Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways. G protein-coupled receptor kinases (GRKs) were originally identified as the kinases that phosphorylate and desensitize agonist-bound G protein-coupled receptors (GPCRs). The phoGRKs are composed of seven isoforms (GRK1–GRK7). Althoug1AR)[Recent studies have revealed that GRKs are involved not only in GPCR desensitization but also in G protein-independent signaling,45. G pr1AR), vasopre1AR), and β2-1AR). GRK/β-a1AR)-51. The 1AR)-55. Thes1AR),57. Thus1AR) or GRK/β1AR),60 by th1AR) and have1AR). As diff1AR) . It demo1AR by GRK2 and GRK3 induces GPCR desensitization and internalization, whereas phosphorylation by GRK5 leads to β-arrestin-dependent ERK activation[The mechanism by which GRKs determine whether to promote GPCR desensitization or G protein-independent signaling remains unclear. Several studies have focused on the GRK subfamily that mediates desensitization or GRK/β-arrestin signaling,47,65,66tivation. It has tivation.It has also been proposed that a differential phosphorylation pattern is essential for determining whether to promote GPCR desensitization or signaling. Butcher et al. found that different tissues and cells exhibit a differential GPCR phosphorylation pattern of the M3 muscarinic receptor. HoweverThus, the conformational changes of GPCRs and phosphorylation pattern of GPCRs could be important for G protein activation, GPCR desensitization, and GRK/β-arrestin-mediated signaling. Although “phosphorylation barcoding” was recently proposed as a key factor for determining whether to promote desensitization or GRK/β-arrestin-mediated signaling, it remains to be elucidated how each GRK phosphorylates specific serine/threonine residues. The identification of the consensus phosphorylation sequences for each GRK would be meaningful to understand how GRKs regulate GPCR desensitization and GRK/β-arrestin-dependent signaling.−-β1-AR TG). They also produced transgenic mice expressing wild-type β1-AR in the heart (WT-β1-AR TG). When these mice were subjected to chronic exposure of isoproterenol, GRK−-β1-AR TG mice showed a significantly higher number of apoptotic cells than WT-β1-AR TG mice. This resulted in decreased cardiac performance in GRK−-β1-AR TG mice. They also demonstrated that epidermal growth factor receptor (EGFR) transactivation by GRK/β-arrestin-mediated, but not G protein-mediated, signaling is important for cardioprotective effects. As the chronic activation of Gs signaling by β1-AR is reported to be cardiotoxic, β-adrenergic blocking agents are beneficial for the treatment of heart failure[Many agonists can usually activate both G protein- and β-arrestin-mediated signaling pathways. A biase failure. They su failure, and car failure. Fibrosi failure,73. This1AR has also been well studied as a model GPCR to analyze biased agonists[1AR, and SII has been frequently used for the study of G protein-independent signaling of AT1AR[1AR could be beneficial for the heart under physiological and pathological conditions. However, it remains to be determined which GRKs are involved in AT1AR-mediated biased signaling and which molecules downstream of GRKs and β-arrestins are responsible for signaling.ATagonists,71. Biasagonists. [Sar1, of AT1AR,64,75,76of AT1AR,75. In cof AT1AR. Thus, bIn addition to the role of GRKs in GRK/β-arrestin-dependent signaling, it has been recognized that GRKs also interact with non-GPCR proteins,77. Non-Several reports have suggested that the interaction of GRK with intracellular non-GPCRs affects signaling pathways. It has been reported that GRK2 negatively regulates CCL2-induced ERK activation by interacting with mitogen-activated protein kinase kinase (MEK). Other s2+-induced opening of the mitochondrial permeability transition pore after ischemic injury, which promoted cardiomyocyte death[Interactions between GRKs and intracellular proteins occurred at various sites including the outer membrane of the mitochondria and nucleus in addition to the plasma membrane and cytosol. For example, GRK2 was shown to localize in the mitochondria and to ite death. It was te death. These rte death. This phte death. Howeverte death. The NLSte death.Some studies have reported the mechanism by which GRKs are activated and promote signaling by non-GPCR proteins. It has Grk2-knockout mice[Recent studies have suggested an in vivo significance of the interaction between GRK and intracellular non-GPCR proteins. GRK2 interacts with Akt and inhibits endothelial NO synthase activity and NO production, resulting in less severe portal hypertension in GRK2-deficient mice after liver injury. GRK5 phout mice because out mice. Furtherout mice. In theiAlthough the abovementioned studies have mainly focused on GRK2 and GRK5, the importance of the interaction of other GRK subfamilies with intracellular proteins remains poorly understood. GRK6 was recently found to mediate the removal of apoptotic cells (engulfment) and the clearance of senescent red blood cells through a new engulfment pathway. InsuffiLpr mice, a murine model of systemic lupus erythematosus (SLE), and the autopsied spleens from SLE patients[It has become clear that GRKs are multifunctional proteins that interact not only with GPCRs but also with intracellular non-GPCR proteins. However, several issues remain to be resolved in future studies. One issue is the mechanism by which GRKs phosphorylate specific serine/threonine residues in GPCRs and non-GPCR proteins. Although GRKs can phosphorylate a large number of proteins, the consensus sequence of the phosphorylation site for each GRK has not been firmly established. The secpatients. The chapatients, schizoppatients, and deppatients. Howeverpatients,113-115.patients. Insteadpatients,118. It patients,118 is bThe authors declare that they have no competing interests.KW wrote a draft, and MN and HK edited it. All authors read and approved the final manuscript.


The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus causing an aggressive T-cell malignancy, adult T-cell leukemia (ATL). Although HTLV-1 has a compact RNA genome, it has evolved elaborate mechanisms to maximize its coding potential. The structural proteins Gag, Pro, and Pol are encoded in the unspliced form of viral mRNA, whereas the Env protein is encoded in singly spliced viral mRNA. Regulatory and accessory proteins, such as Tax, Rex, p30II, p12, and p13, are translated only from fully spliced mRNA. For effective viral replication, translation from all forms of HTLV-1 transcripts has to be achieved in concert, although unspliced mRNA are extremely unstable in mammalian cells. It has been well recognized that HTLV-1 Rex enhances the stability of unspliced and singly spliced HTLV-1 mRNA by promoting nuclear export and thereby removing them from the splicing site. Rex specifically binds to the highly structured Rex responsive element (RxRE) located at the 3′ end of all HTLV-1 mRNA. Rex then binds to the cellular nuclear exporter, CRM1, via its nuclear export signal domain and the Rex–viral transcript complex is selectively exported from the nucleus to the cytoplasm for effective translation of the viral proteins. Yet, the mechanisms by which Rex inhibits the cellular splicing machinery and utilizes the cellular pathways beneficial to viral survival in the host cell have not been fully explored. Furthermore, physiological impacts of Rex against homeostasis of the host cell via interactions with numerous cellular proteins have been largely left uninvestigated. In this review, we focus on the biological importance of HTLV-1 Rex in the HTLV-1 life cycle by following the historical path in the literature concerning this viral post-transcriptional regulator from its discovery to this day. In addition, for future studies, we discuss recently discovered aspects of HTLV-1 Rex as a post-transcriptional regulator and its use in host cellular pathways. Human T-cell leukemia virus type 1 (HTLV-1) is widely accepted as the causative agent of adult T-cell leukemia (ATL) and was discovered almost a decade after the recognition of ATL as a disease . By the gag and pol are separated by pro, which overlaps both the 3′ end of gag and 5′ end of pol. The protein precursors, Gag-Pro and Gag-Pro-Pol, share a common Gag initiator codon located at the 5′ end of gag, and expression is translationally regulated by an in-frame read-through and PRF. PRF is a mechanism frequently used by viruses to alter the translational reading frame by shifting the ribosome at a slippery site  during the early phase of infection. Tax also has a significant influence on host signal transduction, gene expression, and cell cycle regulation by interacting with various cellular proteins and plays a major role in immortalization and leukemogenesis of the host T-cells . On the Rex is an mRNA binding protein, which specifically binds to the Rex responsive element (RxRE) and acts as a post-transcriptional regulator of HTLV-1 mRNA. Since RxRE locates to the U3 and R regions, all HTLV-1 transcripts  have RxRE. The most important function of Rex is selectively binding to unspliced and partially spliced HTLV-1 mRNA in the nucleus and quickly exporting them to the cytoplasm, thereby preventing further splicing and enhancing effective translation of the structure proteins .tax/rex mRNA and retains it in the nucleolus. Therefore, p30II reduces Tax and Rex expression levels , which eventually leads the virus to enter the latent period , is expressed from the minor doubly spliced viral mRNA and retains tax/rex mRNA in the nucleoli, thus preventing their expression and avoiding immune evasion to initiate latency. The time course of HTLV-1 expression was thoroughly investigated by Figure 1).After HTLV-1 entry and integration into the host human genome, proviral expression is initiated and the viral regulatory/accessory proteins function in concert with a precise schedule. Such well-organized regulation of HTLV-1 expression has been investigated by many researchers in the field of molecular and cellular virology and it was also recently confirmed by kinetic calculations . Figure t of Rex . Tax boo+ and activates the electron transport chain. This results in increased mitochondrial production of reactive oxygen species, which induces genetic instability and apoptosis (2+ release from the ER and activation of the nuclear factor of activated T-cells (NFAT), a mitogenic pathway in T-cells. On the other hand, p8, which is cleaved from p12 in the ER, travels to the cell surface and induces T-cell anergy. p8 also increases cell-to-cell viral transmission through the formation of immunological synapses  and suppresses Tax-mediated viral transcription. HBZ also enhances viral replication . On the nic mice . These rOverall, the interactions and positive and negative feedbacks among HTLV-1 Tax, Rex, p30II, and HBZ control the activation and inhibition of HTLV-1 expression, whereas p13, p12, and p8 organize a cellular environment suitable for viral retention.HTLV-1 Rex is a viral RNA binding protein of approximately 27 kDa and is essential for nuclear export of viral mRNA. Rex is also known to stabilize and export unspliced and singly spliced viral mRNA that code structural proteins; thus, Rex is considered essential for viral replication . It has cis-acting repressive sequence (CRS) is another regulatory sequence of HTLV-1 mRNA, located at both ends of HTLV-1 LTRs. gag/pol mRNA and singly spliced env mRNA of HTLV-1 was Rex-dependent, whereas that of alternatively spliced mRNA was not. According to their conclusion, the unspliced and singly spliced HTLV-1 mRNA, containing RxRE/CRS and a functional splice donor site, are nuclear-exported in a Rex/RxRE-dependent manner, whereas the fully spliced mRNA is not, even though it contains a 3′RxRE/CRS. Their results are somewhat different from those of tax/rex mRNA was also nuclear-exported in a Rex/RxRE/CRM1-dependent manner. All together, nuclear export of unspliced and spliced mRNA of HTLV-1 seems to be fine-tuned by nuclear retention activity of CRS and selective nuclear exporting activity of Rex.As a viral post-transcriptional regulator, Rex binds to the RxRE of the viral transcript with high affinity. The RxRE sequence spans 255 nt from the U3 to R region of the 3′LTR and forms a stable secondary structure consisting of four stem loops . RxRE isRex is a phosphoprotein; therefore, its activity is determined by the state of phosphorylation at the several serine/threonine residues . Adachi p24 gag mRNA, was not significantly influenced.The HTLV-1 Rex, a protein of 27 kDa, contains several functional domains which play essential roles to induce the function of Rex as a nuclear–cytoplasmic mRNA transporter. The locations and physiological importance of each Rex domain are well described in several review articles . A highlFigure 2). To date, interaction of Rex with the following cellular proteins have been confirmed: CRM1 as already mentioned, the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), the splicing factor SF2, importinβ, and nucleolar protein B-23. hnRNPs are heterogeneous nuclear RNA (hnRNA) binding proteins associated with pre-mRNA in the nucleus that influence the processing/splicing of pre-mRNA and the transport of mature mRNA. hnRNP A1 was shown to bind to the RxRE sequence of HTLV-1 viral mRNA in competition with Rex . Usually, importinα serves as a bridge between the NLS and the import receptor importinβ. It was demonstrated that the NLS of Rex directly bound to importinβ . The authors found that Rex was nuclear-imported by interactions with importinβ and independent of importinα. Nucleolar phosphoprotein B-23, also known as nucleophosmin (NPM), is a phosphoprotein mainly localized in nucleoli. Previously, it was determined that B-23 bound to the N′-terminal NLS/NoLS of Rex . Both are able to infect human T-cells and induce immortality. In spite of a high similarity in the genome and life cycle, there is a significant difference in pathogenesis between retroviruses. The most outstanding difference is that HTLV-1 induces a severe hematopoietic malignancy (ATL), whereas HTLV-2 does not . It is unclear as to why there is such a significant difference in outcomes from similar genomic structures. Nevertheless, current knowledge indicates that the differences in properties and functions of accessory and regulatory proteins expressed from the pX region of the virus are critical for the distinct pathological differences between the HTLVs.HTLV-1 and HTLV-2 belong to the same genus  and sharFigure 3).Both HTLV-1 and HTLV-2 encode Tax and Rex, the major transcriptional and post-transcriptional regulators. Tax-1 from HTLV-1 shows transforming ability, whereas Tax-2 from HTLV-2 does not. Thus, the different transforming activity of Tax determines the malignant pathology of this virus . Both TaRex-1 encoded in HTLV-1 is a 27-kDa (189 aa) protein, whereas Rex-2 from HTLV-2 consists of 170 aa and its molecular weight ranges between 24 and 26 kDa depending on the phosphorylation-induced conformational changes . Rex-1 aFigure 3). The RxRE motif of HTLV-1 mRNA (RxRE-1) is located in the U3/R region; consequently, all HTLV-1 mRNA have an intact RxRE-1 in the 3′UTR. On the other hand, the RxRE of HTLV-2 (RxRE-2) is located in the R/U5 region and only unspliced mRNA maintains an intact RxRE-2 .A major difference between HTLV-1 and HTLV-2 regarding Rex function may be the position of RxRE in the viral transcripts  by HIV-1]. Both viruses encode transactivators, HTLV-1 Tax and HIV-1 Tat, and post-transcriptional regulators, HTLV-1 Rex and HIV-1 Rev. Although Tax and Tat transactivate their respective LTRs, they act though different mechanisms and cannot be replaced by each other. On the other hand, even though the homology in the sequence of HTLV-1 Rex and HIV-1 Rev is low, they play similar functions through common cellular pathways . Both Rex and Rev have arginine-rich sequences that are necessary for binding to their respective responsive elements. They stabilize unspliced or partially spliced viral mRNA and actively transport them to the cytoplasm for selective translation of viral structural proteins. The functional similarities and differences between HTLV-1 Rex and HIV-1 Rev were extensively investigated from late 1980s to the early 1990s.HTLV-1 and HIV-1 are evolutionally distinct, but both belong to a family of complex retroviruses sharing tropism for human CD4pathways . A majortat mRNA using a stop codon within the intron of tat mRNA, meaning that Rev suppresses splicing and stabilizes unspliced tat mRNA .tat mRNA . Rimsky ough RRE ; howeverough RRE . Interesough RRE . Neverthough RRE ; thus, Rough RRE . Thus, tFigure 4). After translation, the NLSs of both Rex and Rev bind to importinβ and the complexes are then translocated to the nucleus .The arrangements of primary Rex and Rev structures are distinctive; however, both viral RNA binding proteins have NLSs and NESs and also use the same cellular nucleocytoplasmic shuttling machinery  consisting of 3–13 aa, rather than large structural domains of each protein . InteresFigure 5). The cellular proteins listed below potentially interact with Rex. Air1 (ZCCHC7) is a component of the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex, which is involved in nuclear mRNA surveillance , and the extent of the influence of Rex by making use of the cellular pathways for viral benefits. We still do not know the underlying mechanism by which Rex increases The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most HTLV-1/2 infections in Spain have been found in native intravenous drug users carrying HTLV-2. However, the large immigration flows from Latin America and Africa during the last decade may have changed the prevalence and distribution of HTLV-1 and HTLV-2 infections, and hypothetically even open the opportunity for introducing the new HTLV-3 or HTLV-4 variants.A national multicenter, cross-sectional, national study was conducted in June 2009 recruiting consecutive adult outpatients attending 16 different hospitals in Spain. HTLV-1/2 antibodies were screened using an EIA and further confirmed using a commercial Western blot. Samples with indeterminate Western blot patterns were examined using specific PCR assays for discriminating HTLV types 1 to 4.A total of 6,460 patients were studied. Overall, 40% were males and 88% were native Spaniards. The main origin of the immigrant population was Latin America (4.9%), Africa (3.6%) and other European countries (2.8%). A total of 9 individuals were seroreactive for HTLV antibodies . Of these, Western blot confirmed as HTLV-1 in 4  and HTLV-2 in 5 . Another 2 specimens yielded EIA reactivity close to the cut-off and could not be confirmed as positive for any of the HTLV types 1 to 4. All but one HTLV-1 cases were Latin Americans while all HTLV-2 cases were native Spaniards. The single HTLV-1 subject born in Spain was a 31 year-old woman who denied any significant risk behavior for HTLV exposure, including intravenous drug use, sexual promiscuity, transfusions or travel to Latin America. All HTLV-1 carriers but one were asymptomatic. A woman from the Dominican Republic was diagnosed with mild TSP/HAM.The overall prevalence of HTLV-1/2 infections in Spain remains low, although a shift in the relative proportion of HTLV-1 and HTLV-2 seems to have occurred in recent years. The recognition of HTLV-1 in a native Spaniard with lack of recognizable risk factors for HTLV-1 exposure should alert about a spread of HTLV-1 outside classical risk groups. No cases of HTLV-3 nor HTLV-4 infections have been identified so far in Spain.


An extraordinary progress has been made over the last two decades on understanding the components and mechanisms governing plant innate immunity. After detection of a pathogen, effective plant resistance depends on the activation of a complex signaling network integrated by small signaling molecules and hormonal pathways, and the balance of these hormone systems determines resistance to particular pathogens. The discovery of new components of hormonal signaling pathways, including plant nuclear hormone receptors, is providing a picture of complex crosstalk and induced hormonal changes that modulate disease and resistance through several protein families that perceive hormones within the nucleus and lead to massive gene induction responses often achieved by de-repression. This review highlights recent advances in our understanding of positive and negative regulators of these hormones signaling pathways that are crucial regulatory targets of hormonal crosstalk in disease and defense. We focus on the most recent discoveries on the jasmonate and salicylate pathway components that explain their crosstalk with other hormonal pathways in the nucleus. We discuss how these components fine-tune defense responses to build a robust plant immune system against a great number of different microbes and, finally, we summarize recent discoveries on specific nuclear hormonal manipulation by microbes which exemplify the ingenious ways by which pathogens can take control over the plant’s hormone signaling network to promote disease. In nature, plants live in complex environments in which they intimately interact with a broad range of microbial pathogens with different lifestyles and infection strategies. To defend themselves against all these different types of pathogens, plants have evolved sophisticated strategies to perceive their attacker and to translate this perception into an effective immune response. Two tiers of recognition by the innate immune system have been defined . The firvice versa  in phytohormone signaling . In factHere, we review the most recent and outstanding examples regarding hormonal crosstalk at the molecular level, focusing on the jasmonate and salicylate pathways and how the newly identified nuclear components fine-tune defense responses to build a robust plant immune system against a great number of different microbes. We also describe some of the best characterized molecular examples of specific nuclear hormonal manipulation by microbes, which exemplify the ingenious ways by which pathogens can take control over the plant’s hormone signaling network to suppress host immunity.Plant immunity strongly relies on two plant mutually antagonistic hormones, JA and SA . Both hoArabidopsis  TFs MYC2, MYC3, and MYC4 . Fernandsyringae , indicatsyringae . HoweverPseudomonas syringae  genes  3, 6, and 7  genes . PR geneactivity . NPR1 exactivity . Howeveractivity . NPR1 mo6, and 7 . In the 6, and 7 . Strikin6, and 7 . Thus, iThe activation of plant defenses implies allocation and ecological costs . For exaArabidopsis, mitogen activated protein kinase 4 (MPK4) acts as a negative regulator of SA signaling and positive regulator of JA signaling  and ORA59 , including GRX480 and several others of the ROXY class . These pnd ORA59 . For exand ORA59  and has  pathway , indicatAnother important regulatory component is the SA master regulator NPR1 itself, which interacts with TGA TFs that are involved in the activation of SA-responsive PR genes . NuclearErysiphe cichoracearum but repressed the expression of JA-responsive marker gene PDF1.2 and compromised resistance to the necrotrophic pathogen Alternaria brassicicola . The ERF branch is regulated by members of the AP2/ERF family of TFs, such as ERF1 and ORA59 , a gene encoding an isochorismate synthase required for SA biosynthesis , RGA (REPRESSOR OF GA1-3), RGL1 (RGA-like1), RGL2, and RGL3  serve as CK receptors , a mimic of the bioactive jasmonate hormone JA-Ile, by some strains of syringae . COR, assyringae . This lesyringae , indicatsyringae , facilitsyringae , 2008 an pathway . Interes pathway . This inPseudomonas effector AvrPtoB stimulates ABA biosynthesis and ABA responses, which in turn antagonize SA biosynthesis and SA-mediated defenses  metabolism  provides a paradigm to explain the rapid and appropriate level of defense responses to specific signals. Moreover, key hormones regulating growth, such as GA and auxins, are involved in the orchestration of the plant immune response suggesting that developmental and defense signaling networks are closely interconnected. This would explain why activation of costly defenses is often accompanied by significant growth inhibition. Thus, hormone homeostasis and cross-talk seem to be a dominant feature to maximize defenses and to fine-tune growth and protection. Not surprisingly, pathogens and beneficial microbes have learned the necessity of manipulation of plant hormonal pathways to rewire the immune signaling circuitry for their own benefit. Indeed, the importance of hormones in plant immunity is highlighted by the increasing number of pathogens that are predicted to produce phytohormones or phytohormone mimics, and the recent findings indicating that microbial effectors also targets hormonal pathways to promote disease or establish beneficial interactions . Due to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we report on the partial and full-length genomic (FLG) variability of HTLV-1 sequences from 90 well-characterized subjects, including 48 HTLV-1 asymptomatic carriers (ACs), 35 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 7 adult T-cell leukemia/lymphoma (ATLL) patients, using an Illumina paired-end protocol.Blood samples were collected from 90 individuals, and DNA was extracted from the PBMCs to measure the proviral load and to amplify the HTLV-1 FLG from two overlapping fragments. The amplified PCR products were subjected to deep sequencing. The sequencing data were assembled, aligned, and mapped against the HTLV-1 genome with sufficient genetic resemblance and utilized for further phylogenetic analysis.A high-throughput sequencing-by-synthesis instrument was used to obtain an average of 3210- and 5200-fold coverage of the partial (n = 14) and FLG (n = 76) data from the HTLV-1 strains, respectively. The results based on the phylogenetic trees of consensus sequences from partial and FLGs revealed that 86 (95.5%) individuals were infected with the transcontinental sub-subtypes of the cosmopolitan subtype (aA) and that 4 individuals (4.5%) were infected with the Japanese sub-subtypes (aB). A comparison of the nucleotide and amino acids of the FLG between the three clinical settings yielded no correlation between the sequenced genotype and clinical outcomes. The evolutionary relationships among the HTLV sequences were inferred from nucleotide sequence, and the results are consistent with the hypothesis that there were multiple introductions of the transcontinental subtype in Brazil.This study has increased the number of subtype aA full-length genomes from 8 to 81 and HTLV-1 aB from 2 to 5 sequences. The overall data confirmed that the cosmopolitan transcontinental sub-subtypes were the most prevalent in the Brazilian population. It is hoped that this valuable genomic data will add to our current understanding of the evolutionary history of this medically important virus. Human T-cell leukemia virus type I (HTLV-1) is the retrovirus responsible for adult T-cell leukemia/lymphoma (ATLL) and for the chronic neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) gag, pol and env genes flanked by long terminal repeat (LTR) sequences at both the 5′ and 3′ ends. A distinct molecular structure, known as the pX region, that is not present in other retroviruses is found between env and the 3′ LTR. The plus strand of the pX region encodes the regulatory proteins p40 (Tax), p27 (Rex), p12, p13, p30, and p21, which are critical to the viral infectivity in resting primary lymphocytes and to proliferation in infected cells Similar to other retroviruses, HTLV-1 carries a diploid RNA genome comprising 9032 nucleotides that is reverse-transcribed into double-stranded DNA that integrates into the host genome as a provirus 55 person-years, and the residual transfusion risk was 5.0×106 per blood unit transfused. A high prevalence of HTLV-1 infection has been reported in Salvador, a large city in the eastern part of Brazil, with an estimated prevalence of 1.35% among blood donors and 1.76% of the overall population Much of our current understanding of the HTLV-1 genome structure, variability and evolution has come from the conventional Sanger di-deoxy sequencing approach applied to viral partial sequences. According to previously published data on phylogenetic comparisons of partial sequences, seven subtypes of HTLV-1 strains have been described thus far (a–g) DNA sequencing has been dramatically advanced by increasingly high-throughput technology. Recent work has employed this technology to enable the characterization of entire viral populations in human and nonhuman primates Ninety participants were randomly selected from a larger cohort of 233 HTLV-1-infected persons representing 48 (53.3%) asymptomatic carriers (ACs), 35 (38.9%) HAM/TSP patients and 7 (7.8%) ATLL patients. This sub-cohort was part of an ongoing project to profile human T-cells miRNAs in the course of HTLV-1 infection using a deep-sequencing approach. A decision to include 90 samples was made because up to 96 samples can be pooled and sequenced together in a single flow cell. HTLV-1-positive individuals were recruited from the HTLV-1 outpatient clinic at the University of Sao Paulo and the Institute of Infectious Diseases “Emilio Ribas.” All ACs were diagnosed as HTLV-1 carriers at the time of blood donation. Viral infection was identified by the Murex HTLV I + II  and Vironostika HTLVI/II  HTLV enzyme immunoassays, and infection was confirmed by HTLV BLOT 2.4 . The clinical status of HAM/TSP was determined based on the WHO criteria for HTLV-1-associated diseases tax region using previously published primers RNase P gene from humans 2O control) was run with every assay. Standard curves for HTLV-1 tax were generated from MT-2 cells of log10 dilutions (from 105 to 100 copies). The threshold cycle for each clinical sample was calculated by defining the point at which the fluorescence exceeded a threshold limit. Each sample was assayed in duplicate, and the mean of the two values was considered the copy number of the sample. The HTLV-1 proviral load was calculated as the copy number of HTLV-1 (tax) per 1000 cells = (copy number of HTLV-1 tax)/(copy number of RNase P gene/2)×1000 cells. The method could detect 1 copy per 103 PBMCs.DNA was extracted from peripheral blood mononuclear cells (PBMCs) using a commercial kit  following the manufacturer's instructions. The extracted DNA was used as a template to amplify a 97-bp fragment from the HTLV-1 TGA CAA TGA CCA TGA GCC CCA AAT ATC CCC CGG′35′ and HTLV-1 FG_O1R GCT GGA GTC GGG GGG AGT GGT GAA GCT GCC′35′. Fragment B was amplified using the primers HTLV-1 FG_O2S CGC CGG GGC CTA CTT CCT AAC CAC ATC TGG CAA GG′35′ and HTLV-1 FG_O2R TCT CCT GAG AGT GCT ATA GGA TGG GCT GTC GCT GGC TCC TAT′35′. The boldface nucleotides (non-HTLV-1 specific sequences) are tails at the 5′ end of the outer primers and were added to enhance the nested amplification with inner primers. The PCR products were then subjected to nested PCR to amplify the A and B nested fragments. The nested primers for fragment A were HTLV-1 FG_N1S 5′CCA TGA GCC CCA AAT ATC CCC CGG′3 and HTLV-1 FG_N1R 5′GGG GGG AGT GGT GAA GCT GCC′3. The nested primers for fragment B were HTLV-1 FG_N2S 5′GGC CTA CTT CCT AAC CAC ATC TGG CAA GG′3 and HTLV-1 FG_N2R 5′GGA GCC AGC GAC AGC CCA TCC TAT′3. The PCR conditions for outer and inner PCR were as follows: an initial step of 5 min at 94°C; 35 cycles, with 1 cycle consisting of 30 s at 94°C, 30 s at 60°C, and 5 min at 72°C; and a final step of 10 min at 72°C. The amplified DNA fragments from the nested PCR product were separated by gel electrophoresis and purified using Freeze ‘N Squeeze DNA Gel Extraction Spin Columns. Each purified amplicon was quantified using Quant-IT HS reagents , and both amplicons from a single viral genome were pooled together at equimolar ratios.The complete provirus genome was amplified in two large fragments  from 200–300 ng of extracted genomic DNA. The structural and regulatory genes of each sequence were mapped based on comparison with the genomic sequence of B1033-2009 sub-subtype aB from Japan (GenBank accession no. AB513134). Fragment A was amplified using the primers HTLV-1 FG_O1S Each pool was then quantitated, and approximately 1 ng of each was used in a fragmentation reaction mix using a Nextera XT DNA sample prep kit according to the manufacturer's protocol. Briefly, tagmentation and fragmentation of each pool were simultaneously performed by incubation for 5 min at 55°C followed by incubation in neutralizing tagment buffer for 5 min at room temperature. After neutralization of the fragmented DNA, a light 12-cycle PCR was performed with Illumina Ready Mix to add Illumina flowcell adaptors, indexes and common adapters for subsequent cluster generation and sequencing. Amplified DNA was then purified using Agencourt AMPure XP beads (Beckman Coulter), which excluded very short library fragments. Following AMPure purification, the quantity of each library was normalized to ensure equally library representation in our pooled samples. Prior to cluster generation, normalized libraries were further quantified by qPCR using the SYBR fast Illumina library quantification kit (KAPA Biosystems) following the instructions of the manufacturer. The qPCR was run on the 7500 Fast Real-Time PCR System (Applied Biosystems). The thermocycling conditions consisted of an initial denaturation step at 95°C for 5 min followed by 35 cycles of [30 s at 95°C and 45 s at 60°C]. The final libraries were pooled at equimolar concentration and diluted to 4 nM. To denature the indexed DNA, 5 µL of the 4 nM library were mixed with 5 µL of 0.2 N fresh NaOH and incubated for 5 min at room temperature. 990 µL of chilled Illumina HT1 buffer was added to the denatured DNA and mixed to make a 20 pM library. After this step, 360 µL of the 20 pM library was multiplexed with 6 µL of 12.5 pM denatured PhiX control to increase sequence diversity and then mixed with 234 µL of chilled HT1 buffer to make a 12 pM sequenceable library. Finally, 600 µL of the prepared library was loaded on an Illumina MiSeq clamshell style cartridge for paired end 250 sequencing.de novo assembled into contiguous sequences and annotated with CLC Genomics Workbench version 5.5  and the Sequencher program 5.2 . To improve the quality of reads, approximately 10 nucleotides were trimmed from the 3′ end of the reads from each sequence library of each sample. Because the repetitive and identical sequences of both LTR presented the biggest difficulty in assembling HTLV-1 genome sequence data, we decided to only use the 3′ LTR sequence from each sequence for further analysis. The contiguous genomic sequence from each virus strain was extracted from the assembly and used for further analysis. The full designation of samples is 0YYBR_CLNXXX, where 0YY stands for the year of study, BR for Brazil, CLN for clinical status, and XXX for the enrolment number.Fastq files were generated by the Illumina MiSeq reporter for downstream analysis and validated to evaluate the distribution of quality scores and to ensure that quality scores do not drastically drop over each read. Validated fastq files from each viral genome were To increase the reliability of the observed DNA variations, the background error rate was set at 1% meaning that DNA variations detected in at least 1% of the viral sequences within a given sample are genuine variations.gag, pol, env p30 and tax) were calculated using MEGA5 http://tree.bio.ed.ac.uk/) or the freely available Archaeopteryx Java software Open reading frames were individually aligned with prototype variant B1033-2009 using the MAFFT algorithm All consensus genome assemblies generated in this study were submitted to NCBI's GenBank database .3 PBMCs , 177 copies per 103 PBMCs  and 273 copies per 103 PBMCs (153–3279), respectively. The mean time to HTLV-1 diagnosis was 8.8±5.4 years. The characteristics of the 90 patients included in the study are summarized in In total, 90 blood samples from HTLV-1 infected individuals were included in the study. The participants' ages ranged between 31 and 81 years, and the median age was 56 years. Females constituted 68.9% (n = 62) of the study group. The median measurements of CD4 and CD8 lymphocyte percentage by flow cytometry  were 45% and 22%, respectively, in 31% of subjects. The median proviral loads defined in this study in all the ACS, HAM/TSP and ATLL groups were 431 copies per 103 PBMCs. To assess the genetic variability of HTLV-1, the FLG sequences were successfully assembled into a single, high-quality contig in 76 HTLV-1 sequences, with the majority starting approximately at position 727 of the 5′ long terminal repeat (LTR) to the extreme 3′ LTR at position 9113 (average length of 8259 bp). No ambiguities were detected between the two overlapping sequences, indicating that errors due to PCR had little effect on the overall sequences of the proviral genome of these sequences. The read-through translation of the 5 open reading frames indicated that all complete coding sequences obtained in this study were intact and in frame. There were several nucleotide substitutions in the complete coding regions. Among these, we found 29 specific nucleotides that were always simultaneously substituted in 27 participants and were identical to the prototype Japanese ATK   Table 2.tax gene of the sequences with complete coding regions confirmed the presence of the complete set of four polymorphic sites tax A in 73 (96%) samples, but the tax B profile was confirmed in only 3 (4%) strains, namely 012BR ATL003 HC, 012BR ASY032, and 012BR ATL005 HC, which clustered within the tax B sub-subtypes  samples , which belonged to the Japanese HTLV-1 aB sub-subtypes and are represented in this tree by a single branch Figure 1Figure S1), failed to generate full genomic data by our deep sequencing method. The de novo assembly of the compiled genome from each of these sequences had an average sequencing depth ranging from 351–5713. When aligned to the B1033-2009 sequence, eight sequences displayed two contigs separated by a gap of less than 500 bp, suggesting the small size of most gaps .Fourteen HTLV-1 sequences, identified by a phylogenetic tree from partial data as belonging to the HTLV-1 aA (n = 13) or HTLV-1 aB (n = 1) sub-subtypes . On the basis of a phylogenetic analysis of this region  were extracted from the HTLV complete and partial genomes of each subject and aligned with the LTR sequences representing all assigned subtypes and unassigned variants with a minimum length of 500 bp from the HTLV-1 molecular epidemiology database  that were classified as subtypes aA (n = 8), aB (n = 2), aC (n = 2) and 1b (n = 1) and 2 unassigned, for which some of the sequences have the country of sampling stated. Among these, there were 3 recovered from Brazil. The scarcity of these sequences prompted us to generate newer genetic materials of these viruses and to obtain more information on the molecular basis of HTLV-1 sequences in Brazil. To date, no study has employed second-generation sequencing techniques to examine HTLV-1 heterogeneity across entire genomes from different clinical settings. In this work, we used state-of-the-art Illumina MiSeq instrumentation to generate 76 and 14 HTLV-1 complete and partial genome sequences, respectively, from different ACs, HAM/TSP and ATLL clinical sources in a single run. Thus, the sequences described in this study quintuple the publicly available full genome sequence information for HTLV-1 viruses. Analysis indicates that the vast majority of subjects in this study was infected with the cosmopolitan genotype, mostly the transcontinental and more rarely the Japanese sub-subtypes, a finding that is in agreement with previous reports Currently, there are only 15 complete sequences of HTLV-1 sequences in the GenBank and HTLV-1 molecular epidemiology databases  to define their genomic locations. The star symbol indicates proviral load . The pilcrow symbol indicates the overall mean coverage depth.(TIF)Click here for additional data file.


Most species and the PFTs they represent were ubiquitously distributed along the topoedaphic gradient, but with varying abundances. Overstory-understory comparisons suggest that while future species composition of these woody communities is likely to change, PFT composition is not. Canonical correspondence analysis (CCA) ordination and variance partitioning  indicated that woody species and PFT composition in developing woody communities was primarily influenced by intrinsic landscape location variables  and secondarily by plant-induced changes in soil organic carbon and total nitrogen content. The ubiquitous distribution of species and PFTs suggests that woody plants are generally well-suited to a broad range of grassland topoedaphic settings. However, here we only examined categorical and non-quantitative functional traits. Although intrinsic soil properties exerted more control over the floristics of grassland-to-woodland succession did plant modifications of soil carbon and nitrogen concentrations, the latter are likely to influence productivity and nutrient cycling and may, over longer time-frames, feed back to influence PFT distributions. Woody plant encroachment into grasslands has been globally widespread. The woody species invading grasslands represent a variety of contrasting plant functional groups and growth forms. Are some woody plant functional types (PFTs) better suited to invade grasslands than others? To what extent do local patterns of distribution and abundance of woody PFTs invading grasslands reflect intrinsic topoedaphic properties versus plant-induced changes in soil properties? We addressed these questions in the Southern Great Plains, United States at a subtropical grassland known to have been encroached upon by woody species over the past 50-100 years. A total of 20 woody species  were encountered along a transect extending from an upland into a playa basin. About half of the encroaching woody plants were potential N Increases in the abundance of trees and shrubs have been reported in ecosystems world-wide -3. The r2-fixing to non-fixing; from deep rooted to shallow rooted; and from mesophytic to xerophytic in water relations . Thus, the discrete upland cluster, grove and drainage woodland communities represent examples of topoedaphic mediation of woody plant encroachment and community development. In this study we sought to determine if there were fundamental differences in the species and PFT composition of these communities that might shed light on how the shrub encroachment process plays out on upland and lowland portions of grassland landscapes. Our assessments of species and PFT composition and abundance were conducted across a local landscape-scale gradient. As such, they are not confounded by differences in climate, weather or land use history that can occur when comparing species abundance and community structure at multiple sites within a region or across bioclimatic zones. Here we only selected several categorical functional traits to define different PFTs, which we believe represent a wide range of functional traits that are important. However, including more detailed quantitative traits such as seed size or leaf traits may make our case stronger by providing other important aspects of the community assemblage.P. aculeata, the woody species and PFTs in playa communities were also present and typically common in cluster, grove, and woodland communities  have been dying in cluster communities [1] as was evidenced by its absence in our sampling (Table 2). A. farnesiana, representing the same PFT as P. glandulosa, also occurred in clusters, but was not represented in the shrub layer (Table 3) and hence may not persist in the community. However other N2-fixing woody species occur in the shrub layer, so this PFT could still be represented in future vegetation states even with the demise of the current N-fixing PFTs in the tree layer. Prosopis overstory plants were doing well in the other communities (Table 2); and, with the exception of playa communities, was well-represented in the shrub class (Table 3). This suggests that overstory trees of this PFT will be replaced when they die. These observations centered around N2-fixing, deciduous PFTs are consistent with ordination results  and suggest two things: (i) significant changes in the species composition of each of these woody plant communities may be forthcoming; and (ii) species-specific rather than PFT-specific differences are driving the compositional dynamics of the woody communities developing on grassland. In other words, while some species may eventually be lost, the PFTs they represent will not be.The woody communities on this former grassland site are relatively young (< 60-100 y of age) and may still be developing. Their species and PFT composition in the future may, to a large extent, depend upon the extent to which the present-day dominants are represented in the understory shrub layer. Overstory Prosopis plants  perspective  on this former grassland, P. glandulosa can quickly grow roots deeper than the zone utilized by grasses, which enhanced their survival during the earlier stage of their life history [P. glandulosa is an important mechanism of vertical water redistribution. However, the hydraulic lift is temporal dynamic and its effect on understory shrubs varies [P. glandulosa trees [Rooting depth is an important functional trait in savanna ecosystems because niche separation of soil water use at different depth was believed to be an importance mechanism to tree-grass coexistence ,50. In o history . Later is varies . Overallsa trees ,52. Our Relative basal area of evergreen and deciduous woody species are relatively stable along the topoedphic gradient . Faculta2-fixation potential are not necessarily reliable indicators of ecosystem processes related to resource use [The number of woody species in shrub cluster communities and their patterns of occurrence at this site are strongly related to the size/age of the overstory trees . Soils aurce use  or decomurce use . 2-fixing PFTs at our site, P. glandulosa was clearly the most aggressive initial invader of the historical grasslands [2-fixation. In addition, most of the other non N2-fixing woody PFTs at this site are bird-dispersed; and the establishment of livestock-dispersed P. glandulosa provides a perching structure attracting birds disseminating seeds of other woody PFTs [P. glandulosa plants colonizing grasslands may therefore be a secondary or coincidental factor influencing the encroachment of other woody PFTs – a conjecture consistent with our finding that patterns of species and PFT presence/absence and abundance were primarily a function of differences in intrinsic soil physical properties and secondarily related to modifications of SOC and TN by early-establishing shrubs  appears to be important in initiating the transition from grassland to shrubland across the catena gradient at this site, our survey gives little reason to expect that the shrubland communities that subsequently develop are following any particular or predictable assembly rules that might consistently reflect PFT responses to changes in microclimate and soils occurring in the transition from grassland to shrubland or woodland. The lack of strong patterns in woody species or PFT occurrence within the four woody plant communities developing on contrasting topoedaphic settings may reflect the fact that PFTs can exhibit considerable convergence in resource use [The results from our study are consistent with assertions of random community assembly  in that urce use  and that


A left-right patterning study in developing sea urchin shows that the opposing roles of Nodal and BMP signaling in patterning the left-right axis are conserved in deuterostomes. veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the  Bilateral animals that are externally symmetric in appearance often have internal organs that are asymmetric with respect to the left and right sides of their bodies. Two signals, Nodal and BMP, have been shown to establish this asymmetry during vertebrate embryogenesis. We investigate here whether the same mechanisms that establish left-right patterning in vertebrates are conserved in invertebrate animals, specifically in the California purple sea urchin. This sea urchin passes through various stages in its lifecycle before developing to adulthood, including a feeding larva stage in which the tissue that goes on to form the adult, the so-called adult rudiment, forms on the left side. Previous studies have shown that right-sided Nodal signaling in sea urchins prevents the formation of the adult rudiment. In this study, we show that BMP signaling is activated on the left side and is required for the development of this left-sided structure. We also demonstrate that Nodal signaling blocks BMP activity and induces apoptosis of the tissue that forms the adult rudiment on the right side. We propose that the roles of Nodal and BMP signals in establishing left-right asymmetry are evolutionarily conserved. In the past two decades, studies of molecular pathways controlling LR asymmetry have broadened our knowledge regarding the dissimilar and conserved mechanisms among different animal models nodal expression on the left side, BMP transcripts or activities are observed on the right side of the node or LPM. For example, bmp4 is expressed on the right side of Hensen's node in the chick embryo and initiates a right-sided signaling cascade bmp genes are expressed symmetrically in the LPM of the mouse and chick embryos, BMP activity is greater on the right side due to the presence of BMP antagonists on the left side Xenopus embryo dpp-bmp2/4 plays important roles in shell formation and coiling in gastropods Bone morphogenic protein (BMP), another TGFβ family member, is also involved in LR patterning veg2 mesoderm and small micromeres (Smm) forms at the archenteron tip and later separates into left and right pouches in the bilateral gastrula Sea urchins belong to the phylum Echinodermata, which is characterized by a pentaradiate adult body plan. In indirect developing sea urchins, the adults are derived from bilaterally symmetric larvae. The transition from a bilaterally symmetric to pentasymmetric body plan relies on a LR asymmetrical control that results in the formation of an adult rudiment on the left side of the larva . During nodal expression on the right side, which is reversed when compared to vertebrates, prevents the formation of the adult rudiment Our knowledge of the molecular mechanisms concerning LR patterning in sea urchins is relatively limited. It has been shown that sea urchin LR axis specification depends on cell interactions bmp genes are symmetrically expressed in skeletogenic micromeres, but BMP signaling is asymmetrically activated in the left CP-derived HC. Through cell lineage analysis, we detected active BMP signaling in veg2 descendants but not in the Smm. We further provided evidence that BMP signaling is required for left-sided structure development and the expression of several left-sided marker genes. We also demonstrate that right-sided Nodal signaling restricts BMP activity and is involved in the asymmetrical separation and apoptosis of the Smm. We discuss these findings in the context of Nodal and BMP signaling in patterning LR asymmetry in the sea urchin embryo.In this study, we focused on the role of the BMP pathway and examined the molecular basis of LR asymmetry in the sea urchin embryo. We found that bmp ligand genes: bmp2/4, bmp3, and bmp5–8Bmp2/4 is initially transcribed in the oral ectoderm at the blastula stage, but the Bmp2/4 ligand translocates to the aboral side and plays key roles in the aboral ectoderm gene regulatory network bmp3 and bmp5–8 have not been elucidated. Therefore, we performed quantitative PCR and discovered that the bmp3 transcripts are not detectable during the first 3 d of development, whereas bmp5–8 was expressed in the egg and during this period (unpublished data). In situ hybridization demonstrated that bmp2/4 expression shifted from the oral ectoderm to the aboral skeletogenic mesenchyme cells during gastrulation and remained in a few cells at the apex of the pluteus larva  and did not observe asymmetrical LR expression (unpublished data). We then investigated BMP signaling activity by monitoring the phosphorylation and nuclear translocation of its downstream mediator Smad1/5/8 (pSmad). Immunostaining with a pSmad antibody showed that BMP signaling was activated in the aboral ectoderm at the blastula stage, as previously reported bmp2/4 and bmp5–8 are symmetrically expressed, BMP signaling is activated on the left side in the pluteus larva and may play a role in controlling left-sided development in sea urchins.To study the role of BMP signaling in LR asymmetry in sea urchins, we first examined the expression patterns of genes related to the BMP signaling pathway. The sea urchin genome contains three us larva . This exs stages . Both thveg2 descendants veg2 cells and differentiated into the HC. Smm lacking pSmad staining were located in the roof of the archenteron at the gastrula stage and later constituted the base of the left CP in the pluteus larva.The CPs consist of two cell lineages: Smm and veg2 descendants or a Drosophila Vasa antibody that labeled Smm similarly to BrdU    and S2F.us stage .veg2-derived oral and aboral tips and the Smm located on the roof  axial patterning, we used pharmacological treatments to perturb BMP signaling prior to LR axis establishment. A small molecule, dorsomorphin (DM), which selectively inhibits BMP type I receptors and blocks Smad phosphorylation, was previously identified in a screen for compounds that perturb dorsoventral (DV) axis formation in zebrafish d signal . When thd signal , we obsela stage , indicatiated HC . We obseiated HC . This waveg2 descendants, including soxE, pax6, six1/2, eya, and dach, were diminished in DM-treated embryos but remained in the single CP when BMP signaling was elevated  or was retained in the oral ectoderm (16%). These results indicate that BMP is required for nodal expression initiation, although the mechanism remains unknown.We further analyzed LR marker gene expression patterns after BMP signaling was perturbed. The expressions of all examined genes that were normally expressed in the aboral elevated . Smm gen embryos . These r and not  [6],[39]xpressed . We furt blocked . The exphox7 but had little effect on the non-target chordin  input may also be involved in the spatial regulation of nodal expression. Taken together, these data suggest that BMP signaling is both upstream and downstream of Nodal signaling.Based on the lineage and perturbation analyses, we provided a schematic representation of the molecular pathways in LR patterning. veg2 mesoderm contribute to the CPs, past studies were unable to clearly identify genes that are specifically expressed in either lineage. It was also unknown which of the two lineages contributed to the left CP-derived HC. In addition to identifying several lineage-specific genes in the CP and the HC, we also provided evidence to demonstrate that BMP signals act in the left CP together with Nodal signaling to regulate LR patterning. Given that left-sided nodal expression is a conserved feature in chordates Most sea urchin adult tissues derive from the rudiment developed from the left CP. Although it is known that both Smm and the chordin and noggin genes, which encode BMP antagonists lefty genes that encode Nodal antagonists chordin, which restricts BMP signals in the aboral ectoderm chordin, noggin, follistatin, dan, or gremlin in the sea urchin embryo (unpublished data). The second molecular mechanism to explain the mutual antagonism between Nodal and BMP signaling is the direct competition between the two signals for the limited amount of the common effector Smad4. In the mouse embryo, BMP signaling has been shown to set a repressive threshold for Nodal signaling in the LPM by limiting Smad4 availability We demonstrated that elevating either Nodal or BMP signaling resulted in the loss of the other signal. This mutual antagonism between Nodal and BMP signaling has been observed during LR patterning in vertebrates. Nodal signaling inhibits BMP signals in the left LPM of mouse embryos by activating the expression of nodal expression. These results suggest that BMP signaling is required for right-sided nodal expression in the sea urchin embryo. This positive role of BMP signaling on nodal gene expression has also been observed in vertebrates. In the absence of mouse embryonic BMP4, nodal expression is lost in the left LPM bmp2-expressing cells nodal expression. During the late segmentation stages of zebrafish embryos, BMP4 signaling is required to activate the expression of the nodal-related gene cyclops in the left LPM The repressive role of Nodal signaling on BMP in the sea urchin embryo is evident given that increasing or blocking Nodal signaling results in the loss of or bilateral pSmad staining in CPs, respectively. However, the effects of BMP signaling on Nodal are complicated because increasing and blocking BMP signaling both result in the loss of bmp genes are transcribed in the skeletogenic mesenchyme cells near the aboral apex of the larva. These observations suggest that BMP ligands are secreted from these micromere-derived skeletogenic cells to control LR asymmetry. It was previously shown that when the micromere lineage was removed from embryos of sea urchin Hemicentrotus pulcherrimus, the LR placement of the rudiment was randomized Although we observed LR asymmetrical BMP signaling with pSmad staining in the CPs in the sea urchin, vasa, nanos2, and seawinanos2 expression and cell death. These results suggest that Nodal signaling in the right CP represses nanos2 expression in the Smm, which leads to cell death. To our knowledge, the role of Nodal signaling in repressing the expression of germline lineage genes such as nanos has not been reported in other systems. Intriguingly, Nodal is capable of inducing apoptosis in adult ovary during follicular degeneration and human trophoblast cells during normal placentation In sea urchin embryos, Smm form during the fifth cleavage and are regulated by a set of conserved germline lineage genes, including Strongylocentrotus purpuratus) and their gametes were obtained as previously described Rhodomonas lens provided by Pat Leahy was used to feed the larva  for 1 h and then washed twice with 500 µM thymidine. For double labeling, the biotin-avidin system was used to detect the BrdU signal. Antigen retrieval of the incorporated BrdU was conducted by DNA denaturation using 1 N HCl in PBST for 30 min. To block endogenous biotin, the embryos were incubated with 0.01% avidin and 0.001% biotin sequentially. The embryos were then immunolocalized with biotinylated anti-BrdU antibody  and detected with HiLyte Fluor Streptavidin . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed by using the In Situ Cell Death Detection Kit  for 40 min at 37°C.Figure S1Developmental processes and LR asymmetry in the sea urchin. (A) Schematic illustrations of developmental processes from radial symmetric blastula, bilateral symmetric gastrula, left-right asymmetric larva, to pentasymmetric body plan. At the end of gastrulation, two coelomic pouches (CPs) form on each side of the archenteron tip. A distinct LR asymmetry occurs when the hydroporic canal (HC) evaginates from the left CP. The CPs then divide into the axocoel (ax), hydrocoel (hy), and somatocoel (so). The invaginated left oral ectoderm forms the primary vestibule (pv), which makes contact with the enlarged left hydrocoel to form an adult rudiment (ru) on the left side of the larva. A secondary vestibule (sv) also invaginates from the right oral ectoderm. (B–M) LR asymmetry in the sea urchin. At the early pluteus stage , the HC is formed on the left side (B) with a hydropore (hp) opening on the left aboral ectoderm (C). (D) Higher magnification of the HC in the four-arm larva (E). (F) The HC is the first morphological sign of LR asymmetry. (G) Vestibular invagination stage larva. (H) The vestibule and the left hydrocoel become apposed (arrow) at the rudiment initiation stage. (I–J) At the pentagonal disc stage, the five tube-foot primordia (tfp) project against the vestibular floor (arrow). (K) At the advanced rudiment stage, the rudiment with adult plates forms on the left side of the larva. (L) Higher magnification of the juvenile spines (js) that develop at the posterior apex and on the right side of the larva. (M) A juvenile sea urchin with juvenile spines and adult spines (as) after metamorphosis. The observed view is indicated in the bottom right-hand corner of each panel, and the axes are labeled as O, oral; A, aboral; L, left; and R, right.(TIF)Click here for additional data file.Figure S2soxE, pax6, six1/2, eya, and dach at the LG and EPL stages. (C) Double fluorescence ISH analyses of soxE with pax6, six1/2, eya, or dach at the EPL stage. (D) Expression patterns of the germline marker genes at the LG and EPL stages. (E) Expression patterns of foxC and foxF at the LG and EPL stages. (F) Double fluorescence ISH analyses of foxC and nanos2 or foxF. (G) Expression of foxY at different developmental stages. The solid arrowheads indicate Smm, the arrows indicate signals in the aboral tip and HC, and the open arrowheads denote expression in the oral tip of the archenteron and the esophageal domain.Cell lineage analysis and gene expression patterns during CP formation. (A) Smm were labeled with BrdU and Vasa antibody at the LG and EPL stages. (B) Whole mount ISH of (TIF)Click here for additional data file.Figure S3hox7 and pSmad signals in the BMP signaling-perturbed embryos. (D) The embryos were incubated with the vMOs after injecting with the mRNA containing vMO-binding sites upstream of the GFP coding sequence (constructs shown on the right). The effects of the vMOs on the fluorescence intensities of GFP were quantified in embryos treated with different concentrations of the vMOs after injecting GFP mRNA containing the BMP2/4 (E) or Nodal (F) vMO-binding site. Error bars are standard errors of the mean. (G) ISH of chordin and hox7 in 5 µM BMP2/4 vMO- or Nodal vMO-treated embryos. The upper panels (MB) were treated from one-cell to MB stage. The bottom panels (LG) were treated from MB to LG stage. The numbers in the bottom left-hand corners of the photos indicate the phenotype ratios. (H) The embryos were incubated for the indicated time and concentration, and the effects on the HC or CP were assessed. The listed effect represents the phenotype observed in over 90% of embryos. The black bar shows the treatment time used in most experiments. Note that the effective timing for DM and vMO treatment was different (H). DM treatment from 42 to 48 hpf was sufficient to block HC formation, whereas the vMO had no effect when applied in the same period. The different effects might be due to the natures of the two blocking mechanisms. DM inhibits BMP receptor kinase activity and blocks BMP signaling immediately after it penetrates cells. On the other hand, the vMO blocks translation of bmp2/4, and BMP signaling could still be active until the remaining BMP2/4 is degraded.Efficacy of dorsomorphin and vMOs. (A) Western blot analysis using anti-pSmad antibody on mesenchyme blastula (MB) extracts revealed a major band around 60 kDa as predicted (arrow). (B) The pSmad levels decreased upon dorsomorphin treatment. β-tubulin was used as a loading control to quantify the pSmad1/5/8 protein levels. (C) Expression of (TIF)Click here for additional data file.Figure S4Effects of Nodal signaling on LR asymmetry. (A) pSmad and acetylated α-tubulin staining in SB-431542-treated embryos revealed bilateral HC (arrows) in EPL. (B) Expression of right-sided genes (indicated by arrows) following Nodal signaling perturbation. (C and D) Expression of LR marker genes after SB-431542 treatments. The numbers in the bottom left-hand corners of the photos indicate the phenotype ratios. (E) The embryos were incubated for the indicated time and concentration, and the effects on the oral-aboral (OA) axis and HC formation in over 90% of the embryos are listed. The black bar shows the treatment time used in most experiments. The effect of SB inhibitors and Nodal vMO was different in that Nodal vMO did not cause OA defects when treated during MB. The difference might also be due to the differential inhibitory mechanisms: SB inhibitors directly block signaling, whereas vMO blocks translation of the ligand.(TIF)Click here for additional data file.Table S1Gene IDs and primers used to construct clones for probe synthesis in this study.(XLS)Click here for additional data file.Text S1Supplementary methods.(DOC)Click here for additional data file.


Following entry and reverse transcription, HTLV-1 integrates into the host cell genome. The viral promoter activity is directly governed by its chromatin environment. Epigenetic modifications, such as DNA methylation and histone deacetylation, are crucial for transcriptional silencing of the virus. We have previously reported that the cofactor CTIP2 recruits a multienzymatic chromatin-modifying complex, including histone deacetylases and a methyltransferase to the HIV-1 promoter, thereby establishing a heterochromatic environment at the promoter. CTIP2 is recruited to the HIV-1 promoter via its association with the transcription factor Sp1. Here, we investigated the potential role of CTIP2 in transcriptional regulation of the HTLV-1 promoter.Electrophoretic mobility shift assays (EMSAs), transient transfection and chromatin immunoprecipitation (ChIP) assays were performed.Analysis of the nucleotide sequence of the HTLV-1 promoter (LTRHTLV-1) revealed two Sp1 binding sites within the R region. We demonstrated that Sp1 and Sp3 bind in vitro to these sites by EMSAs. We showed, by cotransfection assays in epithelial HEK293T cells and T-lymphoid Jurkat cells, that the cofactor CTIP2 inhibited Tax-mediated transactivation of the HTLV-1 promoter. We demonstrated by ChIP assays the in vivo recruitment of CTIP2 to the LTRHTLV-1 in a latently HTLV-1 infected cell line (TLom1). Conversely, CTIP2 was absent from the proviral genome in a HTLV-1 productive cell line (SLB1). The identification of the multienzymatic complex recruited by CTIP2 to the viral promoter under latent conditions is currently under investigation.Our results demonstrate a repressive role of CTIP2 on LTRHTLV-1 transcriptional activity, suggesting a potential effect of this cofactor in HTLV-1 latency.


HTLV-1 is associated with the onset of various inflammatory diseases such as HTLV-1 Associated Myelopathy / Tropical Spastic Paraparesis, uveitis, infective dermatitis or inflammatory myopathies. Here, we aimed to get new insights into the pathogenesis of HTLV-1 associated inflammatory myopathies (HAIM) by studying muscle biopsy specimens and blood samples from 13 HAIM patients.Mean age of patients was 52.2 years. 7 patients suffered from polymyositis (PM), and 6, from inclusion body myositis (IBM). Histopathological changes were mild to moderate in most patients. Tumor necrosis factor (TNF)-alpha and myeloid dendritic cells were detected in several patients’ biopsies, and Human Leukocyte Antigen (HLA)-ABC, HLA-DR, and matrix metalloproteinase , in most of them. Perforin was frequently detected but there were no apoptotic myonuclei. By means of in situ hybridization, we detected rare HTLV-1 infected infiltrating cells in the muscle tissue of 4 patients. The virus belonged to the cosmopolitan A subtype, transcontinental subgroup. Plasma anti-HTLV-1 antibodies titers were high, but the proviral load was not elevated when compared to asymptomatic HTLV-1 carriers. Myositis-associated autoantibodies were found in patients with HAIM as well as in HTLV-1 infected controls without HAIM, whereas IFN-gamma plasma levels were elevated in HAIM patients.We describe 13 cases of HTLV-1 associated myositis, which show the classical anatomopathologic features of idiopathic myositis, with moderate muscle inflammation and atrophy. Proviral load was not elevated, but anti-HTLV-1 antiboides titer and IFN-gamma plasma levels were raised.


This set point appears to be determined by an equilibrium between virus-driven proliferation and CTL-mediated killing of HTLV-1-infected T cells. However, we do not know what determines the number, the abundance or the pathogenic potential of HTLV-1-infected T cell clones. In addition, the contribution of We hypothesize that the genomic integration site (IS) of the HTLV-1 provirus determines the pattern and intensity of spontaneous proviral expression; the viral gene products in turn determine the rate of proliferation of the infected cells, and the rate of CTL-mediated killing. We aim to identify the factors that determine the integration site targeting, expression and abundance of the HTLV-1 provirus in natural infection.We developed a novel protocol ,2 for hiEach infected individual carries about 25,000 distinct clones of HTLV-1-infected T cells - between 100 and 1000 times more than previously believed.HTLV-1 preferentially integrates into host DNA within 10 to 100 bases of binding sites for specific DNA-binding factors, notably P53 and STAT1. Spontaneous expression of HTLV-1 Tax is associated with antisense orientation of the provirus in the host gene and with specific transcription factor binding sites upstream or downstream of the provirus. Genomic hotspots of HTLV-1 integration are observed in cases of adult T-cell leukaemia/lymphoma (ATLL).in vivo when integrated in an acrocentric chromosome.HTLV-1 preferentially survives in vivo.The results suggest that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection. These results open the way to tests of the molecular mechanisms of targeting, expression and clone proliferation


MHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. Moreover overexpression of the nuclear factor NF-YB, that cooperates with CIITA for the expression of MHC class II genes, results also in inhibition of Tax-2 transactivation. The purpose of this investigation was to assess the cellular and molecular basis of the CIITA-mediated inhibition on Tax-2, and the relative role of NF-YB in this phenomenon.in vivo. A similar approach was used to assess Tax-2-NF-YB interaction. In parallel, deletion fragments of CIITA were tested for the inhibition of Tax-2-dependent HTLV-2 LTR-luciferase transactivation. Subcellular localization of CIITA and Tax-2 was investigated by immunofluorescence and confocal microscopy.By co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was assessed whether the two factors interact in vivo through at least two independent regions, at the 1-252 N-term and at the 410-1130 C-term, respectively. Interestingly only the 1-252 N-term region mediates Tax-2 functional inhibition. CIITA and Tax-2 are localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalize with CIITA in cytoplasm and around the nuclear membrane. The Tax-2 minor remaining nuclear portion also co-localizes with CIITA. Interestingly, when CIITA nucleus-cytoplasm shuttling is blocked by leptomycin B treatment, most of the Tax-2 molecules are also blocked and co-localize with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus.CIITA and Tax-2 interact Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase.These results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB in. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells HTLV-1 (Human T cell Lymphotropic Virus type 1) and HTLV-2 (Human T cell Lymphotropic virus type 2) are closely related human retroviruses that belong to deltaviridae family, subfamily oncovirus type C, characterized by similar genomic organization and common modes of transmission but different disease manifestations . HTLVs a+ T cells [HTLV-1 and HTLV-2 show a differential cellular tropism. HTLV-1 has a preferential tropism for CD4 T cells  while HT T cells -8. HTLV- T cells -12. ConvThe basis of HTLV mediated cellular transformation is not completely understood, but it involves the viral transactivator protein Tax. Tax is essential for HTLV-1- and HTLV-2-mediated immortalization of primary human T cells ,14 and fWe reported that Tax-2 transactivation of the HTLV-2 LTR is strongly inhibited by the host transcription factor CIITA. As a consequence, susceptible T and B human cells do not support HTLV-2 replication when expressing CIITA ,22. SimiAIR-1 locus-encoded class II transactivator CIITA is the master regulator of the expression of Major Histocompatibility Complex class II (MHC-II) genes [The I) genes -27. MHC-I) genes . CIITA iI) genes -34. It iI) genes -38. P-TEI) genes  and we hI) genes . On the I) genes .In this paper we have investigated the intimate molecular nature of the CIITA mediated inhibition of Tax-2. We found that both CIITA and NF-Y interact in vivo with Tax-2. We identified both an N-terminal and a C-terminal region of CIITA interacting with the viral transactivator, although, as stated above, only the N-terminal region is involved in the inhibition of Tax-2 function. Interestingly, in absence of CIITA, endogenous NF-YB can still bind to Tax-2, although, as we have previously shown, this interaction does not results in functional inactivation of Tax-2 on the HTLV-2 LTR promoter. CIITA-NF-YB interaction in vivo is stabilized and/or favoured by the presence of Tax-2. Thus concomitant interaction of Tax-2 with CIITA and NF-YB, most likely in the CIITA-NF-YB molecular complex, is at the basis of the functional inactivation of Tax-2 leading to the inhibition of HTLV-2 retrovirus replication. Further studies of subcellular localization unveiled the co-localization of Tax-2 and CIITA both in the cytoplasm and the nucleus, and the role of CIITA in redirecting, upon binding, Tax-2 molecules mostly in the cytoplasm.These results are discussed within the present knowledge of cell host-pathogen interaction and the identification of the dual role of CIITA as modulator of adaptive immunity and restriction factor against human retroviruses.Full length CIITA (pcDNA3flagCIITA1-1130) and deletion mutants of it  vectors have been described . The fla2). 293T cells were transfected with expressing constructs for the full-length Flag-CIITA or Flag-CIITA deletion fragments using Lipofectamine  following the manufactory protocol. After 24 h, cells were collected, resuspended in lysis buffer  supplemented with 0,1% protease inhibitor mixture  for 45 min on ice and centrifuged for 15 min, 14.000 rpm at 4°C. After preclearing the extracts with 10 μl of 100% mouse Protein A Sepharose 4 fast flow beads  for 30 minutes at 4°C by rotation, 0.5 μl of anti-V5 antibody (Invitrogen) were added and the mixture incubated for 1 hour on ice and then reacted with 50 μl of Protein A Sepharose 4 fast flow overnight at 4°C by rotation. Alternatively cell lysates were immunoprecipitated with 50 μl of anti-Flag M2 Affinity Gel . An aliquot corresponding to 12% of the total cell extract was conserved for proteins expression detection (input). Immunocomplexes were collected by centrifugation, washed five times with the above lysis buffer and once with the lysis buffer containing 500 mM NaCl. The immunocomplexes were detected after SDS-PAGE and Western blotting as described [Human embryo-derived kidney cell line 293T was maintained in DMEM supplemented with 10% FCS and 5 mM glutamine at 37°C and 5% COescribed  with eitHuman 293T cells were seeded on glass coverslips and transiently transfected with 1.5 μg of the indicated expression vectors with Lipofectamine (Invitrogen). 24 h post-tranfection the cells were fixed by incubation with 100% methanol at -20 for 6 min. The cells were washed with PBS and blocked for 1 h in PBS containing 0.5% gelatin (Biorad) and 0.5% bovine serum albumine (Sigma), before overnight incubation at 4°C with monoclonal V5 antibodies (Invitrogen) diluited 1:750 in the blocking solution. Goat anti-mouse IgG2a Fab conjugated to Alexa Fluor 546 (Molecular Probes) was used as secondary antibody. Samples were then mounted in Fluor Save reagent  and analyzed with a laser scanning confocal microscope (Leica) using a 63 × objective and light source wavelengths of 488 and 543 nm.In order to verify whether CIITA-mediated inhibition of Tax-2 could correlate with a direct binding between the two factors, flag-tagged CIITA and V5-tagged Tax-2 were transiently co-expressed in 293T cells. Cell lysates were immunoprecipitated with the anti-V5 antibody and immunocomplexes were examined for the presence of flagCIITA by anti-Flag western blotting. Results clearly indicate that CIITA and Tax-2 strongly interact each other in vivo  of CIITA mediating the interaction with the viral transactivator, several truncated forms of CIITA were tested for their ability to bind Tax-2 by co-immunoprecipitation assay in 293T cells. When either CIITA full length or CIITA fragments were expressed in absence of Tax-2 no specific bands were detected. A non specific band of 55 kD was present in all immunoprecipitates  and increasing amounts of plasmids encoding CIITA wild type pfCIITA 1-1130), N-term  or C-term (pf253-1130) fragments, respectively. It must be noted that in this assay 10 fold less DNA was transfected into the cells as compared to the interaction mapping of Figure -1130, N-Results show that Tax-2-mediated activation of the viral LTR promoter Figure , bar 3 wThus, also in 293T cells CIITA-dependent inhibition of Tax-2 function correlates with the N-term 1-252 region of CIITA. Furthermore, these results strongly suggest that interaction between the N-terminal, but not the C-terminal, part of CIITA, and Tax-2 is responsible of the biological effect of CIITA on the viral transactivator.Previous results from our laboratory have shown that the ubiquitously expressed nuclear transcription factor NF-YB, which interacts and co-operates with CIITA in activating HLA-II genes transcription, could inhibit the HTLV-2 LTR promoter transactivation by Tax-2 in COS-7 cells when over-expressed after transfection . Similarin vivo we performed initially co-immunoprecipation experiments by using lysates of 293T cells co-transfected with myc-tagged NF-YB (mNFYB) and V5-tagged Tax-2. Results presented in Figure in vivo with Tax-2. Although with the limitations of the relatively low expression of the endogenous NF-YB protein with respect to the protein expressed after transient transfection, the interaction of NF-YB with Tax-2 was observed also in this case  and nuclear export (NES) signals that allow the molecule to shuttle between cytoplasm and nucleus. CIITA NES are CRM-1 dependent, as treatment of CIITA-positive cells with the CRM-1 inhibitor leptomycin B (LMB) relocalizes CIITA mostly within the nucleus ,42. HTLVTaken together, the above subcellular localization studies indicate that the physical CIITA-Tax-2 interaction is mirrored by a strong co-localization of the two molecules in cytoplasmic and nuclear subcellular compartments. Moreover and of particular importance, interaction with CIITA makes Tax-2 molecules prone to migrate to cytoplasm where they can no longer exert their transactivating function on the HTLV-2 LTR.in vivo.Host-pathogen interaction is regulated by a series of cellular and molecular mechanisms whose outcome dictates in many instances the subtle equilibrium between control of infection and pathological consequences for the host. This is particular relevant for pathogens like human oncogenic retroviruses such as HTLVs whose infectivity can generate, as clearly demonstrated for HTLV-1, not only severe infections but also neoplastic transformation . It is tin vivo interaction between CIITA and Tax-2. (Figure in vivo between Tax-2 and NF-YB. In fact, and again for the first time, we demonstrated in this investigation that both transfected, thus overexpressed, and endogenous NF-YB could interact with the viral transactivator. Whether this is a direct interaction or requires a third partner is presently under scrutiny. Moreover the binding of endogenous NF-YB with Tax-2 molecules was increased by the presence of CIITA, strongly suggesting that at least a trimeric NF-YB-CIITA-Tax-2 complex is formed in vivo, in the nucleus and this could be a mechanism preventing Tax-2 from correctly transactivate HTLV-2 LTR and thus viral replication. It remains to be established whether the binding of the viral transactivator with CIITA and NF-YB, prevents Tax-2 from interacting with its own promoter or the Tax-2 -LTR promoter complex is formed but is functionally inhibited by a mechanism of steric hindrance.We demonstrated for the first time the existence of . Figure  and showAlthough the Tax-2-dependent HTLV-2 LTR transactivation takes place in the nucleus, it is still possible that additional mechanisms of CIITA-mediated inhibition of Tax-2 function operate outside the nucleus. For this reason, experiments of subcellular Tax-2 localization in the presence of CIITA were performed by immunofluorescence and confocal microscopy. In the absence of CIITA, Tax-2 localized both in the cytoplasm and the nucleus in the great majority of the cells. These results obtained in 293T cells are only partially similar to those obtained by other groups, using other cellular systems, as for example both in HeLa and in HEP-2 cells Tax-2 was mostly localized in the cytoplasm ,45. ThisThe direct interaction of CIITA with Tax-2, a crucial regulator of human oncogenic retrovirus replication, opens new ways for understanding the peculiar mechanisms by which CIITA has evolved its dual function to counteract pathogens' infections. From one side, CIITA triggers the molecular events leading to transcription of MHC class II genes, whose encoded molecules serve as antigen presenting receptors for peptides from all sort of pathogens, including viruses. In so doing CIITA governs the CD4+ T cell triggering leading to optimal activation of immune effector mechanisms, particularly specific antibody production by B cells. Antibody binding is a crucial event for neutralization of extracellular viruses which cannot infect host cells and are driven to degradation. From the other side, the newly acquired function of CIITA as a molecule that directly binds HTLV-2 Tax-2, and physically neutralizes its activating function on viral replication, represents a potent mechanism of intrinsic immunity. Together with previous results of our group demonstrating the inhibition of HIV-1 retrovirus replication by CIITA , the resThe authors declare that they have no competing interests.CO carried out the biochemical studies, immunoassays, participated in the discussion of results and drafted the manuscript. GF carried out immunofluorescence staining assay, participated in the discussion of results and drafted the manuscript. GT participated in the design and coordination of the study and in the discussion of the results. RSA conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


Deltaretrovirus genus. Four types have been identified thus far, with HTLV-1 and HTLV-2 much more prevalent than HTLV-3 or HTLV-4. HTLV-1 and HTLV-2 possess strictly related genomic structures, but differ significantly in pathogenicity, as HTLV-1 is the causative agent of adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis, whereas HTLV-2 is not associated with neoplasia. HTLVs code for a protein named Tax that is responsible for enhancing viral expression and drives cell transformation. Much effort has been invested to dissect the impact of Tax on signal transduction pathways and to identify functional differences between the HTLV Tax proteins that may explain the distinct oncogenic potential of HTLV-1 and HTLV-2. This review summarizes our current knowledge of Tax-1 and Tax-2 with emphasis on their structure, role in activation of the NF-κB (nuclear factor kappa-B) pathway, and interactions with host factors.Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the  The Human T cell leukemia viruses (HTLVs) are complex retroviruses, belonging to the primate T-lymphotropic virus (PTLV) family. HTLVs are classified as Deltaretroviruses, together with bovine leukemia virus (BLV) and simian T-lymphotropic viruses (STLVs). HTLV-1 was originally described in 1980  and was in vitro, suggests that Tax might be important for establishing the leukemic phenotype of ATL, but may become dispensable for its maintenance. In addition, recent studies suggest that other viral gene products may play relevant roles in HTLV-1-mediated transformation and may be responsible of the different HTLV-1 and HTLV-2 pathogenicity, including the antisense HTLV-1 genome transcript HBZ . HTLV-3 shares about 62% identity with HTLV-1 and HTLV-4 shares 62–71% nucleotide similarity with HTLV-1, HTLV-2, and HTLV-3  sequence of Tax-1 is composed of 353 residues, and is organized into functional domains that have been extensively investigated . Lysines K6 and K8 are critical for NF-κB activation and are highly conserved in all Tax proteins. Additional lysines, indicated by additional roman numbers, are present in Tax-2  whereas Tax-3 and Tax-4 contain 11 and 10 lysines, respectively, but not all are conserved at the same position. Alignment of the predicted Tax-4 sequence shows the absence of K10, which is a target of acetylation in Tax-1 and possibly in Tax-2  with lentiviruses encoding Tax-1 or Tax-2B they observed a higher immortalization activity of Tax-2B as compared to Tax-1.Tax-2 transforms rat fibroblasts less efficiently than Tax-1 . On the in vitro transforming activity , whereas the activation of the non-canonical pathway involves signaling molecules that are recognized only by a specific subset of tumor necrosis factor receptors (TNFRs), such as lymphotoxin-β, BAFF, RANKL, and TWEAK  in circulating leukemic cells from ATL patients. This phenomenon is likely due to epigenetic silencing of the plus-strand promoter, e.g., by methylation and/or expression of repressors of the Polycomb family . A pivotIt is likely that further studies aimed at dissecting the functional differences between the Tax proteins will reveal novel functions of host factors that are involved in the signal pathways altered in ATL and may become potential targets for effective therapies against leukemia.The studies of the differences between Tax-1, Tax-2, Tax-3, and Tax-4 interacting cell factors and transactivating activities will provide useful information to the understating of Tax-1 structural transformation that may open a new approach on HTLV studies based on Tax-1 peculiarities and interactions with addition viral products, that are not present in HTLV-2, HTLV-3, and HTLV-4 Tax proteins.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1 persists by driving clonal proliferation of infected T-lymphocytes. A high proviral load predisposes to the inflammatory and malignant diseases associated with HTLV-1. Yet the reasons for the remarkable variation within and between individuals in the abundance of HTLV-1-infected clones remain unknown. We demonstrate that negative selection dominates during chronic infection, favouring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We postulated that this selection occurred mainly during the primary infection. We are testing this hypothesis in an animal model by studying the BLV clonality during the primary infection in cows. By measuring the proviral load, the anti-BLV immune response and the BLV clonality we aim to quantify the impact of the immune response on the rate of infectious spread and on the selection of proviruses inserted in a particular genomic environment. Co-infection with Strongyloides stercoralis or Staphylococcus appears to be another risk factor for the development of HTLV-1 associated diseases. We observed that HTLV-1 clonality is altered by co-infection with these pathogens with an increase of both the number and the abundance of the infected T-cell clones. The genomic characteristics of the proviral integration sites in the most abundant clones differ significantly between co-infected individuals and those with HTLV-1 alone, implying the existence of different selection forces in co-infected patients. The rate of appearance of new clones in patients co-infected with Strongyloides stercoralis is higher than in patients with HTLV-1 alone. By comparing skin lesions and blood samples from patients with Infective Dermatitis associated with HTLV-1 (IDH), we observed a significant proportion of distinct infected clones between the two compartments. The skin lesions seem to be a site for HTLV-1 infectious spread.