The only known human tumor retrovirus, HTLV-1, achieves cellular transformation through mechanisms that do not involve delivery or insertional activation of a cellular oncogene. Oncogenesis is driven through the activities of a viral protein with oncogenic properties. In fact, the activities ascribed to the HTLV-1 Tax protein are varied and likely reflect the role of this protein in both the biology of the virus and in virus-host interaction (44
). In regards to a potential oncogenic function, Tax again displays a great variety of activities, although the exact mechanism of action is unknown. One consistent observation has been that expression of Tax correlates with genomic instability in both in vitro
and ex vivo
models. Furthermore, Tax-dependent genomic instability is observed in preneoplastic cell isolates and is a precondition to cellular transformation (9
). Thus, expression of Tax fulfills the first condition, loss of genomic integrity, of a DNA damage model for cancer (49
A variety of mechanisms have emerged to explain how oncogenes lead to cancer. In the DNA damage model, an oncogene acts through increased replication to induce DNA damage primarily as double-strand breaks caused by collapse of replicative forks (50
). This process would result in genomic instability but would also activate checkpoints via Chk2/Chk1 and subsequently p53-mediated apoptosis/senescence. Overcoming this transformation block is often accomplished through mutation in p53. We present a scenario by which the viral oncogene Tax sequesters DDR factors, thus competing with damage-induced DDR. This competition-based model provides for a mechanistic path to genomic instability through repression of DDR. Interestingly, because we had previously established that Chk2 is also one of the recruited DDR proteins, this model may explain how HTLV-1-infected cells eventually surmount the checkpoint block to cellular transformation.
Suppression of repair by Tax has been proposed as a mechanism for disrupting normal cellular genomic integrity. The consistent observation that Tax-expressing cells display increased mutation frequency in the absence of a direct DNA damage activity drives this repression-of-repair concept. Although a variety of reported Tax activities might indirectly impact the repair process (8
), there is support for more direct interplay. Overexpression of Tax can repress base excision and nucleotide excision repair (13
) potentially as a result of repression of repair genes (12
). Recently, Chandhasin et al.
) clearly demonstrated that repair of double-stranded breaks is impaired in Tax-expressing cells. These authors observed premature cessation of ATM activity and suboptimal recruitment of MDC1 to sites of DNA damage. Our observation that Tax binds to and competitively recruits MDC1 into TSS provides a model that explains these observations. In our model, Tax bridges the formation of a damage-independent DDR foci by recruitment of MDC1. The C-terminal half of Tax binds to MDC1, and the N-terminal half serves as a subcellular localization signal to an as yet undefined chromatin region. Because it has already been established that tethering of MDC1 alone to chromatin can nucleate a DDR and foci formation (38
), it is logical that targeting of MDC1 to chromatin by Tax is sufficient for TSS foci formation and generation of a DDR.
Our characterization of the TSS foci revealed that several classic DDR proteins were present. Specifically, we found, in addition to MDC1, the Tax-dependent recruitment of DNA-PK and BRCA1 into these foci. In addition, we have previously reported on the presence of 53BP1 and Chk2 within these same structures (15
). Interestingly, Nbs1 was not recruited to the Tax foci. Nbs1 is a component of the MRN complex that is an early damage recognition factor. The association of MRN with damaged DNA ends has been linked to constitutive phosphorylation of MDC1 (26
). However, although it is clear from earlier work (38
) that stable association with chromatin can initiate DDR in the absence of damage, it is not clear whether MDC1 alone can recruit MRN. For that matter, these damage-independent DDR have yet to be characterized. Thus, the failure to observe recruitment of Nbs1 into TSS foci is reflective of the fact that these foci are not damage-initiated. In fact, our results suggest that the amplification and persistence of MRN are dependent upon the presence of DNA lesions in addition to MDC1.
We show that both IRIF and TSS exist in separable regions and that these sites compete for DDR proteins such as MDC1. When IR is in excess, MDC1 is recruited away from TSS and to IRIF. However, when recruitment to TSS becomes dominant, the result may be premature dissociation of MDC1 from IRIF. In fact, we show that Tax expression and TSS formation result in a dramatic reduction in IR-induced Nbs1-containing foci. Because the quantitation of IRIF via measurable Nbs1 foci has been shown to characterize the cellular DDR capacity, our results demonstrate that Tax impairs the repair response through a competitive mechanism. Competition with IRIF and control of DDR activities would be expected to: 1) increase fixed damage by bypass of repair, 2) suppress recognition of catastrophic damage and postpone apoptosis, 3) alter the availability of naïve (responsive) DDR factors, thus impairing the ability of the cell to judge extent of damage, and 4) deregulate the DDR activities to benefit the virus via checkpoint control. The formation of TSS is typical of Tax-expressing cells and has been observed in HTLV-1-tranformed cell lines (34
). Therefore, we would expect that competition for recruitment of MDC1 occurs in the context of viral infection. However, the functional impact of DDR sequestration at early infection stages needs to be determined.
We present a novel competition model for a viral oncoprotein to supplant cellular control over DDR to ensure survival of the viral host. This competition-based model allows for a mechanism by which Tax overcomes two barriers to oncogenesis. Sequestration of DDR factors results in repression of repair, resulting in increased mutation frequency and genomic instability. This loss in genomic integrity signals the bypass of the first barrier to cancer development. Typically under these circumstances checkpoint activation would engage p53-mediated apoptosis and cell death and presentation of the second barrier. However, competitive sequestration of DDR factors, which include Chk2, would be expected to suppress checkpoint activation and delay apoptosis. The benefits to the virus are selective survival of the host cell, whereas the trade-off is promotion of oncogenesis. Clearly, other virus-host events contribute to long term infection prior to cellular transformation.