP. However, the differences in SNP distribution observed between HTLV-1-infected individuals and controls suggest that these SNPs in the promoter region of CD209 may be associated with the risk of HTLV-1 infection. Thus, the variations observed at positions −336 and −139 in the lectin DC-SIGN may be of crucial importance in the susceptibility/transmission of HTLV-1 infection.In conclusion, the variations detected in the promoter region of the


Trophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs). While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer. The signals that control cell fate determination and coordinate the development of the organs needs to be exquisitely regulated in both time and space. Activation of RTKs by their cognate trophic factors is among the signals that are critically involved in morphogenesis, inducing signaling pathways that control cellular processes such as cell proliferation, differentiation, migration and survival . Thus, tRTKs are single spanning transmembrane proteins possessing an intrinsic kinase activity. Upon ligand binding, the kinase is activated and autophosphorylates itself on tyrosine residues located within the cytoplasmic tail, creating docking sites for proteins containing phosphotyrosine-binding domains and forming the starting-point for a variety of different signaling cascades that regulate cell physiology. In particular, Ras-Erk/MAP kinase and phosphatidylinositide-3 kinase (PI3K)-AKT pathways represent two critical signaling cascades induced upon the activation of RTKs by trophic factors .The deregulation of approximately fifty percent (30 of 58) of the genes known to encode RTKs are associated with human tumors . SeveralRTKs coordinate a wide variety of biological processes and are therefore subjected to multiple levels of control. Multiple modes of action have been described to inhibit RTK signaling. In In Drosophila, activation of epidermal growth factor receptor (EGFR) homologue, DER is strictly regulated. DER is a receptor tyrosine kinase required for developmental processes throughout life cycle  and diffAlthough we do not know yet whether a mammalian Argos equivalent exists, the mammalian protein structurally related to Kekkon1, LRIG1 (leucine-rich repeats and immunoglobulin-like domain 1) has been described. Interestingly, LRIG1 has been shown to inhibit mammalian EGFR activation by a different mechanism see .Even though evidences for RTK regulation by ligand sequestration come from studies made in Drosophila, they suggest new possible strategies for the design of novel anti-oncogene agents. Future studies are required to develop new reagents that can neutralize RTK ligands, which overexpression is involved in the development of different malignancies.During the last years, a new mechanism of RTK negative regulation was described . In contInhibition of RTK autophosphorylation can be achieved by protein tyrosine phosphatases (PTPs). Several studies have shown that PTPs specifically dephosphorylate certain subsets of phosphorylated tyrosines on RTKs that have multiple phosphorylation sites, indicating a certain degree of selectivity . Severalin vitro and in vivo. Binding of SAP requires Trk receptor activation and phosphorylation of the tyrosine 674, which is located in the activation loop of the kinase domain. Moreover, overexpression of SAP attenuates tyrosine phosphorylation of Trk receptors and suppress NGF-induced neurite outgrowth  receptor, TrkA. SHP-1 interacts with TrkA at tyrosine 490 and controls both the basal NGF-stimulated level of TrkA activity in developing peripheral neurons . Anotherutgrowth .During the last years Mitogen-inducible gene 6  was identified as a feedback inhibitor of different RTKs. Several studies indicate that Mig6 can attenuate mitogen signaling induced by EGF, Heregulin (HRG-β) and Hepatocyte growth factor (HGF/ MET) . The molErbB2 gene containing a truncated extracellular domain. Herstatin has been shown to disrupt receptor dimerization and reduce ErbB2 receptor phosphorylation  family of proteins has emerged as a major class of trophic factor-inducible antagonists of RTK signaling. In particular, Sprouty proteins appear to specifically inhibit the Ras-Raf-Erk1/2 pathway, leaving the PI3K and other MAPK pathways intact . The negSprouty genes during normal development and multiple modes of action of the Sprouty proteins in the regulation of RTK-induced responses. As a negative regulator, Sprouty itself is subject to tight control at multiple levels. Specifically, growth factors increase the levels of the Sprouty transcripts, regulate the recruitment of Sprouty proteins to the plasma membrane and modulate Sprouty activity through rapid and transient tyrosine phosphorylation (Y55)  . In parton (Y55)  However,on (Y55) . Therefoon (Y55) . This nofgf genes). This newly identified antagonist encodes a putative Type I transmembrane protein that is conserved across zebrafish, mouse and human (Sef (hSef) gene alter the subcellular localization of this protein and diversify the repertoire of RTKs to be inhibited  is another attenuator that has been implicated in negative signaling by RTKs. This phosphatidylinositol phosphatase is implicated in negative signaling that specifically inhibits PI3K-Akt signaling pathway triggered by RTKs. This pathway is a key regulator of cell proliferation, motility and survival. The activity of Akt is regulated by PI3K via the synthesis of phosphatidyl inositol 3, 4, 5-triphosphate (PIP3). PTEN antagonizes PI3K by degrading PIP3 to phosphatidyl inositol 4, 5-biphosphate (PIP2). Deregulation of Akt through loss of functional PTEN has been implicated in the progression of different tumors . In agreDown-regulation of RTKs is an irreversible mechanism of inhibition that regulates the extent of the signal by removing activated receptors from the plasma membrane. Once activated, RTKs are ubiquitinated, internalized and targeted for degradation to the lysosomal compartment or locally destroyed in proteasomes. Many of these processes are regulated by ubiquitination, a post-traslational modification where the small protein ubiquitin is covalently attached to a target protein. While poly-ubiquitination marks proteins for proteasomal degradation, mono- or multi-ubiquitination is sufficient to direct endocytosis and lysosomal degradation of membrane receptors .Central to the process of receptor down-regulation are the roles of Cbl and Nedd families of ubiquitin-protein ligases, which act through limiting signal propagation independently of new transcriptional events . RecentlConsistent with its role in the down-regulation of RTKs, dominant negative mutants of c-Cbl lacking ubiquitin ligase activity have been identified in mouse tumors . In addiRecent studies have also linked RTK ubiquitination to receptor endocytosis . A numbeLrig1 gene in mice resulted in fertile animals that develop skin defects, suggesting involvement in EGFR signaling regulation  protein as an endogenous inter-actor of c-Cbl . LRIG1 igulation . Based ogulation .Based on the premise that the LRR domain is the critical interacting domain between LRIG1 and the EGF/ErbB receptor family, Finally, Decorin represents another example of the negative regulation of EGF/ErbB receptors by proteins containing leucine-rich repeats. Decorin is a secreted proteoglycan molecule that acts as an inhibitor of mammalian EGFRs . In partOver activation of RTK signaling is a common feature of cellular transformation and malignancy. Based in this concept, several groups began investigating the role of negative regulators of trophic factor-mediated signaling in cancer. They described that the expression of specific RTK attenuators is down-regulated in different types of human cancer. These studies led to the identification of specific tumor-suppressor genes, whose dysfunction or their down-regulation results in promoting malignancy. In this section, we highlight recent progresses in understanding the defective attenuation of RTK signaling in cancer and discuss their potential for the development of effective therapeutic approaches. In particular, we provide evidences for a role of the protein attenuators: PTEN, Mig6, LRIG1 and Sprouty in human cancer.Pten is embryonic lethal in mice. However, Pten +/− mice show hyperplastic-dysplastic features and are highly susceptible to develop epithelial tumors . In melanoma, PTEN mutations also appear to be associated with late stages of the disease  . Severall tumors . Recentl disease . Differe disease . Several disease . Interes disease .ErbB2 receptor and the subsequent inhibition of its downstream PI3K-Akt signaling pathway  and Spry2 (96%) have been observed. These findings were confirmed by real time PCR . DiffereRecent advances in the understanding of the mechanisms involved in down-regulating RTKs and restricting their signaling has been summarized above. The overall picture that emerges from these studies indicates that the mechanisms of regulation occur at numerous levels, including ligand binding, receptor autophosphorylation, induction of inhibitory proteins that counteract downstream signaling pathways and receptor endocytosis and degradation. Another important concept is related to the fact that negative feedback loop is one of the mechanisms that has evolved to provide an effective way of controlling RTK-mediated signaling.Conclusions derived from the work outlined in this review indicate that RTK activity is tightly controlled through the coordinated action of several negative protein regulators that function at multiple levels of the signaling cascade, and at different time-points after receptor engagement. Recent evidence also demonstrates that certain inhibitors have multiple mechanisms of action that depend on the cellular context and the identity of the RTK inhibited.In addition to protein attenuators, microRNAs (miRNAs) have emerged as an abundant class of small (approx. 22-nucleotides) non-protein-coding RNAs that play an important role in the negative regulation of gene expression, controlling the translational efficiency of target mRNAs . MicroRNin vivo function.Many important aspects of RTK signaling inhibition still remain unanswered. One significant issue that requires a more detailed investigation is the exact identification of the signaling pathways regulated by these inhibitory molecules and, particularly, their Pten in the mouse central nervous system can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD (The elucidation of the mechanisms that control RTK activation is today seen as one of the major challenges in biomedical science. Neurotrophic factor signaling through their RTK receptors play critical roles in the development of the nervous system, in the survival and maintenance of specific subpopulations of differentiated neurons. While the over activation of RTK signaling due to impaired deactivation of RTK signaling is associated with cancer, it is also possible that alterations in these control mechanisms could contribute to the pathogenesis of neurodevelopmental diseases and neuro-degenerative disorders. In particular, individuals with germline PTEN mutations display brain disorders including macrocephaly, seizures and mental retardation . Interesuman ASD . TherefoFinally, a more complete understanding of these emergent mechanisms will have wide implications in medicine, particularly in the identification of tumor-suppressor markers and in the design of efficient therapeutic approaches to human diseases.


Adult T-cell leukaemia (ATL) is caused by the human T-cell lymphotropic virus type 1 (HTLV-1). HTLV-1 has elaborated strategies to persist and replicate in the presence of a strong immune response. In this review, we summarise these mechanisms and their contribution to T-cell transformation and ATL development. Deltaretrovirus genera of the Orthoretrovirinae subfamily. HTLV-1 is the first discovered human retrovirus, isolated in the early 1980s from peripheral blood samples of a patient with cutaneous T-cell lymphoma . HTLV-2 was identified in cell lines derived from a patient with atypical hairy cell leukaemia although further studies failed to confirm the association of HTLV-2 with lymphoproliferative diseases. HTLV-3 and -4 subtypes have recently been identified in bushmeat hunters in Central Africa; infection by these two viruses being presently not associated to any pathology (Human T-lymphotropic virus type-1 (HTLV-1) belongs to the lymphoma . HTLV-1 athology . BLV is Most HTLV-1 carriers remain infected lifelong without developing any major clinical manifestation. After several decades, only a small proportion  of HTLV-1-infected subjects will progress to ATL. The term ATL includes a spectrum of diseases that are referred to as smoldering, chronic, lymphoma and acute. ATL patients have atypical lymphoid cells with multilobulated nuclei  in their peripheral blood. ATL cells are consistently monoclonal with respect to proviral integration and originate from initial polyclonal/oligoclonal expansion of HTLV-1-infected cells. Leukaemia may progress from a smouldering phase to chronic and acute clinical manifestations. Acute and lymphoma subtypes show aggressive and rapidly fatal clinical courses with a median survival time of about 1 year. Although tumour cells are sensitive to conventional chemotherapy, patients rapidly relapse and become resistant to further treatment. Chronic and smouldering stages have a more indolent course and do not require chemotherapy  flanked by two long terminal repeats (LTRs). The long terminal repeats (LTR) are subdivided into three regions  that contain the cis-acting elements essential for viral gene expression: transcription factor-binding sites, transcription start and termination sites, polyadenylation and splicing sites. A region called pX, which is located between the env gene and the 3′-LTR, contains at least four partially overlapping reading frames (ORFs) encoding accessory proteins , the post-transcriptional regulator REX (ORF III) and the TAX transactivator (ORF IV). In addition, HBZ is encoded from the 3′ LTR in the complementary strand of the genome , HTLV-1 expresses multiple gene products by using both strands of its proviral genome and complex events of mRNA splicing . The HTLin vitro, stimulate colony formation in soft agar and produce tumours in transgenic mouse models. Within the cell, TAX exerts its functions by complex formation with more than a hundred cellular proteins (TAX (p40) activates viral transcription by interacting with triplicate enhancer elements (TxRE) located in the 5′-LTR. TAX is also a major factor mediating viral persistence and disease development. The oncogenic potential of TAX is supported by its ability to immortalise cells proteins . AltogetκB and AP1 are thought to have predominant roles in T-cell proliferation and transformation. Molecular mechanisms sustaining these TAX activities have been reviewed recently /IL2 receptor (IL2R), IL9, IL13 and IL15/IL15R as well as members of the tumour necrosis factor receptor family. Thereby, TAX provides stimulatory signals resulting from the binding of ligands to their receptors and promotes cell proliferation as well as the initiation and maintenance of transformation , TAX interacts with hDLG, which binds to the adenomatous polyposis complex (APC) tumour suppressor and enforces G0/G1 cell cycle arrest. To override regulatory functions of hDLG, TAX induces its hyperphosphorylation , located at the 3′-end of sense viral mRNAs. REX promotes the export of the unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm and inhibits splicing and transport of doubly spliced RNAs from the pX region (REX (p27) is an RNA-binding post-transcriptional regulator that binds specifically to its X region .I, p13II, p21II, p30II) are encoded by different pX mRNAs formed by alternative splicing events. p12 localises to the endoplasmic reticulum (ER) and regulates calcium-dependent signalling thereby activating nuclear factor of activated T-cell (NFAT)  production (The recently identified HBZ (HTLV-1 bZIP) factor acts as a negative regulator of TAX-mediated viral transactivation by heterodimerising with CREB, CREB2 and p300/CBP , neuropilin 1 (NRP1) and surface heparan sulfate proteoglycans (HSPGs) are cell surface receptors for HTLV-1 and are required for efficient entry (Although HTLV-1 infects many cell types + T-cells in a biphasic manner (de novo-produced virus from infected DCs (cis-infection).Transmission of HTLV-1 occurs through transfer of infected cells: from mother to child during breast-feeding, via sexual intercourse and through exposure to infected blood products or sharing of needles and syringes . Efficieex vivo  are required for efficient infectivity, viral spread and persistence in vivo . In conthbz mRNA to upregulate E2F1 gene transcription might supplant TAX-mediated release of E2F to promote continuous cell proliferation in latter steps of viral persistence  HTLV-1 persistence relies on a subtle equilibrium regulated by the expression of viral proteins stimulating cell-growth in infected cells and their elimination by cytotoxic T-lymphocytes; (2) TAX and HBZ cooperate to promote infected cell proliferation in the early and late steps of infection and (3) silencing of viral expression allow escape from immune response.in vitro and in vivo. TAX has both clastogenic and aneuploidic effects that are thought to contribute to its oncogenic potential . This process can lead to faulty chromosome segregation and ensuing aneuploidy. Mammalian cells have evolved the mitotic spindle assembly checkpoint (SAC) that is the guardian of genome euploidy. TAX binds to MAD1, an integral constituent of the SAC machinery, and impairs its activity. TAX thus promotes aneuploidy by interacting with proteins that monitor chromosomal segregation during mitosis .β that is involved in base excision repair and inhibits nucleotide excision repair induced by UV irradiation , is activated. The DDR signalling network is often constitutively activated in premalignant lesions. This pathway is orchestrated by ATM, ATR and DNA-PK kinases whose substrates  delay cell cycle progression, promote DNA repair or even elicit permanent proliferative arrest  and apoptosis in case of extended, irreparable, DNA lesions. tability . Our unpGrowth arrest and apoptosis controlled by the DDR pathway may appear contradictory with the ability of the virus to sustain continuous cell proliferation. Nevertheless, permanent activation of DDR signalling creates a selection pressure that may be breached by cells deficient for ATM, CHK2, p53 or 53BP1 (in vivo (HTLV-1 has developed sophisticated mechanisms to ensure persistence and, perhaps as a bystander effect, pathogenesis in vivo . As infe


Transformation of infected cells involves HTLV-1's oncoprotein Tax, which perturbs cell cycle regulation and modulates cellular gene expression. The latter function is also a hallmark of microRNAs, a rather new layer in the regulation of gene expression. Affecting e.g. proliferation, microRNAs constitute a potential target for viral interference on the way to persistence and transformation. Hence, we explored the interconnections between HTLV-1 and cellular microRNAs.Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of a severe and fatal lymphoproliferative disease of mainly CD4κB-mediated transactivation of its promoter; a single NF-κB site proximal to the transcription start point was necessary and sufficient for this to happen. An in silico analysis of potential target genes revealed candidates that might be coregulated by two or more of the aforementioned overexpressed microRNAs.We report that several microRNAs – miRs 21, 24, 146a, 155 and 223 – are deregulated in HTLV-1-transformed cells. They are all upregulated except for miR-223, which is downregulated. Each of those microRNAs has ties to cancer. Their expression pattern forms a uniform phenotype among HTLV-transformed cells when compared to HTLV-negative control cells. In particular, miR-146a expression was found to be directly stimulated by Tax via NF-These data demonstrate that cellular microRNAs are deregulated in HTLV-1-transformed T cells. In the case of miR-146a, this could be directly attributed to HTLV's oncoprotein Tax. Interference with cellular microRNAs may be crucial to maintaining persistence or may facilitate transformation of host cells. Lifelong persistence ensues, which, after decades, can entail an aggressive neoplastic disease, adult T cell leukemia/lymphoma (ATLL). Another HTLV-1-associated disease presents as progressive neurodegeneration termed HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP)  upstream of the escribed . Briefly3, pcTax  or 2 μg For evaluation of differences in expression levels of HTLV-1-/Tax-positive and -negative samples, the Mann-Whitney U-test was applied using SPSS version 12.0.2 . Stimulated CEM cells were excluded from the calculation. Analysis of correlations between provirus copy number and microRNA expression employed the Spearman-Rho test.The authors declare that they have no competing interests.KP carried out quantification of RNAs and luciferase assays, performed the statistical and database analyses and wrote the manuscript. GS carried out luciferase assays. RG conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.Correlation analysis of provirus copy number and microRNA expression levels. Provirus copy number (PL) in seven cell lines  was determined as described in materials and methods. Correlations between PL and microRNA expression levels were then evaluated using the Spearman-Rho test. Resulting correlation coefficients, P values and the number of analyzed samples are given in the table.Click here for filePrimers and probes. Supplementary table S1 lists primers used for cloning miR-146a promoter and its NF-κB deletion mutants. Furthermore, primers and probes used in RT-PCR and qPCR analyses are given unless obtained from commercial sources.Click here for fileQuantification and comparison of U44 expression in HTLV-1-/Tax-positive and -negative cells. In addition to RNU6B (U6) transcripts, RNU44 (U44) was quantified in the samples indicated. Quantification was performed as described in materials and methods. To assess variation of expression values, i.e., the usefulness of U6 and U44 as normalization controls, the mean and coefficient of variation (CV) was determined. The CV was calculated as standard deviation devided by the mean. Smaller CV values indicate higher overall expression stability.Click here for fileMicroRNA expression normalized to U6, U44 or U6/U44 geometric mean. MicroRNA expression was normalized to both U6, U44 and their geometric mean in the following samples: Abgho, Nilu, Eva, Xpos, ATL-3, JuaW, StEd, Champ, PaBe, HuT-102, C91-PL, MT-2, CEM, PBMC and CD4+ T cells. Afterwards, differences in expression levels in HTLV-/Tax-positive vs. -negative cells was evaluated using the Mann-Whitney test. Note that the sample set is not identical to the one in Figure Click here for file


Human T-cell leukaemia virus (HTLV-1) and bovine leukaemia virus (BLV) entry into cells is mediated by envelope glycoprotein catalyzed membrane fusion and is achieved by folding of the transmembrane glycoprotein (TM) from a rod-like pre-hairpin intermediate to a trimer-of-hairpins. For HTLV-1 and for several virus groups this process is sensitive to inhibition by peptides that mimic the C-terminal α-helical region of the trimer-of-hairpins.We now show that amino acids that are conserved between BLV and HTLV-1 TM tend to map to the hydrophobic groove of the central triple-stranded coiled coil and to the leash and C-terminal α-helical region (LHR) of the trimer-of-hairpins. Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics. Conversely, a BLV LHR-mimetic peptide antagonized BLV envelope-mediated membrane fusion but failed to inhibit HTLV-1-induced fusion. Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides. Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor. Finally, the structural predictions assisted the design of a more potent antagonist of BLV membrane fusion.A conserved region of the HTLV-1 and BLV coiled coil is a target for peptide inhibitors of envelope-mediated membrane fusion and HTLV-1 entry. Nevertheless, the LHR-based inhibitors are highly specific to the virus from which the peptide was derived. We provide a model structure for the BLV LHR and coiled coil, which will facilitate comparative analysis of leukaemia virus TM function and may provide information of value in the development of improved, therapeutically relevant, antagonists of HTLV-1 entry into cells. Bovine Leukemia Virus (BLV) and Human T-Cell Leukemia Virus Type-1 (HTLV-1) are closely related deltaretroviruses that cause aggressive lymphoproliferative disorders in a small percentage of infected individuals . In ordeThe amino-acid sequences of the HTLV-1 and BLV envelope glycoproteins are strikingly similar  and, in Accumulating evidence advocates a conserved mechanism of retroviral envelope-mediated membrane fusion -15. SU bDespite the sequence homology and conserved modular structure, there are notable differences in primary sequence, size, and function of the HTLV-1 and BLV envelope proteins. It is likely that these differences contribute in a substantial way to the species-specificity, and the distinctive patterns of tissue tropism and pathogenesis that are observed for these viruses ,3. Conseleash and α-helical region (LHR) of HTLV-1 TM are inhibitory to envelope-mediated membrane fusion. Prototypic α-helical TM-mimetic inhibitory peptides have also been characterized for a number of highly divergent enveloped viruses, including HIV and paramyxoviruses  phosphine (TCEP) reducing agent (Pierce), and subsequent biotinylation was carried out with EZ-LinkA two-fold serial dilution of peptide in DMSO was performed, and added in duplicate to 96-well microplates. Filtered PBS was added to give a total volume of 200 μl and a final DMSO concentration of 1.5 % in all wells. The plates were incubated at room temperature for 1 hr and the relative solubility of peptides was established by measuring forward scattered light using a NEPHELOstar laser-based microplate nephelometer (BMG LABTECH). Wells containing PBS and 1.5 % DMSO only were used as blanks. Data analysis was carried out using ActivityBase, and peptides giving readings up to and including 3-fold higher than the average reading for the DMSO control were considered to be in solution at the concentrations specified.env-RRE and pRSV-Rev using the Genejuice™ transfection reagent (Novagen) in accordance with the manufacturer's instructions. 24 h later, 3 × 105 effector cells were added to 7 × 105 untransfected HeLa target cells in six-well dishes (Nunc). Where appropriate, the co-culture was incubated in the presence of peptides at the concentrations specified. To assess the ability of the peptides to inhibit fusion induced by virally expressed BLV envelope, 2 × 105 BLV-infected FLK cells were used as effectors and added to 8 × 105 uninfected HeLa cells. After incubation at 37°C for 16 h, cells were washed twice with PBS and fixed in PBS + 3% paraformaldehyde. Assays were performed in triplicate and the number of syncytia (defined as multinucleated cells with 4 or more nuclei) from 10 low-power fields (LPF) per replicate was scored by light microscopy; some assays were stained using Giemsa. A syncytium formation value of 100% is defined as the number of syncytia formed in the absence of peptide but in the presence of 1.5% DMSO. The peptide concentration required to give 50% inhibition (IC50) of syncytium formation was calculated using GraphPad Prism 4.Syncytium interference assays were performed by standard methods ,31. Brie6CC motif common to oncoretroviral fusion proteins. The crystal structure of the HTLV-1 six-helix-bundle has been solved and the structure spans these regions of homology [Although there are considerable differences in the amino acid sequence of class-1 fusion proteins from diverse viral groups there is exceptional conservation of secondary and tertiary structure. To compare the class-1 fusion proteins from the related retroviruses BLV and HTLV-1, the predicted coiled-coil regions of the BLV TM were identified using the program LearnCoil-VMF  and the homology .Using the crystal structure of the HTLV-1 TM as a template, we mapped on the coiled coil and LHR the location of amino acid residues that are conserved between the ectodomain of HTLV-1 and BLV TM Figure . Using tcr-400, which mimics the LHR of the HTLV-1 TM is a potent inhibitor of envelope-catalysed membrane fusion [A synthetic peptide, Pe fusion . This pee fusion -22. These fusion ,23  early promoter, and immediately upstream of the human immunodeficiency virus Rev-response element (RRE). The RRE forms a region of extensive secondary structure in the mRNA that is recognized by Rev and the resulting ribonucleoprotein complex is subsequently exported out of the nucleus. The BLV envelope expression construct was examined for envelope-induced membrane fusion in syncytium formation assays. Briefly, HeLa cells were either transfected with pCMV-BLVenv-RRE or pRSV-Rev individually, or cotransfected with equal amounts of both vectors. These cells were then used as effector cells to induce syncytia when co-cultured with non-transfected cells. Neither vector induced syncytium formation when transfected alone, but cotransfection of effector cells with pCMV-BLVenv-RRE and pRSV-Rev resulted in the widespread formation of large syncytia ; this may, in part, be due to the nuclear retention of the envelope transcripts as observed for HIV-1 and HTLV-1. Therefore, we developed an envelope expression vector whereby BLV a Figure . FurtherBLV-391 includes residues Cys391 to Gln419 of BLV Env and spans a region that is equivalent to the HTLV-1 LHR-derived peptide Pcr-400 . These results can be explained only in part by the decrease in peptide solubility at concentrations above 11 μM that is associated with the loss of the three N-terminal amino acid residues  the alanine-substituted peptides were severely compromised in their ability to inhibit membrane fusion  improvement in peptide potency when compared to PBLV-391. The peptide PBLV-R403A is more than twice as potent as PBLV-391 in syncytium interference assays, with a calculated IC50 of 1.56 ± 0.05 μM compared to 3.49 μM ± 0.03 μM for PBLV-391  against HTLV-1. The structure and model of the HTLV-1 and BLV TM suggests a plausible explanation for this observation in that, relative to the HTLV-derived peptide, the BLV peptide displays a smaller surface area available for interaction with the core coiled-coil. In addition, non-conserved residues within the HTLV-1 peptide may contribute disproportionately to the stability of the interaction between the HTLV-1 peptide and the core coiled-coil. The model and accumulated data also underscore the importance of a deep pocket that is situated towards the membrane-proximal end of the trimer-of-hairpins and is conserved between leukaemia viruses. The peptide inhibitors engage this pocket and this interaction appears to contribute substantially to the stability of peptide association with the coiled coil and is required for optimal inhibitory activity. The data provides further validation of the BLV coiled coil and LHR model and reveals that conserved hydrophobic amino acid side-chains within the helical and non-helical regions mediate interaction of the peptide inhibitors with their target.A recurring theme in the interaction of the C-terminal helix of the trimer-of-hairpins with the coiled coil of viral fusion proteins is the interaction of non-polar side chains with deep pockets on the coiled coil ,35,36,42f HTLV-1 . In part [cr-400 . Leucine Pcr-400  and suggAn intriguing finding of this study is that, directed by analysis of the model structure, an improved inhibitor of BLV envelope-mediated membrane fusion can be generated by the substitution of a single amino acid residue, Arg403, with alanine. A similar observation has been made for the Ile412 residue of the HTLV-1 fusion inhibitors . InteresBLV is prevalent among cattle throughout many regions of the world . The comOur data further define a membrane-proximal region of TM that is conserved between BLV and HTLV-1, which has potential as an anti-HTLV-1 drug target. This study demonstrates that comparative analysis of BLV and HTLV-1 induced membrane fusion will provide significant insight into envelope function and ultimately will be of value in the quest for compounds that block HTLV-1 entry into cells.The authors declare that they have no competing interests.DL performed the experiments and helped to draft the manuscript, AS provided technical expertise in molecular modeling, DvA provided assistance and technical expertise in structural analysis, DWB designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.