HTLV-1 Tax and the DNA damage response: inhibition of multiple pathways

As discussed in a previous post, Human T-Cell Leukaemia/Lymphoma Virus type 1 (HTLV-1) is the causative agent of Adult T-cell leukaemia/lymphoma (ATL/L or ATL) as well as HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), the latter being a neuroinflammatory disease.

The transformation of cells infected with HTLV-1 is mediated primarily via expression of the viral transactivator protein (Tax) encoded within the pX region of the viral genome which induces not only oncogene induced senescence (OIS) in both infected cells and cells expressing Tax in the absence of other viral proteins as a result of increased DNA replication but also genomic instability, and thus potentially apoptosis.  As discussed before, the induction of autophagy by Tax via increased expression of Bcl-3, recruitment of Beclin-1 and Bif-1 to lipid rafts in a IKKα/β/γ dependent manner, and upregulation of AMPK not only inhibits the intrinsic but also the extrinsic, TRAIL induced, apoptotic pathway, thus potentially inhibiting apoptosis induced as a consequence of increased DNA replication.

The expression of Tax in HeLa cells induces OIS which is accompanied by an arrest in G1 phase of the cell cycle via hyperactivated NF-κB, mediated in part by two Cyclin Dependent Kinase (CDK) inhibitors, p21CIP1/WAF1 (p21) and p27Kip1 (p27), that can be inhibited by the expression of the viral HBZ protein as well as the expression of ΔN-IκBα, both of which inhibit NF-κB. Since HBZ also inhibits p53 via inhibiting the acetylation of p53 by binding p300/CBP as well as P/CAF these data suggest that the induction of OIS is dependent on hyperactivated NF-κB and maybe on p53 inhibition. In both HeLa and Jurkat cells p53 is either mutated or inactivated, but data obtained from HeLa, H1299, and Saos-2 cells indicate that Tax expression inhibits p53 which (in my opinion) might lead to increased expression of an antiapoptotic protein, Mcl-1, that localises to the mitochondria and is stabilised in Tax expressing Jurkat cells by TRAF-6 via IKKα/β/γ activation whilst decreasing the expression of Bim and Bid.

The induction of OIS can be seen as a cellular response to prevent the progression of tumours since senescent cells exhibit a cell cycle arrest as a result of the induction of the DNA damage response by increased DNA replication.

Upon the induction of DNA damage, a signalling pathway involving the activation of specific kinases -ATM, ATR, or DNA-PK - and the phosphorylation of downstream effectors, in particular γH2AX induces the recruitment of DNA repair proteins to sites of DNA damage, leading to the ordered assembly of DNA repair foci as well as the phosphorylation of p53, allowing not only the repair of damaged DNA but also p53 dependent regulation of gene expression. DNA damage repair (DDR) pathways can be initiated during all phases of the cell cycle, with the Non-Homologous End Joining (NHEJ) pathway present in all phases of the cell cycle and Homologue Repair HR) pathway limited to S and G2 phase of the cell cycle. In general, the DDR is activated during DNA replication in Sp hase due tot he induction of transient DNA damage and this activation does not induce prolonged S phase arrest or senescent. In contrast, prolonged induction of the DDR during S phase induces an arrest in G2 phase of the cell cycle via Checkpoint Kinase-2 (CHK-2) dependent signalling, characterised by the accumulation of γH2AX positive foci that also contain other components of the DDR such as MRN and NBS1.

Following UV irradiation of clonal rat embryo fibroblasts (CREFs), Tax expressing CREFs only exhibit an initial arrest in G1 phase followed by an acceleration of entry into S phase when compared with control cells expressing the backbone vector. Furthermore, CREF-tax cells also maintain a higher abundance of UV induced thymidine dimers and fail to induce the formation of γH2AX and phosphorylated Replication Protein (RPA) positive foci, a hallmark of DDR induction, indicating that the expression of Tax attenuates the DDR. 

Immunofluorescence analysis of gamma-irradiated cells expressing Tax further indicated that Tax indeed sequesters and/or inhibits various components of the DDR, including MDC-1, CHK-1/-2, as well as p53, leading to an inhibition of the DDR as well as preventing cell cycle arrest and OIS. In addition to preventing the initiation of the DDR, Tax also induces the activation of Wild-type p53-induced phosphatase 1 (Wip-1) thereby attenuating γH2AX dependent assembly of DNA damage repair foci by (premature) dephosphorylation of γH2AX and ATM. It should be noted that in gamma-irradiated CREF tax does not inhibit the initial formation of ATM and NBS1 positive foci, suggesting that the initial phosphorylation of H2AX at Ser-139 should not be inhibited. Tax expression however prevents the proper accumulation of ATM and NBS1 by sequestering the ATM/NBS1/ complex or alternatively preventing the recruitment of MDC1, which is a prerequisite for the formation of stable DNA repair foci.

Tax however does not only inhibit the ATM dependent pathway but also decreases the expression of Ku80 and thus inhibit the recruitment of DNA-PK to sites of DNA damage, leading to an increased formation of micronuclei, although Ku70 is not affected. Interestingly the depletion of ku80 by shKu80 in HEK-293T cells and six human carcinoma cell lines (LNcaP, K562, MDA-MB-231, MCF-7, EC9706, and K150) has been shown to inhibit cell proliferation and sensitise cells to Mitomycin-C and γ-irradiation induced apoptosis.

Paradoxically, the expression of Tax has been proposed to increase NHEJ mediated DNA repair whilst inhibiting the HR pathway in  a Jurkat cell line stably expressing Tax.

In addition to promoting the induction of DNA breaks by increased DNA replication, Tax also increases the accumulation of Reactive Oxygen Species (ROS) by interacting with and inhibition of ubiquitin-specific protease 10 (USP10), a component of stress granules. As mentioned before, the expression of Tax decreases the formation of stress granules in HTLV-1 infected and cells transfected with Tax. Indeed in Jurkat cells, the expression of Tax inhibits the formation of stress granules in response to Arsenite whilst inducing ROS and in human BJ fibroblasts Tax not only increases ROS levels but also DNA damage (as measured by Comet assay) and -interestingly- senescence. Tax might increase the sensitivity to ROS induced DNA damage by inhibiting Ku80 since Ku80 deficient cells are not only showing a decrease in the NHEJ DNA damage repair pathway but also in DNA damage repair mediated by the Base Excision Repair (BER) pathway which can be compensated for by overexpression of PARP-1. Indeed, Tax has been shown to inhibit BER mediated DNA repair which has originally being linked to the increase in the expression of PCNA. Also, since ROS can induce autophagy and autophagy senescence it remains to be seen if these two processes are connected or not.

HTLV-1 Tax and the DDR: multiple points of interaction and the connection to
the induction of autophagosome formation

In this context it is interesting that the treatment of Tax positive MT-2 and Hut-102 with Everolimus, an inducer of autophagy, not only decreases Tax levels but also increases senescence, indicating that the Tax mediated attenuation of the DNA damage response in conjunction with a decrease in autophagic flux prevents senescence; indeed, Everolimus has been shown not only to induce autophagy but also induce G1 arrest in Mantle Cell Lymphoma cells.  

In addition to the induction of autophagosome formation, attenuation of the DDR, and NF-κB hyperactivation, X-box binding protein 1 (XBP1) has been shown to be a binding partner of Tax and together with Tax a transactivator of the viral LTR. As discussed earlier, XBP1 is a component of the ER stress response and the accumulation of the spliced form of XBP-1, sXBP1, is a hallmark of the induction of the ER stress response following the accumulation of misfolded proteins within the ER or the induction of the ER stress response following lipid depletion. In the case of HeLa cells transiently transfected with Tax, the author of those lines showed that Tax expression not only leads to an accumulation of Tax in nuclear bodies but also in the perinuclear region. In transiently transfected HeLa cells nuclear Tax is absent following treatment with Etoposide concomitant with an increase in Tax localised in the perinuclear region. Since Tax expression prevents Etoposide induced apoptosis, it is likely that Tax prevents apoptosis by inhibiting the activation of caspases via increased expression of cFLIP. If the binding of Tax to XBP1 inhibits ER stress induced apoptosis however remains to be seen.

In conclusion, the expression of Tax not only is necessary for the transformation of primary cells by inducing cell proliferation but also by inhibiting apoptosis and senescence via inhibiting the DDR as well (potentially) autophagy and the ER stress response.

Further reading

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Saggioro D, Majone F, Forino M, Turchetto L, Leszl A, & Chieco-Bianchi L (1994). Tax protein of human T-lymphotropic virus type I triggers DNA damage. Leukemia & lymphoma, 12 (3-4), 281-6 PMID: 8167559 

Torino, M., Leszl, A., Chieco-Bianchi, L., Saggioro, D., Majone, F., & Turchetto, L. (1994). Tax Protein of Human T-Lymphotropic Virus Type I Triggers DNA Damage Leukemia and Lymphoma, 12 (3), 281-286 DOI: 10.3109/10428199409059600 

Zhi H, Yang L, Kuo YL, Ho YK, Shih HM, & Giam CZ (2011). NF-κB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ. PLoS pathogens, 7 (4) PMID: 21552325 

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