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Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag-PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P 2 than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P 2 via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P 2 and phosphatidylserine enhance myr(-)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(-)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number of human proteins, thus potentially instituting the basis for cross-reactions. We found a potential for cross-reactivity between the virus and the human proteome and described an epitope platform to be used in order to avoid it, thus obtaining effective, specific, and safe immunization. Potential advantages for mRNA and peptide-based vaccine formulations are discussed. more...

Human T cell leukemia virus type 1 (HTLV-1) as the first human retrovirus is currently a serious endemic health challenge. Despite the use of assorted molecular or serological assays for HTLV-1 detection, there are several limitations due to the lack of a confirmatory test that may affect the accuracy of the results. Herein, a novel label-free biosensor for the detection of HTLV-1 Tax gene has been reported. An electrochemical facile ecofriendly synthesis method has been demonstrated based on a synthesis of nanocomposite of reduced graphene oxide, polypyrrole, and gold nanoparticles (rGO-PPy-(l-Cys)-AuNPs) deposited on the surface of screen-printed carbon electrode. Electrochemical techniques were used to characterize and study the electrochemical behavior of the rGO-PPy-(l-Cys)-AuNPs, which exhibited a stable reference peak at 0.21 V associated with hybridization forms by applying the differential pulse voltammetry. The designed DNA biosensor presented a wide linear range from 0.1 fM to 100 µM and a low detection limit of 20 atto-molar. The proposed biosensor presented in this study provides outstanding selectivity, sensitivity, repeatability, and reproducibility., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.) more...

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells., Competing Interests: The authors have declared that no competing interests exist. more...

Human T-cell lymphotropic virus type 1 (HTLV-1) infection is linked to adult T-cell leukemia-lymphoma (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and several other disorders. ATLL occurs in approximately 5% of the 15-20 million people infected by HTLV-1 in the world. In general, ATLL is resistant to chemotherapy, which underlines the need for new and effective therapeutic strategies. Previous studies highlighted the role of viral enzymes, responsible for viral replication, and regulatory proteins such as Tax and HBZ in the progression of HTLV-1-associated diseases. There are conflicting reports on the efficacy of current enzyme inhibitors, mainly developed against human immunodeficiency virus (HIV), for treatment of HTLV-1 infection. New treatment approaches including monoclonal antibodies show promising results and exert significant cytotoxic effects on ATLL cells. This manuscript reviews the recent developments in molecular targeting for treatment of HTLV-1 infection., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.) more...

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication., (© 2018 Wu et al.) more...

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αβ TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αβ TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cβ FG loop projecting from the Vβ/Cβ interface; 2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR-binding sites for pMHC and CD3., (© 2018 Rangarajan et al.) more...

The human T cell leukemia virus I basic leucine zipper protein (HTLV-1 HBZ) maintains chronic viral infection and promotes leukemogenesis through poorly understood mechanisms involving interactions with the KIX domain of the transcriptional coactivator CBP and its paralog p300. The KIX domain binds regulatory proteins at the distinct MLL and c-Myb/pKID sites to form binary or ternary complexes. The intrinsically disordered N-terminal activation domain of HBZ (HBZ AD) deregulates cellular signaling pathways by competing directly with cellular and viral transcription factors for binding to the MLL site and by allosterically perturbing binding of the transactivation domain of the hematopoietic transcription factor c-Myb. Crystal structures of the ternary KIX:c-Myb:HBZ complex show that the HBZ AD recruits two KIX:c-Myb entities through tandem amphipathic motifs (L/V)(V/L)DGLL and folds into a long α-helix upon binding. Isothermal titration calorimetry reveals strong cooperativity in binding of the c-Myb activation domain to the KIX:HBZ complex and in binding of HBZ to the KIX:c-Myb complex. In addition, binding of KIX to the two HBZ (V/L)DGLL motifs is cooperative; the structures suggest that this cooperativity is achieved through propagation of the HBZ α-helix beyond the first binding motif. Our study suggests that the unique structural flexibility and the multiple interaction motifs of the intrinsically disordered HBZ AD are responsible for its potency in hijacking KIX-mediated transcription pathways. The KIX:c-Myb:HBZ complex provides an example of cooperative stabilization in a transcription factor:coactivator network and gives insights into potential mechanisms through which HBZ dysregulates hematopoietic transcriptional programs and promotes T cell proliferation., Competing Interests: The authors declare no conflict of interest. more...

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h 1 DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h 1 DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h 2 DNA and h 3 DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

Female genital epithelial cells cover the genital tract and provide the first line of protection against infection with sexually transmitted pathogenic viruses. These cells normally are impervious to HIV-1. We report that coinfection of cells by HIV-1 and another sexually transmitted virus, human T-lymphotropic virus 1 (HTLV-1), led to production of HIV-1 that had expanded cell tropism and was able to directly infect primary vaginal and cervical epithelial cells. HIV-1 infection of epithelial cells was blocked by neutralizing antibodies against the HTLV-1 envelope (Env) protein, indicating that the infection was mediated through HTLV-1 Env pseudotyping of HIV-1. Active replication of HIV-1 in epithelial cells was demonstrated by inhibition with anti-HIV-1 drugs. We demonstrated that HIV-1 derived from peripheral blood of HIV-1-HTLV-1-coinfected subjects could infect primary epithelial cells in an HTLV-1 Env-dependent manner. HIV-1 from subjects infected with HIV-1 alone was not able to infect epithelial cells. These results indicate that pseudotyping of HIV-1 with HTLV-1 Env can occur in vivo Our data further reveal that active replication of both HTLV-1 and HIV-1 is required for production of pseudotyped HIV-1. Our findings indicate that pseudotyping of HIV-1 with HTLV-1 Env in coinfected cells enabled HIV-1 to directly infect nonpermissive female genital epithelial cells. This phenomenon may represent a risk factor for enhanced sexual transmission of HIV-1 in regions where virus coinfection is common. IMPORTANCE Young women in certain regions of the world are at very high risk of acquiring HIV-1, and there is an urgent need to identify the factors that promote HIV-1 transmission. HIV-1 infection is frequently accompanied by infection with other pathogenic viruses. We demonstrate that coinfection of cells by HIV-1 and HTLV-1 can lead to production of HIV-1 pseudotyped with HTLV-1 Env that is able to directly infect female genital epithelial cells both in vitro and ex vivo Given the function of these epithelial cells as genital mucosal barriers to pathogenic virus transmission, the ability of HIV-1 pseudotyped with HTLV-1 Env to directly infect female genital epithelial cells represents a possible factor for increased risk of sexual transmission of HIV-1. This mechanism could be especially impactful in settings such as Sub-Saharan Africa and South America, where HIV-1 and HTLV-1 are both highly prevalent., (Copyright © 2018 Tang et al.) more...

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 μM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

Regulation of expression of HTLV-1 gene products from integrated proviruses plays an important role in HTLV-1-associated disease pathogenesis. Previous studies have shown that T cell receptor (TCR)- and phorbol ester (PMA) stimulation of chronically infected CD4 T cells increases the expression of integrated HTLV-1 proviruses in latently infected cells, however the mechanism remains unknown. Analysis of HTLV-1 RNA and protein species following PMA treatment of the latently HTLV-1-infected, FS and SP T cell lines demonstrated rapid induction of tax/rex mRNA. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability while the stability of unspliced or singly spliced HTLV-1 RNAs did not increase. Tax/rex mRNA in the HTLV-1 constitutively expressing cell lines exhibited high basal stability even without PMA treatment. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression at least in part through increased tax/rex mRNA stability., (Copyright © 2017 Elsevier Inc. All rights reserved.) more...

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500-2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag-Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles. more...

Interference with the expression and/or functions of the multifunctional tumor suppressor BRCA1 leads to a high risk of breast and ovarian cancers. BRCA1 expression is usually activated by the estrogen (E2) liganded ERα receptor. Activated ERα is considered as a potent transcription factor which activates various genes expression by 2 pathways. A classical pathway, ERα binds directly to E2-responsive elements (EREs) in the promoters of the responsive genes and a non-classical pathway where ERα indirectly binds with the appropriate gene promoter. In our previous study, HTLV-1Tax was found to strongly inhibit ERα induced BRCA1 expression while stimulating ERα induced ERE dependent genes. TPA is a strong PKC activator which found to induce the expression of HTLV-1. Here we examined the effect of TPA on the expression of BRCA1 and genes controlled by ERE region in MCF-7 cells and on Tax activity on these genes. Our results showed strong stimulatory effect of TPA on both BRCA1 and ERE expression without treatment with E2. Tax did not show any significant effect on these TPA activities. It seems that TPA activation of BRCA1 and ERE expression is dependent on PKC activity but not through the NFκB pathway. However, 53BP1 may be involved in this TPA activity because its overexpression significantly reduced the TPA stimulatory effect on BRCA1 and ERE expression. Additionally, our Chip assay results probably exclude possible involvement of ERα pathway in this TPA activity because TPA did not interfere with the binding of ERα to both BRCA1 promoter and ERE region. more...

The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. more...

Introduction: The human T-cell lymphotropic virus (HTLV) 1 and 2 cause various clinical disorders associated with degenerative diseases. Blood transfusion is a primary mechanism of transmission that is associated with the use of cellular components such as red blood cells. , Objective: To describe the epidemiology of HTLV 1 and 2 in blood donors in Colombia from 2001-2014. , Materials and Methods: A retrospective analysis was performed using screening, reactivity and positivity for HTLV 1 and 2 data collected from 2001 to 2014 by Colombian blood banks and consolidated by the Instituto Nacional de Salud. Using this information, transfusion-associated infectivity was also estimated. , Results: From 2001 to 2014, 60.2% of blood collected in Colombia was screened for HTLV 1 and 2 and had a cumulative reactivity of 0.30%. This was 20 times higher in Chocó (6.28%), where blood collection ended in 2004. Blood screening for HTLV reached 94.9% in 2014 with a positive concordance of 14.7%, and an estimated 406 unscreened, potentially infectious blood units were released. The majority of the unscreened blood units (215 units, 53%) came from Antioquia, a non-endemic department. , Conclusion: These results suggest that HTLV 1 and 2 infections are distributed in different areas of the country that were not previously classified as endemic. These findings support the importance of the universal screening of blood units to minimize the risk of infection through transfusion for this event. more...

Background: indeterminate Western blot (WB) patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) infection, even in non-endemic areas., Objectives: (a) to define the prevalence of indeterminate WB among different populations from Argentina; (b) to evaluate if low proviral load (PVL) is associated with indeterminate WB profiles; and (c) to describe mutations in LTR and tax sequence of these cases., Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3%) were WB indeterminate and of those 15 (31.3%) were PCR+. Quantitative real-time PCR (qPCR) was performed to 52 HTLV-1+ samples, classified as Group 1 (G1): 25 WB+ samples from individuals with pathologies; Group 2 (G2): 18 WB+ samples from asymptomatic carriers (AC); and Group 3 (G3): 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003) was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE) 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03)., Conclusions: indeterminate WB results confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined. more...

Human T-cell leukemia virus type 1 (HTLV-1) protease is an attractive target when developing inhibitors to treat HTLV-1 associated diseases. To study the catalytic mechanism and design novel HTLV-1 protease inhibitors, the protonation states of the two catalytic aspartic acid residues must be determined. Free energy simulations have been conducted to study the proton transfer reaction between the catalytic residues of HTLV-1 protease using a combined quantum mechanical and molecular mechanical (QM/MM) molecular dynamics simulation. The free energy profiles for the reaction in the apo-enzyme and in an enzyme - substrate complex have been obtained. In the apo-enzyme, the two catalytic residues are chemically equivalent and are expected to be both unprotonated. Upon substrate binding, the catalytic residues of HTLV-1 protease evolve to a singly protonated state, in which the OD1 of Asp32 is protonated and forms a hydrogen bond with the OD1 of Asp32', which is unprotonated. The HTLV-1 protease-substrate complex structure obtained from this simulation can serve as the Michaelis complex structure for further mechanistic studies of HTLV-1 protease while providing a receptor structure with the correct protonation states for the active site residues toward the design of novel HTLV-1 protease inhibitors through virtual screening., (Published by Elsevier Ltd.) more...

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

Background: Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiological agent of tropical spastic paraparesis/HTLV-associated myelopathy (HAM/TSP) that can be identified in around 0.25%-3.8% of the infected population. Disease progression can be monitored by the proviral load and may depend on genetic factors, however, it is not well understood why some HTLV-1 infected people develop the disease while others do not. The present study attempts to assess the molecular diversity of gp46 glycoprotein in HAM/TSP patients and Health Carrier (HC) individuals., Methods: Blood samples were collected from 10 individuals, and DNA was extracted from PBMCs to measure the HTLV-1 proviral load. The gp46 coding sequences were amplified PCR, cloned and sequenced. The molecular characterization was performed using bioinformatics tools., Results: The median HTLV-1 proviral load of HC (n = 5) and HAM/TSP (n = 5) patients was similar (average 316,227 copies/106 PBMCs). The gp46 molecular characterization of 146 clones (70 HC and 76 HAM/TSP) revealed an overall diversity, within HC and HAM/TSP clones, of 0.4% and 0.6%, respectively. Five frequent mutations were detected among groups (HAM/TSP and HC clone sequences). A single amino acid (aa) substitution (S35L) was exclusive for the HC group, and three gp46 substitutions (F14S, N42H, G72S) were exclusive for the HAM/TSP group. The remaining frequent mutation (V247I) was present in both groups (p = 0.0014). The in silico protein analysis revealed that the mutated alleles F14S and N42H represent more hydrophilic and flexible protein domains that are likely to be less antigenic. The Receptor Binding Domain is quite variable in the HAM/TSP group. Two other domains (aa 53-75 and 175-209) that contain multiple linear T-cell epitopes showed genetic diversity in both HAM/TSP and HC groups. Further analysis revealed 27 and 13 T-cell epitopes for class I HLA alleles and class II HLA alleles, when analyzing the entire gp46., Conclusions: The most common gp46 mutations were not associated clinical status because they were found in only one individual, except for the V247I mutation, that was found at viral clones from HAM/TSP ad HC individuals. Because of this, we cannot associate any of the gp46 found mutations with the clinical profile. more...

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type 1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions. more...

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage. more...

Human T-cell leukemia virus type 1 (HTLV-1) has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems. Recent studies on HTLV-1 using state-of-the-art cryo-electron microscopy and fluorescence-based biophysical approaches explored questions related to HTLV-1 particle size, Gag stoichiometry in virions, and Gag-Gag interactions in living cells. These results provided new and exciting insights into fundamental aspects of HTLV-1 particle assembly-which are distinct from those of other retroviruses, including HIV-1. The application of these and other novel biophysical approaches promise to provide exciting new insights into HTLV-1 replication. more...

Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry. more...

A new kind of silver micro-dendrites with no surfactants protected has been synthesized for the separation and detection of DNA merely by earth gravity as the density of silver is larger than that of water. Through this approach, the DNA of human T-lymphotropic virus type I (HTLV-I) can be detected down to 10 pM with the detection range from 10 pM to 100 nM. more...

Background: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells., Results: The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions., Conclusions: In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry. more...

Understanding the energetic and structural response to multiple mutations in a protein-protein interface is a key aspect of rational protein design. Here we investigate the cooperativity of combinations of point mutations of a T cell receptor (TCR) that binds in vivo to HLA-A2 MHC and a viral peptide. The mutations were obtained from two sources: a structure-based design study on the TCR alpha chain (nine mutations) and an in vitro selection study on the TCR beta chain (four mutations). In addition to combining the highest-affinity variants from each chain, we tested other combinations of mutations within and among the chains, for a total of 23 TCR mutants that we measured for binding kinetics to the peptide and major histocompatibility complex. A wide range of binding affinities was observed, from 2- to 1000-fold binding improvement versus that of the wild type, with significant nonadditive effects observed within and between TCR chains. This included an amino acid-dependent cooperative interaction between CDR1 and CDR3 residues that are separated by more than 9 A in the wild-type complex. When analyzing the kinetics of the mutations, we found that the association rates were primarily responsible for the cooperativity, while the dissociation rates were responsible for the anticooperativity (less-than-additive energetics). On the basis of structural modeling of anticooperative mutants, we determined that side chain clash between proximal mutants likely led to nonadditive binding energies. These results highlight the complex nature of TCR association and binding and will be informative in future design efforts that combine multiple mutant residues. more...

Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability. more...

Background: Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation., Results: We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function., Conclusion: We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into the regulation of Rex-1 function. more...

A synthetic peptide based on the leash and alpha-helical region (LHR) of human T cell leukemia virus type 1 envelope is a potent inhibitor of viral entry into cells. The inhibitory peptide targets a triple-stranded coiled-coil motif of the fusion-active transmembrane glycoprotein and in a trans-dominant negative manner blocks resolution to the trimer-of-hairpins form. The LHR-mimetic is, therefore, functionally analogous to the C34/T20-type inhibitors of human immunodeficiency virus. Previous attempts to shorten the bioactive peptide produced peptides with severely attenuated activity. We now demonstrate that truncated peptides often suffer from poor solubility and impaired coiled coil binding properties, and we identify features that are critical to peptide function. In particular, the alpha-helical region of the LHR-mimetic is necessary but not sufficient for inhibitory activity. Moreover, two basic residues are crucial for coiled-coil binding and efficient inhibition of membrane fusion. By retaining these basic residues and a region of main chain peptide contacts with the coiled coil, a core LHR-mimetic was obtained that retains both the inhibitory properties and solubility profile of the parental peptide. Variants of the core peptide inhibit both membrane fusion and infection of cells by free viral particles, but unexpectedly, infection by virions was more susceptible to inhibition by low activity inhibitors than syncytium formation. The core inhibitor provides a valuable lead in the search for smaller more bio-available peptides and peptido-mimetics that possess anti-viral activity. Such molecules may be attractive candidates for therapeutic intervention in human T cell leukemia virus type 1 infections. more...

Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity compared to that of the human immunodeficiency virus type 1 (HIV-1) NC protein. HTLV-1 NC contains two zinc fingers, each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of the HTLV-1 NC protein. The results indicate that the HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR) DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR DNA. These results suggest that, despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart. more...

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia. HTLV-1 Tax1 transforming protein interacts with several PDZ domain-containing proteins, and the interaction is associated with the transforming activities of Tax1 as well as persistent HTLV-1 infection. In this study, we show that Tax1 interacts with the tumor suppressor Scribble containing PDZ domains. Unlike other Tax1-interacting PDZ domain proteins, the PDZ domain-binding motif (PBM) of Tax1 was not required for the interaction with transiently expressed Scribble in 293T cells, but it was essential for the interaction with endogenous Scribble. Endogenous Scribble in 293T cells was primarily localized at the plasma membrane and colocalized with Tax1 but not Tax1C lacking PBM, whereas transiently expressed Scribble was localized in the cytoplasm and colocalized with Tax1C as well as Tax1, thus suggesting that Tax1 is recruited to the site of endogenous Scribble, such as the plasma membrane, in a PBM-dependent manner, and thereafter it interacts with Scribble in a PBM-independent and PBM-dependent manner. Endogenous Scribble was diffusely localized at the plasma membrane of HTLV-1-uninfected T-cell lines, whereas it colocalized with Tax1 as small and large aggregate at the plasma membranes. These results suggest that Tax1 through two binding sites induce aberrant clustering of Scribble, thereby altering the functions in HTLV-1-infected cells, which may thus play a role in persistent HTLV-1 infection and the pathogenesis. more...

The Tax1 oncoprotein encoded by Human T-lymphotropic virus type I is a major determinant of viral persistence and pathogenesis. Tax1 affects a wide variety of cellular signalling pathways leading to transcriptional activation, proliferation and ultimately transformation. To carry out these functions, Tax1 interacts with and modulates activity of a number of cellular proteins. In this review, we summarize the present knowledge of the Tax1 interactome and propose a rationale for the broad range of cellular proteins identified so far. more...

Background: 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 alpha-helical region of the trimer-of-hairpins., Results: 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 alpha-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., Conclusion: 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. more...

Background: The HTLV-I oncoprotein, Tax, is a pleiotropic protein whose activity is partially regulated by its ability to interact with, and perturb the functions of, numerous cellular proteins. Tax is predominantly a nuclear protein that localizes to nuclear foci known as Tax Speckled Structures (TSS). We recently reported that the localization of Tax and its interactions with cellular proteins are altered in response to various forms of genotoxic and cellular stress. The level of cytoplasmic Tax increases in response to stress and this relocalization depends upon the interaction of Tax with CRM1. Cellular pathways and signals that regulate the subcellular localization of Tax remain to be determined. However, post-translational modifications including sumoylation and ubiquitination are known to influence the subcellular localization of Tax and its interactions with cellular proteins. The sumoylated form of Tax exists predominantly in the nucleus while ubiquitinated Tax exists predominantly in the cytoplasm. Therefore, we hypothesized that post-translational modifications of Tax that occur in response to DNA damage regulate the localization of Tax and its interactions with cellular proteins., Results: We found a significant increase in mono-ubiquitination of Tax in response to UV irradiation. Mutation of specific lysine residues (K280 and K284) within Tax inhibited DNA damage-induced ubiquitination. In contrast to wild-type Tax, which undergoes transient nucleocytoplasmic shuttling in response to DNA damage, the K280 and K284 mutants were retained in nuclear foci following UV irradiation and remained co-localized with the cellular TSS protein, sc35., Conclusion: This study demonstrates that the localization of Tax, and its interactions with cellular proteins, are dynamic following DNA damage and depend on the post-translational modification status of Tax. Specifically, DNA damage induces the ubiquitination of Tax at K280 and K284. Ubiquitination of these residues facilitates the dissociation of Tax from sc35-containing nuclear foci, and stimulates nuclear export of Tax through the CRM1 pathway. more...

Infection of T-cells by human T-cell leukemia virus type 1 (HTLV-1) causes a lymphoproliferative malignancy known as adult T-cell leukemia (ATL). ATL is characterized by abnormal lymphocytes, called flower cells, which have cleaved and convoluted nuclei. Tax, encoded by the HTLV-1 pX region, is a critical nonstructural protein that plays a central role in leukemogenesis; however, the mechanisms of HTLV-1 oncogenesis have not been clarified fully. In this review, we summarize current thinking on how Tax may affect ATL leukemogenesis. more...

Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections. more...

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology. more...

Human T-lymphotropic virus 1 (HTLV-1) has infected human beings for thousands of years, but knowledge about the infection and its pathogenesis is only recently emerging. The virus can be transmitted from mother to child, through sexual contact, and through contaminated blood products. There are areas in Japan, sub-Saharan Africa, the Caribbean, and South America where more than 1% of the general population is infected. Although the majority of HTLV-1 carriers remain asymptomatic, the virus is associated with severe diseases that can be subdivided into three categories: neoplastic diseases (adult T-cell leukaemia/lymphoma), inflammatory syndromes (HTLV-1-associated myelopathy/tropical spastic paraparesis and uveitis among others), and opportunistic infections (including Strongyloides stercoralis hyperinfection and others). The understanding of the interaction between virus and host response has improved markedly, but there are still no clear surrogate markers for prognosis and there are few treatment options. more...

The tetraspanin superfamily proteins play important roles in organizing membrane protein complexes, modulating integrin function, and controlling T cell adhesion. Tetraspanins such as CD82 contain two extracellular loops with its N terminus, C terminus, and inner loop exposed to the cytoplasm. The matrix (MA) domain of human T cell lymphotrophic virus, type 1 (HTLV-1), Gag interacts with the cytoplasmic face of the plasma membrane and is concentrated at tetraspanin-enriched microdomains. To understand the basis of this association, we generated site-directed mutations in the various domains of CD82 and used coimmunoprecipitation and colocalization approaches to examine interactions with HTLV-1 MA. The large extracellular loop of CD82, which is important for interactions with integrins, was not required for the association with HTLV-1 MA. The cytoplasmic N terminus and C terminus of CD82 were also dispensable for CD82-MA interactions. In contrast, mutations of conserved amino acids in the inner loop of CD82 or of palmitoylated cysteines that flank the inner loop diminished CD82 association with MA. HTLV-1 MA also interacted with the inner loop of CD81. Thus, association of HTLV-1 Gag with tetraspanin-enriched microdomains is mediated by the inner loops of CD81 and CD82. more...

Whereas adaptive immunity has been extensively studied, very little is known about the innate immunity of the host to HTLV-I infection. HTLV-I-infected ATL patients have pronounced immunodeficiency associated with frequent opportunistic infections, and in these patients, concurrent infections with bacteria and/or parasites are known to increase risks of progression to ATL. The Toll-like receptor-4 (TLR4) activation in response to bacterial infection is essential for dendritic cell maturation and links the innate and adaptive immune responses. Recent reports indicate that TLR4 is targeted by viruses such as RSV, HCV, and MMTV. Here we report that HTLV-I has also evolved a protein that interferes with TLR4 signaling; p30 interacts with and inhibits the DNA binding and transcription activity of PU.1 resulting in the down-regulation of the TLR4 expression from the cell surface. Expression of p30 hampers the release of pro-inflammatory cytokines MCP-1, TNF-alpha, and IL-8 and stimulates release of anti-inflammatory IL-10 following stimulation of TLR4 in human macrophage. Finally, we found that p30 increases phosphorylation and inactivation of GSK3-beta a key step for IL-10 production. Our study suggests a novel function of p30, which may instigate immune tolerance by reducing activation of adaptive immunity in ATL patients. more...

Human T-cell leukemia virus and simian T-cell leukemia virus (STLV) form the primate T-cell lymphotropic viruses group. Human T-cell leukemia virus type 1 and type 2 (HTLV-1 and HTLV-2) encode the Tax viral transactivator (Tax1 and Tax2, respectively). Tax1 possesses an oncogenic potential and is responsible for cell transformation both in vivo and in vitro. We and others have recently discovered the existence of human T-cell lymphotropic virus type 3. However, there is currently no evidence for the presence of a Tax protein in HTLV-3-infected individuals. We show that the serum of an HTLV-3 asymptomatic carrier and the sera of two STLV-3-infected monkeys contain specific anti-Tax3 antibodies. We also show that tax3 mRNA is present in the PBMCs obtained from an STLV-3-infected monkey, demonstrating that Tax3 is expressed in vivo. We further demonstrate that Tax3 intracellular localization is very similar to that of Tax1 and that Tax3 binds to both CBP and p300 coactivators. Using purified Tax3, we show that the protein increases transcription from a 4TxRE G-free cassette plasmid in an in vitro transcription assay. In all cell types tested, including transiently transfected lymphocytes, Tax3 activates its own promoter STLV-3 long terminal repeat (LTR), which contains only two Tax Responsive Elements (TREs), and activates also HTLV-1 and HTLV-2 LTRs. In addition, Tax3 also activates the NF-kappaB pathway. We also show that Tax3 possesses a PDZ-binding sequence at its C-terminal end. Our results demonstrate that Tax3 is a transactivator, and that its properties are more similar to that of Tax1, rather than of Tax2. This suggests the possible occurrence of lymphoproliferative disorders among HTLV-3-infected populations. more...

Human T cell leukemia virus type 1 (HTLV-1), the cause of adult T cell leukemia (ATL), induces clonal expansion of infected T-cells in nonleukemic individuals and immortalizes T cells in vitro. The resistance against apoptotic stimuli of these cells hints at a viral survival function in addition to a proliferation-stimulating activity. Here we describe the up-regulation of the antiapoptotic HIAP-1/CIAP-2 gene as a consistent phenotype of HTLV-1-transformed and ATL-derived cultures and its stimulation by the viral oncoprotein Tax. Cotransfections revealed a 60-fold increase of HIAP-1 promoter activity mediated by Tax mainly via nuclear factor-kappaB (NF-kappaB) activation. To address the relevance of virally increased HIAP-1 levels for the survival of HTLV-1-transformed cells, its expression was RNA interference (RNAi) suppressed using a lentiviral transduction system. This resulted in a dramatic reduction of cell growth, a strong induction of apoptosis rates, and increased caspases 3/7 activity, which is known to be suppressed by HIAP-1. Thus, the Tax-mediated HIAP-1 overexpression is required to suppress endogenous apoptosis and, therefore, is essential for the survival of HTLV-1-transformed lymphocytes. Moreover, this points to HIAP-1 as an important target of the HTLV-1-mediated NF-kappaB activation. more...

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent. more...

Recently, the putative finding of ancient human T cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) DNA sequences in association with a 1500-year-old Chilean mummy has stirred vigorous debate. The debate is based partly on the inherent uncertainties associated with phylogenetic reconstruction when only short sequences of closely related genotypes are available. However, a full analysis of what phylogenetic information is present in the mummy data has not previously been published, leaving open the question of what precisely is the range of admissible interpretation. To fulfill this need, we re-analyzed the mummy data in a new way. We first performed phylogenetic analysis of 188 published LTR DNA sequences from extant strains belonging to the HTLV-1 Cosmopolitan clade, using the method of statistical parsimony which is designed both to optimize phylogenetic resolution among sequences with little evolutionary divergence, and to permit precise mapping of individual sequence mutations onto branches of a divergence network. We then deduced possible phylogenetic positions for the two main categories of published Chilean mummy sequences, based on their published 157-nucleotide LTR sequences. The possible phylogenetic placements for one of the mummy sequence categories are consistent with a modern origin. However, one of these placements for the other mummy sequence category falls very close to the root of the Cosmopolitan clade, consistent with an ancient origin for both this mummy sequence and the Cosmopolitan clade. more...

HERV-FRD is a human endogenous retrovirus that entered the human genome 40 million years ago. Its envelope gene, syncytin-2, was diverted by an ancestral host most probably because of its fusogenic property, for a role in placenta morphogenesis. It was maintained in a functional state in all primate branches as a bona fide cellular gene, submitted to a very low mutation rate as compared to infectious retrovirus genomes. The structure of the syncytin-2 protein thus provides a good insight into that of the oldest mammalian retroviral envelope. Here, we report the crystal structure of a central fragment of its "fossil" ectodomain, allowing a remarkable superposition with the structures of the corresponding domains of present-day infectious retroviruses, in spite of a more than 60% divergent sequence. These results suggest the existence of a unique structural solution selected by these proteins for their fusogenic function. more...

In the absence of the human immunodeficiency virus type 1 (HIV-1) Vif protein, the host-cell cytidine deaminases APOBEC3F and -3G are co-packaged along with virion RNA. Upon infection of target cells, nascent single-stranded DNA can be edited extensively, invariably giving rise to defective genomes called G-->A hypermutants. Although human T-cell leukemia virus type 1 (HTLV-1) replicates in the same cell type as HIV-1, it was shown here that HTLV-1 is relatively resistant to the antiviral effects mediated by human APOBEC3B, -3C, -3F and -3G. Nonetheless, a small percentage of genomes (0.1 more...

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation. more...