HTLV-1 (Human T-cell lymphotropic virus type 1) is a complex human delta retrovirus that currently infects 10–20 million people worldwide. While HTLV-1 infection is generally asymptomatic, 3%–5% of infected individuals develop a highly malignant and intractable T-cell neoplasm known as adult T-cell leukemia/lymphoma (ATL) decades after infection. How HTLV-1 infection progresses to ATL is not well understood. Two viral regulatory proteins, Tax and HTLV-1 basic zipper protein (HBZ), encoded by the sense and antisense viral transcripts, respectively, are thought to play indispensable roles in the oncogenic process of ATL. This review focuses on the roles of Tax and HBZ in viral replication, persistence, and oncogenesis. Special emphasis is directed towards recent literature on the mechanisms of action of these two proteins and the roles of Tax and HBZ in influencing the outcomes of HTLV-1 infection including senescence induction, viral latency and persistence, genome instability, cell proliferation, and ATL development. Attempts are made to integrate results from cell-based studies of HTLV-1 infection and studies of HTLV-1 proviral integration site preference, clonality, and clonal expansion based on high throughput DNA sequencing. Recent data showing that Tax hijacks key mediators of DNA double-strand break repair signaling—the ubiquitin E3 ligase, ring finger protein 8 (RNF8) and the ubiquitin E2 conjugating enzyme (UBC13)—to activate the canonical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and other signaling pathways will be discussed. A perspective on how the Tax-RNF8 signaling axis might impact genomic instability and how Tax may collaborate with HBZ to drive oncogenesis is provided.

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21^CIP1/WAF1 and p27^KIP1, which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21^CIP1/WAF1-/p27^KIP1-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27^KIP1 protein and p21^CIP1/WAF1 mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis. Transcription factors of the NF-κB/Rel family are critical for the proliferation of lymphocytes and the expression of genes that mediate inflammatory and immune responses. They are often aberrantly activated in human cancers, especially leukemia, where they confer survival and proliferation advantages. Through the study of the trans-activator/oncoprotein, Tax, of the human T-lymphotropic virus type 1 (HTLV-1), we have found that persistent and potentially oncogenic activation of NF-κB triggers a defense mechanism that commits cells into senescence, an irreversible state of cell cycle arrest. This checkpoint is turned on by hyper-activated p65/RelA and is mediated by two cyclin-dependent kinase inhibitors, p21 and p27, in a p53- and pRb-independent manner. It is often impaired in cancer cells with constitutively active NF-κB. Our results anticipate that the anti-sense protein of HTLV-1, HBZ, which down-regulates NF-κB and HTLV-1 trans-activation by Tax, would mitigate or prevent Tax-induced senescence. This prediction has been borne out experimentally. Thus, Tax promotes robust HTLV-1 replication, potent NF-κB activation and senescence, while HBZ attenuates Tax-driven viral replication and NF-κB activation to allow proliferation of infected cells and persistent infection. Finally, our data support the notion that inactivation of the senescence checkpoint facilitates chronic NF-κB hyper-activation, a critical step in leukemia development.

Human T-lymphotropic virus type I (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 viral transactivator/oncoprotein, Tax, activates viral transcription and usurps regulatory mechanisms that are critical for cell growth and division to facilitate viral replication. The effects that Tax exerts on cells include potent NF-k B activation, cell cycle perturbation and cell transformation. How Tax influences ATL development is incompletely understood at present. While Tax-expression is needed at the early stages of cellular transformation, at later times most ATL cells do not express tax; therefore, genetic and epigenetic changes in HTLV-1-infected cells are believed to play an important role in the etiology of ATL. This review attempts to integrate recent literature on the biological activities of Tax and the properties of HTLV-1 transformed T-cells and ATL cells, and speculate on what cellular changes may collaborate with Tax to effect cell transformation and ATL development.

The human T‐cell leukemia virus type 1 (HTLV‐1) is a complex deltaretrovirus linked to adult T‐cell leukemia/lymphoma (ATLL), a fatal CD4 + malignancy in 3–5% of infected individuals. The HTLV‐1 Tax regulatory protein plays indispensable roles in regulating viral gene expression and activating cellular signaling pathways that drive the proliferation and clonal expansion of T cells bearing HTLV‐1 proviral integrations. Tax is a potent activator of NF‐κB, a key signaling pathway that is essential for the survival and proliferation of HTLV‐1‐infected T cells. However, constitutive NF‐κB activation by Tax also triggers a senescence response, suggesting the possibility that only T cells capable of overcoming NF‐κB‐induced senescence can selectively undergo clonal expansion after HTLV‐1 infection. Tax expression is often silenced in the majority of ATLL due to genetic alterations in the tax gene or DNA hypermethylation of the 5′‐LTR. Despite the loss of Tax, NF‐κB activation remains persistently activated in ATLL due to somatic mutations in genes in the T/B‐cell receptor (T/BCR) and NF‐κB signaling pathways. In this review, we focus on the key events driving Tax‐dependent and ‐independent mechanisms of NF‐κB activation during the multistep process leading to ATLL.

Human T lymphotropic virus type I (HTLV-I) infection is largely latent in infected persons. How HTLV-1 establishes latency and reactivates is unclear. Here we show that most HTLV-1-infected HeLa cells become senescent. By contrast, when NF-κB activity is blocked, senescence is averted, and infected cells continue to divide and chronically produce viral proteins. A small population of infected NF-κB-normal HeLa cells expresses low but detectable levels of Tax and Rex, albeit not Gag or Env. In these “latently” infected cells, HTLV-1 LTR trans-activation by Tax persists, but NF-κB trans-activation is attenuated due to inhibition by HBZ, the HTLV-1 antisense protein. Furthermore, Gag-Pol mRNA localizes primarily in the nuclei of these cells. Importantly, HBZ was found to inhibit Rex-mediated export of intron-containing mRNAs. Over-expression of Rex or shRNA-mediated silencing of HBZ led to viral reactivation. Importantly, strong NF-κB inhibition also reactivates HTLV-1. Hence, during HTLV-1 infection, when Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-κB hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-κB activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins. Most HTLV-1-infected individuals are asymptomatic. It is thought that the proviral DNA is transcriptionally inert and HTLV-1 replicates through mitotic expansion of host cells. The evolving provirus integration patterns in HTLV-1 carriers, however, suggest new infection occurs continuously. Whether or how HTLV-1 establishes latency and reactivates is unclear. We show that HTLV-1 infection in culture can lead to two alternative outcomes — productive infection accompanied by senescence or latent infection followed by clonal expansion — based on the relative expression of regulatory proteins: Tax, Rex, and HBZ. HTLV-1 latency is established by HBZ, and reactivation is achieved by Rex through regulating nuclear export of viral mRNAs. Elucidating mechanisms underlying HTLV-1 latency and reactivation can facilitate virus control to prevent progression to disease.

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells. Activation of the NF-κB family of transcription factors by the HTLV-1 oncoprotein, Tax, is causally linked to adult T cell leukemia (ATL) development in HTLV-1-infected individuals, but the underlying mechanisms are not fully understood. NF-κB activation requires the phosphorylation of its inhibitor, IκBα, by IκB kinase (IKK), which marks IκBα for degradation. In this study, we demonstrate that Tax inappropriately activates a ubiquitin E3 ligase, RNF8, and ubiquitin E2 conjugating enzymes, Ubc13:Uev1A/Uev2, to assemble long lysine 63-linked polyubiquitin (K63-pUb) chains, which function as signaling platforms for polyubiquitin-binding TGFβ-activated kinase 1 (TAK1) and IKK to congregate and become activated. Because TAK1 mediates the activation of multiple downstream signaling pathways, the mechanism described here can explain the complex effect of Tax on cell signaling. The major functions of RNF8 are to signal cellular DNA damage repair (DDR) and cell division by assembling K63-pUb chains at the site of DNA damage and cell cleavage. As such, the inappropriate activation of RNF8 and the over-abundance of K63-pUb chains in Tax-expressing cells may explain how Tax causes DNA damage and cell division defect.