Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma. The HTLV-1 Tax constitutively activates nuclear factor-κB (NF-κB) to promote the survival and transformation of HTLV-1-infected T cells. Despite extensive study of Tax, how Tax interacts with host factors to regulate NF-κB activation and HTLV-1-driven cell proliferation is not entirely clear. Here, we showed that overexpression of Poly (rC)-binding protein 1 (PCBP1) promoted Tax-mediated IκB kinase (IKK)-NF-κB signaling activation, whereas knockdown of PCBP1 attenuated Tax-dependent IKK-NF-κB activation. However, Tax activation of HTLV-1 long terminal repeat was unaffected by PCBP1. Furthermore, depletion of PCBP1 led to apoptosis and reduced proliferation of HTLV-1-transformed cells. Mechanistically, PCBP1 interacted and co-localized with Tax in the cytoplasm, and PCBP1 KH3 domain was indispensable for the interaction between PCBP1 and Tax. Moreover, PCBP1 facilitated the assembly of Tax/IKK complex. Collectively, our results demonstrated that PCBP1 may exert an essential effect in Tax/IKK complex combination and subsequent NF-κB activation, which provides a novel insight into the pathogenetic mechanisms of HTLV-1.

HTLV-1 is an oncovirus causing ATL and other inflammatory diseases such as HAM/TSP and HU in about 5% of infected individuals. It is also known that HTLV-1-infected cells maintain a disease-free, immortalized, latent state throughout the lifetimes of about 95% of infected individuals. We believe that the stable maintenance of disease-free infected cells in the carrier is an intrinsic characteristic of HTLV-1 that has been acquired during its evolution in the human life cycle. We speculate that the pathogenesis of the virus is ruled by the orchestrated functions of viral proteins. In particular, the regulation of Rex, the conductor of viral replication rate, is expected to be closely related to the viral program in the early active viral replication followed by the stable latency in HTLV-1 infected T cells. HTLV-1 and HIV-1 belong to the family Retroviridae and share the same tropism, e.g., human CD4+ T cells. These viruses show significant similarities in the viral genomic structure and the molecular mechanism of the replication cycle. However, HTLV-1 and HIV-1 infected T cells show different phenotypes, especially in the level of virion production. We speculate that how the activity of HTLV-1 Rex and its counterpart HIV-1 Rev are regulated may be closely related to the properties of respective infected T cells. In this review, we compare various pathological aspects of HTLV-1 and HIV-1. In particular, we investigated the presence or absence of a virally encoded \"regulatory valve\" for HTLV-1 Rex or HIV-1 Rev to explore its importance in the regulation of viral particle production in infected T cells. Finally, wereaffirm Rex as the key conductor for viral replication and viral pathogenesis based on our recent study on the novel functional aspects of Rex. Since the activity of Rex is closely related to the viral replication rate, we hypothesize that the \"regulatory valve\" on the Rex activity may have been selectively evolved to achieve the \"scenario\" with early viral particle production and the subsequent long, stable deep latency in HTLV-1 infected cells.

Adult T cell leukemia/lymphoma (ATLL) is a CD4-positive peripheral T cell lymphoma caused by human T cell lymphotropic virus type 1 (HTLV-1). Although ATLL is quite difficult to be cured, up-regulation of cellular immunity such as HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) has been proved to be important to obtain long-term survival. At present, no efficacious method to activate ATLL-specific cellular immunity is available. This study aimed to investigate whether live attenuated varicella-zoster virus (VZV) vaccination to ATLL can activate HTLV-1 Tax-specific cellular immune response. A total of 3 indolent- and 3 aggressive-type ATLL patients were enrolled. All aggressive-type patients had the VZV vaccination after completing anti-ATLL treatment including mogamulizumab, which is a monoclonal antibody for C-C chemokine receptor 4 antigen, plus combination chemotherapy, whereas all indolent-type patients had the VZV vaccination without any antitumor treatment. Cellular immune responses including Tax-specific CTLs were analyzed at several time points of pre- and post-VZV vaccination. After the VZV vaccination, a moderate increase in 1 of 3 indolent-type patients and obvious increase in all 3 aggressive-type patients in Tax-specific CTLs percentage were observed. The increase in the cell-mediated immunity against VZV was observed in all indolent- and aggressive-type patients after VZV vaccination. To conclude, VZV vaccination to aggressive-type ATLL patients after mogamulizumab plus chemotherapy led to the up-regulation of HTLV-1 Tax-specific CTLs without any adverse event. Suppression of regulatory T lymphocytes by mogamulizumab may have contributed to increase tumor immunity in aggressive-type ATLL patients. Japan Registry of Clinical Trials number, jRCTs051180107.

HTLV-1 is a human retrovirus responsible for adult T-cell leukemia (ATL) and certain other clinical disorders. The viral Tax oncoprotein plays a central role in HTLV-1 pathogenicity, mainly due to its capacity of inducing the transcriptional activity of various transcriptional factors like NFқB. Eucalyptus camaldulensis (Ec) is considered as a traditional medicinal plant with valuable therapeutic effects. Here we evaluated the activity of its ethanolic leave extract on different Tax activities by testing its influence on Tax-induced activity of NFқB and HTLV-1 LTR in Jurkat cells. Our results showed that Ec inhibited Tax induced activation of NFқB -, SRF- dependent promoters and HTLV-1 LTR. Ec extract has no effect on the binding of Tax to NFқB while it strongly prevented the degradation of IҝBα induced by Tax probably as a result of preventing the link between Tax and IKKγ. In addition, increasing the cellular level of P-TEFb-cyclinT1 significantly reduced the inhibitory effect of Ec on Tax activities, probably by preventing the interaction between Tax and P-TEFb-cyclin T1. The 40%-MeOH fraction of this extract, which is rich with polyphenols, offered the highest inhibitory effect against Tax activities. Further studies are required for the isolation and identification of active component/s in this extract which may be developed in the future as preventive/curing drugs for HTLV-1 related diseases.

BACKGROUND: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences.
RESULTS: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis.
CONCLUSIONS: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL.IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection.

Activation of the nuclear factor kappa B (NF-ϰB) transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. While the main function of NF-ϰB is to promote the host\'s immune response, the NF-ϰB pathway is frequently dysregulated by invading viral pathogens. Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL) and an inflammatory disease named tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). HTLV-1 encodes an oncoprotein, Tax, which plays a significant role in the initiation of cellular transformation and the elicitation of the host\'s inflammatory responses. Here, we review current thinking on how Tax may affect both diseases through activation of NF-ϰB signaling.

Persistent activation of NF-κB is a prerequisite for development of adult T cell leukemia-lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes a viral transforming protein named Tax, which constitutively activates the canonical IκB kinases (IKK), the central regulator of NF-κB signaling. However, the role of the non-canonical IκB kinases, TBK1 and IKKε, in the pathogenesis of HTLV-1-associated leukemia has not been evaluated. We here show that TBK1/IKKε are crucial pro-survival molecules by maintaining persistent activity of Stat3. Consistent with this finding, silencing Stat3 by the specific shRNA or by the chemical inhibitor ruxolitinib results in drastic impediment of leukemia cell growth. We further find that in HTLV-1-transformed T cells expressing Tax, TBK1 co-localizes with the canonical IκB kinases and Tax in the lipid raft microdomains. The wild type Tax, but not the Tax mutant defective in activating the canonical IKK, promotes the lipid raft translocation of TBK1. This phenomenon correlates with Tax activation of both NF-κB and Stat3. Tax does not interact directly with TBK1/IKKε, and it rather engages a molecular crosstalk between the canonical IKKs and TBK1/IKKε. Our data, therefore, demonstrate a key role of TBK1/IKKε in the survival and proliferation of HTLV-1-transformed T cells and implicate a potential therapy targeting TBK1/IKKε and Stat3 in controlling HTLV-1-mediated oncogenesis.

The retroviral oncoprotein Tax from human T cell leukemia virus type 1 (HTLV-1) induces persistent activation of IκB kinase (IKK)/NF-κB signaling, an essential step for initiating HTLV-1 oncogenesis. The regulation of the IKK/NF-κB signaling in HTLV-1-transformed T cells remains incompletely understood. In the present study, we showed that the autophagy molecule Beclin1 not only executed a cytoprotective function through induction of autophagy but also played a pivotal role in maintaining Tax-induced activation of two key survival factors, NF-κB and Stat3. Silencing Beclin1 in HTLV-1-transformed T cells resulted in diminished activities of NF-κB and Stat3 as well as impaired growth. In Beclin1-depleted cells, Tax failed to activate NF-κB and Stat3 at its full capacity. In addition, we showed that Beclin1 interacted with the catalytic subunits of IKK. Further, we observed that selective inhibition of IKK repressed the activities of both NF-κB and Stat3 in the context of HTLV-1-transformation of T cells. Our data, therefore, unveiled a key role of Beclin1 in maintaining persistent activities of both NF-κB and Stat3 in the pathogenesis of HTLV-1-mediated oncogenesis.

The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL.