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.

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.

The HTLV-1 oncoprotein Tax is a potent activator of classical and alternative NF-κB pathways and is thought to promote cell proliferation and transformation via NF-κB activation. We showed recently that hyperactivation of NF-κB by Tax triggers a cellular senescence response (H. Zhi et al., PLoS Pathog. 7:e1002025, 2011). Inhibition of NF-κB activation by expression of I-κBα superrepressor or by small hairpin RNA (shRNA)-mediated knockdown of p65/RelA rescues cells from Tax-induced rapid senescence (Tax-IRS). Here we demonstrate that Tax-IRS is driven by the transcriptional activity of NF-κB. Knockdown of IKKγ, the primary Tax target, by shRNAs abrogated Tax-mediated activation of both classical and alternative NF-κB pathways and rendered knockdown cells resistant to Tax-IRS. Consistent with a critical role of IKKα in the transcriptional activity of NF-κB, IKKα deficiency drastically decreased NF-κB trans -activation by Tax, although it only modestly reduced Tax-mediated I-κBα degradation and NF-κB nuclear localization. In contrast, although IKKβ knockdown attenuated Tax-induced NF-κB transcriptional activation, the residual NF-κB activation in IKKβ-deficient cells was sufficient to trigger Tax-IRS. Importantly, the phenotypes of NIK and TAK1 knockdown were similar to those of IKKα and IKKβ knockdown, respectively. Finally, double knockdown of RelB and p100 had a minor effect on senescence induction by Tax. These data suggest that Tax, through its interaction with IKKγ, helps recruit NIK and TAK1 for IKKα and IKKβ activation, respectively. In the presence of Tax, the delineation between the classical and alternative NF-κB pathways becomes obscured. The senescence checkpoint triggered by Tax is driven by the transcriptional activity of NF-κB, which depends on activated IKKα and p65/RelA.

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The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) -a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling -to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.