HTLV-1 Tax: Senescence, Autophagy 1  

and the DNA damage response 2  

Torsten Wurm PhD 3  

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Summary 19  

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of ATL 20  

and the HTLV-1 encoded Tax protein has been shown to induce oncogene 21  

induced senescence (OIS), apoptosis, and to be sufficient to transform primary 22  

cells. Stabilization of Mcl-1 by HTLV-1 Tax might not only prevent OIS 23  

associated decrease in cell proliferation, but also apoptosis and the induction of 24  

mitophagy in accordance with observations that the expression of Tax inhibits the 25  

fusion of autophagosomes with the lysosome despite inducing the formation of 26  

autophagosomes via hyperactivation of NF-κB as well as the recruitment of 27  

Beclin-1 to lipid rafts. Inducing the expression and stabilization of cFLIP by Tax 28  

in a Ku70 dependent and independent manner not only inhibits apoptosis but 29  

also necrosis in addition to potentially inhibiting the formation of the necrosome. 30  

Inhibiting autophagic flux might also prevent the secretion of inflammatory 31  

cytokines, which is not only autophagy dependent but might also be dependent 32  

on the activation of caspase-3 and contributing to the proliferation of HTLV-1 33  

infected and Tax expressing cells by preventing senescence. Deacetylated 34  

Ku70 has also been demonstrated to localise to the nucleus where it binds sites 35  

of DNA damage by forming a complex with Ku80, increasing the amount of 36  

deacetylated Ku70 in the cytoplasm due to preventing p300/CBP dependent 37  

acetylation of Ku70 might the NHEJ pathway which is also induced by inhibiting 38  

autophagy. 39  

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Keywords 41  

HTLV-1 Tax, Senescence, Autophagy, DNA Damage response 42  

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Introduction 63  

Human T-cell lymphotropic virus type 1 (HTLV-1) was first discovered in the early 64  

1980s independently by two groups in the US and in Japan (Poiesz et al., 1981; 65  

Yoshida et al., 1982). HTLV-1 is a complex deltaretrovirus, with four known 66  

types, HTLV-1 to 4, with HTLV-1 being the most pathogenic of the group and the 67  

first human retrovirus being associated with cancer, Adult T-Cell 68  

leukaemia/lymphoma (ATL/L or ATL) (Mahieux & Gessain, 2007), although ATL 69  

itself was first described in 1977 by Takasuki (Uchiyama et al., 1977). In general, 70  

HTLV-1 infects about 5-20 million people worldwide, predominantly in Japan, 71  

Africa, Latin America and the Caribbean, out of which about 3-5% of all infected 72  

people develop ATL in their lifetime, often decades following the initial infection 73  

(Gessain & Cassar, 2012; Ishitsuka & Tamura, 2014). 74  

During viral replication, Tax activates the transcription of viral proteins via 75  

recruiting the cellular transcription factors CREB and ATF to the LTR promoter 76  

(Semmes et al., 1996) and mediates the initial steps of cell transformation 77  

(Tanaka et al., 1990) in transgenic mice (Hasegawa et al., 2006; Ruddle et al., 78  

1993) and CD4+ T lymphocytes as well as human fibroblasts (Endo et al., 2002; 79  

Sieburg et al., 2004) in the absence of other viral proteins. 80  

In contrast to Tax-2 (derived from HTLV-2), Tax-1 localises predominantly to the 81  

nucleus (Avesani et al., 2010; Turci et al., 2006), and both Tax-1 and Tax-2 are 82  

acetylated, ubiqutinated, and SUMOylated (Zane & Jeang, 2012). In addition to 83  

CREB and ATF, Tax-1 interacts with a number of host factors including 84  

 

 

components of the PI3K, Akt, and NF-κB pathways (reviewed in (Romanelli et al., 85  

2013) (Fig.1), contributing to the oncogenic properties of Tax-1. 86  

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Cellular senescence is a stable cell cycle arrest characterised by an active 88  

metabolism caused by a variety of stressors, including activation of the DNA 89  

damage response, epigenetic changes, and senescence-associated secretion 90  

phenotype (Young et al., 2009). Senescent cells are however not necessarily 91  

arrested irreversible since in the case of replicative senescence cells can be 92  

proliferative in the presence of active telomerase and senescent tumour cells can 93  

proliferate following treatment with chemotherapeutic drugs or following radiation 94  

(reviewed in (Evan & d'Adda di Fagagna, 2009)). Oncogene-induced senescence 95  

(OIS) is induced in cells undergoing DNA hyper-replication and associated with 96  

the induction of the DNA damage response (DDR) as a result of increased DNA 97  

replication (Di Micco et al., 2008; Di Micco et al., 2006) (reviewed in (Sulli et al., 98  

2012)) as indicated by the presence of damaged DNA in cells undergoing OIS 99  

(Di Micco et al., 2006). 100  

The expression of Tax in HeLa cells induces OIS which is accompanied by an 101  

arrest in G1 phase of the cell cycle via hyperactivated NF-κB, mediated in part by 102  

two Cyclin Dependent Kinase (CDK) inhibitors, p21CIP1/WAF1 (p21) and p27Kip1 103  

(p27) thus modulating the activity of both Cdk4 and Cdk6 (Schmitt et al., 1998; 104  

Zhang et al., 2009), stimulating the G1 to S transition as well as inducing 105  

senescence in HTLV-1 infected HeLa and SupT1 cells (Yang et al., 2011). Both 106  

 

 

the induction of p21 and p27 expression as well as senescence can be inhibited 107  

by the expression of the viral HBZ protein as well as the expression of ΔN-IκBα, 108  

both of which inhibit NF-κB (Zhi et al., 2011). Further to the stimulating the G1 to 109  

S transition, Tax increases the expression of PCNA, thus increasing DNA 110  

replication (Lemoine et al., 2000; Ressler et al., 1997). Furthermore, ATL cells 111  

exhibit increased levels of H-RasV12 (Tanaka et al., 1999), suggesting that OIS 112  

in HTLV-1 infected cells is mediated in part by increased levels of H-RasV12 as a 113  

result of increased DNA damage due to DNA hyper-replication as well as the 114  

induction of ROS and RNS. Indeed the expression of Tax in normal human BJ 115  

fibroblasts, Jurkat cells, and primary human CD4+ T lymphocytes induces not 116  

ROS but also senescence that can be relieved by treating cells with a ROS 117  

scavenger (Kinjo et al., 2010). 118  

Paradoxically, the expression of Tax can prevent senescence and promote cell 119  

proliferation, suggesting that the expression of Tax can prevent senescence at 120  

least in subset of cells. One mechanism of antagonizing OIS is to inhibit 121  

autophagy (Gewirtz, 2013; Young et al., 2009). Indeed, the expression of Tax in 122  

various cell lines not only interferes with the DDR, but also with autophagy, thus 123  

counteracting senescence and promoting cell proliferation. 124  

In this review the interference of Tax with components of the autophagy pathway 125  

and the DNA damage response pathway induced by increased levels of ROS 126  

and RNS due to decreased mitophagy are discussed and a model is presented 127  

which proposes that the inhibition of autophagy contributes to the formation of 128  

 

 

micronuclei and thus to the accumulation of chromosomal aberrations. Also, the 129  

role of Tax mediated inhibition of necrosis via increased expression and 130  

stabilization of cFLIP is highlighted, leading to a model in which Tax mediated 131  

inhibition of autophagy not only contributes to oncogenesis by inducing 132  

chromosomal aberrations but also protecting cells from drug-induced necrosis. 133  

Mcl-1 stabilization: preventing oncogene associated senescence, 134  

apoptosis and mitophagy? 135  

The expression of Tax in Jurkat as well as in TLOM-1 and MT-2 cells stabilises 136  

the antiapoptotic Mcl-1 protein by inducing the (nondegradative) K63-linked 137  

polyubiquitination of Mcl-1 via IKKα/β/γ and TRAF-6 activation whilst decreasing 138  

the expression of the pro-apoptotic Bid and Bid proteins as well as inhibiting Bax, 139  

thus inhibiting Etoposide induced apoptosis in Tax positive HTLV-1 transformed 140  

but not Tax negative ATL cell lines (Choi & Harhaj, 2014; Muhleisen et al., 2014). 141  

Since both the induction of OIS and the deletion of Mcl-1 have been linked to the 142  

induction of autophagy-associated apoptosis (Germain & Slack, 2011; 143  

Wajapeyee et al., 2008), it might be possible that Tax mediated downregulation 144  

of p53 not only prevents the induction of OIS by inhibiting p21 induced 145  

senescence but possibly also by inhibiting p53 dependent induction of autophagy 146  

by downregulation the expression of genes associated with autophagy induction 147  

such as DRAM-1, AMPK, PTEN and Sestrins’ that activate autophagy mainly 148  

through inhibition of mTOR and/or promoting the formation of the phagophore 149  

(Rufini et al., 2013)). In this context it is interesting that the treatment of Tax 150  

 

 

positive MT-2 and Hut-102 with Everolimus, an inducer of autophagy, not only 151  

decreases Tax levels but also increases senescence similar to Everolimus 152  

treated Mantle Cell Lymphoma cells (Darwiche et al., 2011; Rosich et al., 2012), 153  

suggesting that the expression of Tax prevents senescence (promoting cell 154  

proliferation) as well as its degradation by inhibiting autophagic flux. In addition, 155  

by stabilising Mcl-1, Tax might prevent mitophagy but not ER and/or lipid raft 156  

associated autophagy since Mcl-1 predominantly degrades mitochondrial Beclin-157  

1 (Germain et al., 2011; Germain & Slack, 2011). Stabilization of Mcl-1 by Tax 158  

might therefore inhibit the induction of Bax dependent apoptosis by forming a 159  

complex with Bim and BH-3 mimetics (Lopez & Tait, 2015) in addition to 160  

destabilizing Beclin-1, preventing the induction of apoptosis as well as mitophagy 161  

similar to fludarabine resistant B cell leukemia cells (Sharma et al., 2013) by 162  

inducing the fusion of mitochondria (Perciavalle et al., 2012) and thus 163  

antagonizing mitophagy in addition to stabilising the respiratory complex 164  

(Andersen & Kornbluth, 2012; Elgendy et al., 2014; Perciavalle et al., 2012). If 165  

however Tax expressing cells exhibit an increase in mitochondrial mass due to 166  

mitochondrial fusion and thus an increase in respiratory rate remains to be seen. 167  

Alternatively, inhibition of mitophagy might lead to an increase in the production 168  

of ROS and RNS similar to yeast (Kurihara et al., 2012) due to the accumulation 169  

of defective mitochondria (Filomeni et al., 2015) contributing to the induction of 170  

DNA damage by an increase in ROS and RNS. 171  

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HTLV-1 Tax and autophagy: inhibition of autophagic flux a contributing 173  

factor to inhibition of the antiviral response and increased topoisomerase 174  

sensitivity? 175  

Autophagy is generally characterised as a mechanism by which substrates such 176  

as proteins, organelles, or pathogens are delivered to lysosomes for degradation, 177  

contributing to cellular homeostasis and clearance of pathogens. 178  

Three different types of autophagy are distinguished, chaperone-mediated 179  

autophagy (CMA) microautophagy and macroautophagy, the latter commonly 180  

refereed to as autophagy being the best characterised and most studied form of 181  

autophagy. The degradation of autophagic cargo involves the formation of 182  

double-membrane vesicles, sequestration of proteins or organelles, and the 183  

fusion of these vesicles with lysosomes in a highly regulated process. Defects in 184  

autophagy in knockout mice hetero-or homozygous for essential autophagy 185  

genes (ATG) have been associated with an increase in oncogenesis when 186  

compared with wt mice, suggesting that autophagy contributes to the 187  

suppression of genomic defects associated with the development of tumours 188  

(reviewed in (Galluzzi et al., 2015)), probably by degradation of dysfunctional 189  

mitochondria via mitophagy (Green et al., 2011; Takahashi et al., 2013b), the 190  

removal of protein aggregates (Komatsu et al., 2007; Mathew et al., 2009), and 191  

the removal of micronuclei (Rello-Varona et al., 2012; Vessoni et al., 2013). 192  

Physiologically, in mammalian cells autophagy is induced by inhibiting the 193  

mammalian target of Rapamycin (mTOR) complex -1 (mTORC1), which triggers 194  

 

 

a signal transduction cascade involving the phosphorylation of proteins that form 195  

the ULK-1/-2 complex (Jung et al., 2009) including phosphorylation of the 196  

catalytic subunit of phosphatidylinositol 3-kinase (PIK3C3/VPS34) and Beclin 1 197  

(reviewed in (Mah & Ryan, 2012) (Fig.2A). Following the recruitment of LC3-I, 198  

the phagophore is elongated, forming the nascent LC3-I autophagosome 199  

(omegasome), which matures into the LC3-II positive autophagosome. The 200  

mature autophagosome then either forms the amphisome by fusing with 201  

endosome prior fusing with the lysosome, or alternatively fuses directly with the 202  

lysosome, becoming the autolysosome, in a RabGTPase, Beclin1 and UVRAG 203  

dependent step prior to the degradation of the content (reviewed in (Mah & Ryan, 204  

2012; Parzych & Klionsky, 2014; Xie et al., 2015) (Fig. 2B). 205  

Tax and autophagy 206  

Jurkat cells which have been infected with HTLV-1 by co-culturing them with a 207  

HTLV-1 transformed cell line, MT-2, exhibit an increase in NF-κB activity as 208  

measured by the luciferase activity following the transfection with a pNF-κB-luc 209  

reporter plasmid, similar to a Tax-positive Jurkat-TaxP cell line stably expressing 210  

Tax (Wang et al., 2013). Upon examination of the changes in gene expression in 211  

the latter using Microarray analysis, approx. 48 genes exhibited a change in gene 212  

expression of at least twofold with 10 genes associated with oncogenesis, 213  

differentiation, proliferation, cell development being unregulated and 38 genes 214  

down regulated in the presence of Tax. Interestingly one of the genes, Bcl-3, 215  

was upregulated not only in Jurkat-TaxP cells but also significantly in MT-2 cells, 216  

 

 

accompanied by nuclear translocation of Bcl-3 in Jurkat-TaxP (Saito et al., 2010). 217  

These results indicate that Tax not only induces the translocation of NF-κB but 218  

also the expression and nuclear translocation of Bcl-3, which is a negative 219  

inhibitor of NF-κB as well as inhibiting p53 (Kashatus et al., 2006) thus potentially 220  

contributing to Tax induced resistance to p53 dependent apoptosis by preventing 221  

the expression of pro-apoptotic genes, inducing the expression of Bcl-xL via NF-222  

κB, as well as inducing cell proliferation (Saito et al., 2010; Tsukahara et al., 223  

1999). Binding of Bcl-3 however to both Tax and transducers of regulated CREB 224  

activity (TORC)-3 inhibits Tax induced gene expression from the HTLV-1 LTR by 225  

displacing p300 from the HTLV-1 LTR (Hishiki et al., 2007; Kim et al., 2008), thus 226  

downregulation the expression of viral genes. 227  

Regarding the induction of autophagy, the overexpression of Bcl-3 in HeLa cells 228  

co-transfected with Tax induces the formation of autophagosomes following 229  

Rapamycin or Pifithrin-α treatment as well as in mock treated cells whereas 230  

under starvation the formation of autophagosomes decreased (Wang et al., 231  

2013), indicating that the formation of autophagosomes is independent of p53 232  

and that starvation might induce apoptosis due to the inhibition of NF-κB by Bcl-3 233  

(Fabre et al., 2007). In the same study, the authors found that the formation of 234  

autophagosomes also increases in MT2 and MT4 cells following the treatment 235  

with an inhibitor of NF-κB, Bay 11-7082, that inhibits IκBα phosphorylation, 236  

suggesting that the inhibition of the NF-κB signalling pathway is sufficient to 237  

induce autophagosome formation (Wang et al., 2013). Additionally, the 238  

 

 

expression of a Tax mutant, TaxM22, incapable of inducing the activation of NF-239  

κB also fails to induce the formation of autophagosome, indicating that inducing 240  

the expression of Bcl-3 via NF-κB activation is necessary for the induction of 241  

autophagy, whereas the expression of TaxM47 not only activates NF-κB but also 242  

increases the formation of autophagosomes similar to wt Tax (Wang et al., 2013). 243  

Paradoxically, Bcl-3 might also antagonize NF-κB and thus NF-κB induced 244  

formation of the autophagosome and that additionally, Tax mediated activation of 245  

the IKKβ kinase complex activates autophagy by recruiting a complex containing 246  

Beclin-1 and Bif-1 to lipid raft microdomains (Ren et al., 2015). In this model, Tax 247  

interacts with both complexes directly, thereby promoting the assembly of LC3-II 248  

positive autophagosomes (Fig.2B). As discussed below, it is however not clear if 249  

the formation of autophagosomes by forming a complex of Tax with lipid rafts and 250  

Beclin-1 also inhibits the fusion of autophagosomes with the lysosome (Fig.2B). 251  

In addition autophagosome formation is also induced by IKK induced 252  

phosphorylation of AMPK and the subsequent inactivation of mTOR induces the 253  

formation of autophagosomes independent of PI3-K and direct inhibition of 254  

mTOR (Baldwin, 2012; Chen & Debnath, 2013; Criollo et al., 2010), suggesting 255  

that the inability of TaxM22 to induce the formation of autophagosomes is due to 256  

the inability of TaxM22 to bind IKK and activate NF-κB. 257  

Increasing the formation of autophagosomes has been shown to increase the 258  

replication of HTLV-1 in HEK-293T cells transfected with a HTLV-1 molecular 259  

clone (K30) whilst the expression of Tax in HeLa and Jurkat cells as well as in 260  

 

 

other established T cell lines such as PTX4-1 or PL9-1 not only increases the 261  

formation of autophagosomes -as measured by the increase in LC3-II- but at the 262  

same time also inhibiting the fusion with the lysosome as indicated by the 263  

accumulation of Neutrophil Cytosolic Factor 4(NCF)/p40phox (Ren et al., 2015), 264  

with similar results reported in primary human CD4+ cells transduced with HTLV-265  

2 derived a Tax (Tax-2) (Ren et al., 2012; Ren et al., 2015). Accordingly, the 266  

fusion of the autophagosome with the lysosome is inhibited in HEK-293T cells 267  

transfected with the HTLV-1 molecular clone as well as in MT1, MT2, and 268  

HUT102 cells (Tang et al., 2013), suggesting that even in the context of the 269  

presence of other viral proteins autophagic flux is inhibited. It should be noted 270  

that in approx. 60% of ATL patients, HTLV-1 infected T lymphocytes do not 271  

express Tax as a result of the accumulation of mutations which render Tax 272  

inactive (Takeda et al., 2004) suggesting that in patients presenting themselves 273  

with ATL the inactivation of Tax might contribute to a restoration of autophagic 274  

flux. Autophagy however might be inhibited in these cases by the expression of 275  

the antisense HBZ protein, since HBZ has been reported to inhibit starvation 276  

induced autophagy via activation of the mTOR pathway by increased 277  

phosphorylation of the S6 Kinase and inhibition of GADD34, thus inhibiting 278  

GADD34 mediated dephosphorylation of TSC2 (Mukai & Ohshima, 2014). 279  

Although it is well established that Tax does induce the formation of 280  

autophagosomes whilst inhibiting the fusion of the autophagosomes with the 281  

lysosome (as evidenced by the accumulation of LC3-II), both the consequences 282  

 

 

of autophagy deregulation (in particular for mitophagy and apoptosis) and the 283  

mechanism of autophagy inhibition are less well characterised. Based on studies 284  

using either fusion inhibitors (Bafilomycin-A or Chloroquine) or transfecting cells 285  

with siLAMP2 the inhibition of autophagy increases the production of infectious 286  

HTLV-1 particles by approx. 3-fold as measured by p19Gag ELISA, suggesting 287  

that autophagy constitutes part of the antiviral response (Tang et al., 2013). 288  

Inhibiting autophagy by the application of PI3K class III inhibitors or transfection 289  

of siBeclin-1 as well as shTax indeed results in impaired proliferation of HTLV-1 290  

infected SLB1 and MT2 cells as well as a decrease in LC3-II levels, suggesting 291  

that the induction of autophagy is required for the proliferation of HTLV-1 infected 292  

cells (Tang et al., 2013). 293  

One mechanism of Tax induced blocking of the fusion of the autophagosome 294  

with the lysosome might be by sequestering Beclin-1 to lipid rafts thus inhibiting 295  

the formation of a complex containing UVRAG, VPS34 and Beclin-1, which is 296  

required for the fusion of the mature autophagosome with the lysosome, and 297  

stabilising the Rubicon/Vps34 complex thus inhibiting autophagy (Zhong et al., 298  

2009) similar to HEK 293T cells expressing the coronaviral PLP2 protein (Chen 299  

et al., 2014b). Sequestering Bif-1 might not only facilitate the formation of 300  

autophagosomes (Takahashi et al., 2007) and inhibit the fusion of the mature 301  

autophagosome with the lysosome but also prevent the induction of Bax/Bak 302  

mediated apoptosis (Takahashi et al., 2005), linking the induction of 303  

autophagosome formation to the prevention of apoptosis and thus promoting the 304  

 

 

survival of HTLV-1 infected or Tax expressing cells (Fig.2C). The expression of a 305  

Tax mutant not capable of binding Beclin-1, treatment with Tat-Beclin (Shoji-306  

Kawata et al., 2013), or overexpression of Beclin-1 therefore might allow the 307  

formation of the autolysosome as well as the disruption of the Tax/Beclin-1/Bif-1 308  

complex, decreasing the proliferation of HTLV-1 infected cells similar to the 309  

treatment with siBeclin-1. 310  

Because Tax induced autophagy has also been implicated in the degradation of 311  

phosphorylated IKKβ (Wada et al., 2009), it might be possible that the formation 312  

of autophagosomes via direct recruitment of Beclin-1 and Bif-1 is also necessary 313  

for the inhibition of NF-κB and inhibition of antiviral signalling via TRAF6 in 314  

general , whereas early in infection the activation of NF-κB mediated signalling is 315  

necessary for cell transformation and that inhibition of autophagy – or more 316  

precisely the fusion of the autophagosomes with the lysosome- counteracts Tax 317  

induced senescence. Unfortunately, so far it has not been shown if the induction 318  

of autophagy by Bcl-3 is accompanied by an increase in autophagic flux as 319  

measured by the degradation of p62/SQSTM-1 although the accumulation of 320  

NCF/p40phox in HeLa cells transfected with Tax strongly suggests that 321  

autophagic flux might be inhibited (Ren et al., 2015) despite increase in Bcl-3 322  

levels. It should be also be noted that cytosolic -but not nuclear- HTLV-1 Tax 323  

interacts with both HDAC-6 and USP10, preventing the formation of stress 324  

granules (Legros et al., 2011; Takahashi et al., 2013a). Since the deacetylation of 325  

microtubuli by HDAC-6 has been shown to be required for the fusion of 326  

 

 

autophagosomes with lysosomes (Seguin et al., 2014), the expression of Tax-1 327  

(at least the cytosolic form) might increase the clearance of stress granules by 328  

autophagy (Legros et al., 2011) induced via the recruitment of Beclin-1 to lipid 329  

rafts as well as facilitating the fusion of the autophagosome with the lysosome via 330  

interaction with HDAC-6. The application of HDAC-6 inhibitors such as 331  

Voronistat or Belinostat therefore might not only facilitate the formation of the 332  

autolysosome but also inhibit HTLV-1 replication via autophagy induction similar 333  

to HIV-1 (Campbell et al., 2015) as well as promoting the formation of stress 334  

granules via deregulating autophagy (Seguin et al., 2014) although further 335  

studies are needed. 336  

Interestingly in primary human primary human CD4+ cells transduced with Tax2-337  

GFP and treated with Chloroquine or 3-MA, caspase-3 is activated and apoptosis 338  

induced, suggesting that the inhibition of autophagy decreases the survival and 339  

immortalization of Tax2 expressing cells (Ren et al., 2012), similar to Pepstatin A 340  

or E-64D treated RD-A cells infected with Enterovirus-71 (Xi et al., 2013). Since 341  

in Tax2 expressing cells p53 is inhibited to a lesser degree compared to Tax1 342  

expressing cells (Mahieux et al., 2000) it might however be possible that the 343  

induction of p53 by Tax2, but not Tax1, induces both autophagy (via DRAM-1 344  

and Sestrin mediated formation of the phagophore) and apoptosis (by increasing 345  

the expression of pro-apoptotic genes such Bik); indeed although both the stable 346  

expression of both Tax1 and Tax2 in Jurkat cells induces resistance to Etoposide 347  

and Camptothecin, serum-starved Tax2 expressing Jurkat cells are less resistant 348  

 

 

to Etoposide or Camptothecin induced apoptosis (Sieburg et al., 2004) which 349  

might or might not be dependent on p53. Since both Etoposide and 350  

Camptothecin induced apoptosis increases following the inhibition of autophagy 351  

(Hollomon et al., 2013; Xie et al., 2011; Zhang et al., 2014), it might be possible 352  

that the failure to inhibit autophagy in addition to p53 mediated pro-apoptotic 353  

signalling by Tax2 contributes to decreased sensitivity of HTLV-2 infected cells to 354  

treatment with topoisomerase inhibitors. 355  

Tax and cFLIP 356  

HTLV-1 Tax also inhibits apoptosis induced by TRAIL and CD95L by increasing 357  

intracellular levels of cFLIP in a NF-κB and IKK dependent manner and pre-358  

treatment of Tax expressing U251 astroglioma cells with inhibitors preventing the 359  

formation of autophagosomes such as LY294002 (inhibiting PI3K) or with 3-MA 360  

sensitizes cells to death receptor mediated apoptosis (Jeong et al., 2008; 361  

Krueger et al., 2006; Wang et al., 2014) indicating that the induction of 362  

autophagosome assembly by Tax is a prerequisite for protecting cells against 363  

CD95L and TRAIL induced apoptosis in both U251 cells and Jurkat cells stably 364  

transfected with a estrogen receptor–Tax fusion protein as well as in HTLV-1 365  

infected cell lines (MT-2, SLB-1, and HuT-102) (Krueger et al., 2006). Generally, 366  

cFLIP inhibits DISC induced activation of caspase-8 by binding to pro-caspase-8 367  

thereby inhibiting apoptosis and RIP-1 induced necroptosis (He & He, 2013; Lee 368  

et al., 2009). Indeed, the expression of Tax in Jurkat cells induces the expression 369  

of not only cFLIPs but also cFLIPL (Krueger et al., 2006) preventing the cleavage 370  

 

 

of Bid into the active –truncated- form, tBid (Krueger et al., 2006). Since both Tax 371  

and cFLIP inhibit autophagic flux either by inhibiting the fusion of the 372  

autophagosome with the lysosome (Tax) or the formation of LC3-II positive 373  

autophagosomes by binding Atg3 (cFLIP) (Lee et al., 2009), both Tax and cFLIP 374  

might work synergistically to inhibit autophagic flux in activated CD3+ T 375  

lymphocytes (He & He, 2013) which in HTLV infected and Tax expressing Jurkat 376  

cells is dependent on the activation of the T cell receptor by anti-CD3 antibodies 377  

(Krueger et al., 2006). Additionally, as well as inducing the formation of 378  

autophagosomes, the ability of Tax to activate IKK and subsequently 379  

hyperactivation of NF-κB (Wang et al., 2014) by inducing the formation of 380  

autophagosomes, is required for NF-κB induced increase in cFLIP. Because 381  

autophagosomes are also providing a scaffold for the assembly of the necrosome 382  

(Basit et al., 2013), it might be possible that cFLIP not only inhibits the DISC 383  

complex but also the necrosome formation on autophagosomes induced by Tax 384  

by inhibiting the formation of LC3-II positive autophagosomes via binding to 385  

ATG3 (He & He, 2013) (Fig.2D). The importance of Tax induced expression of 386  

cFLIP is further highlighted that the treatment of HTLV-1 infected cells with 387  

Rocoglamide, a herbal inhibitor of eIF4E, inhibits not only the expression of 388  

cFLIP (by inhibiting the MEK-ERK-MNK1 pathway) but also sensitises Tax 389  

expressing cells to TRAIL and CD95L induced apoptosis (Bleumink et al., 2011). 390  

In a similar way, the expression of other viral proteins binding Atg3 –such as 391  

KSHV vFLIP (Lee et al., 2009)- or containing a LC3 binding motif – such as 392  

 

 

Influenza Virus M2 (Beale et al., 2014) – might prevent necroptosis and 393  

apoptosis in cFLIP deficient cells infected with HTLV-1 or expressing Tax. 394  

Inhibition of the necrosome by Tax also differs from MCMV vIRA, HSV-1 ICP6, 395  

and HSV-2 ICP10 proteins which prevent the formation of the active necrosome 396  

in necroptosis-sensitive human cells by forming complexes with caspase-8 via 397  

binding to the DED domain (Guo et al., 2015) (reviewed in (Mocarski et al., 398  

2015). 399  

Treatment of Tax expressing U231 astroglioma cells with either Chloroquine or a 400  

combination of Chloroquine and MG132 not only decreases cell viability following 401  

activation of the CD95L or TRAIL pathway but also decreases the amount of Tax 402  

as well as inducing the cleavage of caspase-3 even in the absence of TRAIL or 403  

CD95L activation of the DR pathway (Wang et al., 2014), suggesting that Tax 404  

expression does induce the activation of caspase-9 and -3 as well as preventing 405  

apoptosis. This leads to a model where Tax expression in absence of external 406  

stimuli does not completely inhibit neither autophagic flux nor the activation of 407  

caspase-3 and -9 but rather balances autophagy and caspase activation. 408  

Interestingly, caspase-9 has been postulated to activate the HTLV-1 LTR by 409  

forming an Sp1-p53 complex following treatment with stress inducing (Torgeman 410  

et al., 2001) as well as DNA damaging agents (Abou-Kandil et al., 2011). Taken 411  

together these results suggest that the expression of Tax might induce the 412  

activation of the cellular caspase-9 maybe via Bik whilst preventing the activation 413  

of caspase-8 mediated signalling pathways by inducing the degradation of 414  

 

 

caspase-8 by p62/SQSTM-1 dependent selective autophagy and by increasing 415  

the expression of cFLIP (Supplemental Fig. 1). Despite the activation of cellular 416  

caspases, apoptosis might be inhibited due to the stabilization of Mcl-1, thus 417  

preventing mitochondrial depolarization. If however the induction of cFLIP by Tax 418  

indeed prevents the formation of mature autophagosomes via competing with 419  

LC3 for binding ATG3 has not been demonstrated. 420  

Targeting the formation of the mature autophagosome in HTLV-1 infected or Tax 421  

expressing cells therefore might activate Bik and thus induce apoptosis via the 422  

intrinsic pathway by activating Bax as demonstrated by Chen et al. (Chen et al., 423  

2014a). Further to inhibiting RIP1 mediated induction of necroptosis, Tax has 424  

also been reported to inhibit RIP1 mediated induction of Interferon-β by inhibiting 425  

the phosphorylation of IRF-7, thus inhibiting RIG-1 dependent antiviral signalling 426  

induced by dsRNA or Poly (I:C) (Hyun et al., 2015). If however this solely 427  

dependent on binding of Tax directly to RIP1 or is also dependent on cFLIP has 428  

not been demonstrated. Additionally, Tax has been reported to inhibit antiviral 429  

signaling via the induction of SOCS1 in a NF-κB dependent manner 430  

(Charoenthongtrakul et al., 2011) (suggesting that Tax inhibits antiviral signaling 431  

via multiple mechanisms. 432  

In addition to Tax mediated increase in cFLIP gene expression, the 433  

deacetylation of cytoplasmic Ku70 by Tax via binding to of P/CAF (Jiang et al., 434  

1999) and/or p300/CBP (Ramirez & Nyborg, 2007) might stabilise a cytoplasmic 435  

complex consisting of cFLIP, pro-caspase-8 and FADD by inhibiting the 436  

 

 

polyubiquitination of cFLIP (Kerr et al., 2012) (Fig.2D). 437  

Lastly, apoptosis induced due to the inhibition of autophagy, might be inhibited by 438  

Tax induced accumulation of deacetylated Ku70 that counteracts Bik mediated 439  

induction of the intrinsic pathway by forming a stable complex with Bax. In any 440  

case, so far the role of Ku70 in preventing either extrinsically or intrinsically 441  

induced apoptotic pathways in Tax expressing cells and cells infected with HTLV-442  

1 has not been studied. 443  

Autophagy and the DNA damage response: a role for Tax? 444  

The DNA damage response is critical for the detection and repair of single 445  

stranded (ss) or double stranded (ds) DNA breaks as well as DNA lesions or 446  

mismatched bases. DNA breaks are recognized by the Mre11, NBS1 and Rad50 447  

(MRN) complex (reviewed in (Williams et al., 2010)) resulting in the 448  

phosphorylation of H2AX at Ser-139 by phosphatidylinositol-3-OH-kinase-like 449  

family of protein kinases (PIKKs), namely ATM, ATR and DNA-PK (Stiff et al., 450  

2004). Serine 139 phosphorylated H2AX (γH2AX) is important in the recruitment 451  

of downstream factors such as Rad51 and Brca1 to sites of DNA lesions (Celeste 452  

et al., 2002). In the past years, many different viruses have been shown to 453  

increase levels of γH2AX and modulate the DNA damage response (DDR) 454  

including both oncogenic and non-oncogenic viruses (reviewed in (Lilley et al., 455  

2007; Luftig, 2014; Nikitin & Luftig, 2011). 456  

Inhibiting the DDR pathway however does not always induce cell death. As 457  

mentioned above, the induction of the DDR by increased DNA replication induces 458  

 

 

cellular senescence in a p21 dependent pathway. Inhibition of the DDR however 459  

also leads to the formation of micronuclei, which are cleared by autophagy if not 460  

resolved by NHEJ (see below). The induction of the DDR can also induce 461  

autophagy, which itself either might promote or inhibit apoptosis (Fig3A). 462  

Tax, DDR, and autophagy 463  

Induction of the DDR by viral oncoproteins can be achieved, among others, by 464  

two mechanisms; first by inducing DNA breaks directly or second by attenuating 465  

the DDR via sequestering proteins required for the execution of the repair 466  

pathway into “pseudo DNA repair foci”, sites where DNA repair factors are 467  

sequestered in the absence of DNA damage (Nikitin & Luftig, 2012; Soutoglou & 468  

Misteli, 2008). In the case of HTLV-1, Tax has been shown to employ both 469  

mechanisms (reviewed in (Boxus & Willems, 2012). HTLV-1 Tax induces dsDNA 470  

breaks by generating ROS (Kinjo et al., 2010) and RNS (Chaib-Mezrag et al., 471  

2014) as well as generating replication dependent dsDNA breaks by accelerating 472  

DNA replication (Boxus et al., 2012). In addition, HTLV-1 Tax attenuates the DNA 473  

damage response by sequestering ATM, DNA-PK, Chk-1/-2 and p53 into Tax 474  

containing nuclear speckles or “pseudo-foci” that are also positive for γH2AX 475  

(Belgnaoui et al., 2010; Durkin et al., 2008; Park et al., 2004; Park et al., 2006) as 476  

well as by repression of Ku80 gene expression (Ducu et al., 2011) (Fig.3B) thus 477  

inhibiting not only the DDR but also the segregation of chromosomes (reviewed 478  

in (Marriott et al., 2002)). In this model, the expression of Tax induces the 479  

formation of ROS by accelerating DNA replication via interacting with the MCM2-480  

 

 

7 helicase complex (Boxus et al., 2012), increasing the DNA replication and 481  

progression of S phase, as well as increasing the expression of PCNA (Gatza et 482  

al., 2003; Lemoine et al., 2000; Ressler et al., 1997). 483  

In contrast to ATM and DNA-PK, Tax does not affect the activation of the ATR 484  

dependent pathway (Haoudi et al., 2003), suggesting that the both the alternative 485  

(A)-NEHJ and ATR mediated HR pathway might be functionally intact, although 486  

both (nuclear) Ku70 (Rai et al., 2010) and Wild-type p53-induced phosphatase 1 487  

(Wip-1) (see below) might attenuate the ATR response (Lu et al., 2005; Lu et al., 488  

2008). Indeed, GFP-Tax expressing CHO xrs6 -/- cells (the equivalent of Ku80) 489  

display an increase in chromosome fusion (Majone & Jeang, 2012) suggesting 490  

that Ku70/Ku80 dependent pathways are induced. 491  

Inhibition of autophagy by Tax: attenuating the DDR? 492  

Although autophagy is process occurring in the cytoplasm of cells, nuclear 493  

proteins which are polyubiquitinylated can be recognised and degraded by 494  

autophagy by binding nuclear p62/SQSTM-1 prior of being exported to the 495  

cytoplasm (Pankiv et al., 2010) and Beclin-1 deficient immortalised mammary 496  

epithelial cells accumulate damaged DNA (Karantza-Wadsworth et al., 2007). 497  

Since the inhibition of autophagy has been associated with a higher incidence of 498  

ROS due to the accumulation of damaged proteins and organelles, autophagy 499  

deficiency has been linked to the induction of DNA lesions via an increase in 500  

ROS production (reviewed in (Filomeni et al., 2015)). 501  

In addition to induce the formation of ROS upon inhibition of autophagy 502  

 

 

(Kageyama & Komatsu, 2012; Lee et al., 2012), the deletion of Atg7 or Atg5 in 503  

primary mouse embryonic fibroblast cells also induces a defect in HR mediated 504  

pathway by promoting the degradation of phosphorylated Chk-1 via the 505  

proteasome without affecting the recruitment of ATR, ATM, or Rad51 to sites of 506  

DNA damage, favoring error-prone NHEJ which might contribute to the 507  

accumulation of DNA damage in autophagy deficient cells and knockdown of 508  

Beclin-1, UVRAG or Atg5 induces the formation of 53BP1 positive foci, indicating 509  

the activation of NHEJ (Park et al., 2014). The inhibition of DNA-PK in Atg5-/- or 510  

Atg7-/- MEF using chemical inhibitors not only inhibits NHEJ but also induces 511  

apoptosis following treatment with Etoposide or exposure to IR (Liu et al., 2015). 512  

In line with these results, deleting FIP200 in MEF impair the DDR and increases 513  

the sensitivity ionizing radiation induced ROS (Bae & Guan, 2011), suggesting 514  

that one of the main functions of autophagy is preventing the induction of DNA 515  

lesions by inhibiting the formation of ROS via regulation of mitophagy, thus 516  

decreasing DNA damage indirectly (reviewed in (Filomeni et al., 2015)). Once 517  

however DNA lesions are induced by ROS, sensor proteins such as PARP1 and 518  

ATM not only sense DNA damage but also activate the DDR and linking the DDR 519  

with autophagy. In the case of MEF treated with Doxorubicin, PARP1 is 520  

hyperactivated in MEF, thus leading to the depletion of ATP and NAD+, resulting 521  

in the activation of AMPK as well as increased expression of Bnip-3, Cathepsin-522  

B/-L, and Beclin-1 (Munoz-Gamez et al., 2009), similar to MEF treated with 523  

Methylnitronitrosoguanidine (MNNG) (Zhou et al., 2013). In the case of ATM, it 524  

 

 

has been demonstrated that following the treatment of MCF7 and HeLa S3 cells 525  

with H2O2, ATM translocates from the nucleus into the cytoplasm, where it 526  

induces autophagy by repressing mTORC1 via LKB dependent and independent 527  

pathways (depending on the concentration of H2O2) involving the activation of 528  

AMPK via ATM dependent phosphorylation of Tuberous sclerosis complex-2 529  

(TSC-2) (Alexander et al., 2010); treatment with Etoposide however did not 530  

initiate the induction of autophagy suggesting that ATM induced autophagy is 531  

selective. KU55933 treatment of HeLa cells however inhibits the induction of 532  

autophagy via the mTORC-1 pathway, MAPK14, and Beclin-1/PI3KIII pathway 533  

(Liang et al., 2013), suggesting that ATM activation can initiate autophagy via 534  

multiple pathways. In addition to TSC-2, the induction of both Sestrin-1/-2 and 535  

REDD via ATM inhibits Akt kinase mediated inhibition of AMPK (Cam et al., 536  

2014), thus counteracting the activation of Akt kinase by DNA-PK and Akt 537  

dependent inhibition of TSC-2 (Inoki et al., 2002) as well as AMPK in both 538  

Topotecan treated NHDF and MEF cells; however, although both mTORC-1 and 539  

the ribosomal p70 S6 Kinase are activated following Topotecan treatment, the 540  

induction of autophagy flux has not been demonstrated yet. 541  

Constitutive activation of ATM following the induction of DNA damage and 542  

treatment with HDAC inhibitors also results in an intranuclear activation of NEMO 543  

and translocation of the ATM/NEMO complex to the cytoplasm where in 544  

subsequent steps the IKK complex is degraded and the p65/p50 complex is 545  

activated, leading to NF-κB hyperactivation and apoptosis (Rosato et al., 2010; 546  

 

 

Wu & Miyamoto, 2008; Wu et al., 2006). Although it has not been demonstrated, 547  

inducing ATM and/or PARP1 by Tax may also promote the activation of NF-κB 548  

by translocation of the ATM/NEMO complex to the cytoplasm, potentiating the 549  

formation of autophagosomes similar to treatment with HDAC inhibitors whilst 550  

preventing apoptosis. 551  

In the case of Tax expressing or HTLV-1 infected cells a contribution of either 552  

PARP1 or ATM activation to induction of autophagy has not been demonstrated. 553  

Following UV irradiation of clonal rat embryo fibroblasts (CREFs), Tax expressing 554  

CREFs only exhibit an initial arrest in G1 phase followed by an acceleration of 555  

entry into S phase when compared with control cells expressing the backbone 556  

vector (Dayaram et al., 2013). Furthermore, CREF-tax cells also maintain a 557  

higher abundance of UV induced thymidine dimers and fail to induce the 558  

formation of γH2AX and phosphorylated Replication Protein (RPA) positive foci, 559  

a hallmark of DDR induction, indicating that the expression of Tax attenuates the 560  

ATM dependent HR pathway (Baydoun et al., 2012). Immunofluorescence 561  

analysis of gamma-irradiated cells expressing Tax further indicated that Tax 562  

indeed sequesters and/or inhibits various components of the DDR, including 563  

MDC-1, Chk-1/-2, as well as p53, leading to an inhibition of the DDR (Belgnaoui 564  

et al., 2010; Durkin et al., 2008; Ramadan et al., 2008). In addition to preventing 565  

the initiation of the DDR, Tax also induces the activation of Wip-1 thereby 566  

attenuating γH2AX dependent assembly of DNA damage repair foci by 567  

(premature) dephosphorylation of γH2AX and ATM (Cha et al., 2010). If the 568  

 

 

activation of Wip-1 by Tax also inhibits the induction of autophagy is not clear, 569  

but it has been demonstrated that Wip-1 does inhibit the formation of 570  

autophagosomes probably by dephosphorylatingAMPK in bone marrow derived 571  

macrophages (Le Guezennec et al., 2012), potentially inhibiting Tax induced 572  

formation of autophagosomes partially either by dephosphorylating ATM or 573  

AMPK (Fig.3B). 574  

PTEN (Phosphatase and Tensin homolog) is a tumor suppressor protein with 575  

various cytoplasmic and nuclear functions, including regulation of apoptosis in 576  

response to DNA damage and cytokine secretion (Furumoto et al., 2006; Gil et 577  

al., 2006), which in response to DNA damage induced by various treatments 578  

such as Etoposide, Topotecan, or Cisplatin, or following oxidative stress induced 579  

by H2O2 (Chen et al., 2015a; Shen et al., 2007) translocates to the nucleus in a 580  

ATM dependent manner (Chen et al., 2015a). The nuclear translocation of PTEN 581  

has been demonstrated to induce formation of autophagosomes and the 582  

degradation of p62/SQSTM-1 via phosphorylation of Jun that induces the 583  

expression of Sestrin-2 and the activation of AMPK in A549 and HeLa cells 584  

(Chen et al., 2015a). 585  

The expression of Tax in both CV-1 and Jurkat cells in contrast prevents the 586  

activation of PTEN by downregulating the expression of PTEN via 587  

hyperactivation of NF-κB and sequestering p300/CBP by p65 (Fukuda et al., 588  

2012). Since Tax however also induces the expression of Wip-1, it might be 589  

possible that in addition to downregulating the expression of PTEN, Tax might 590  

 

 

inhibit the activation of PTEN dependent autophagy by preventing PTEN 591  

phosphorylation of by ATM following the induction of DNA damage. 592  

Since the expression of Tax suppresses the HR mediated repair pathway, it has 593  

been speculated that Tax induces the error prone (conservative or C-) NHEJ 594  

pathway (Baydoun et al., 2012), which is not only dependent on DNA-PK but also 595  

on Ku80 and Ku70 (Guirouilh-Barbat et al., 2007). HTLV-1 Tax however has 596  

been proposed to inhibit the C-NHEJ pathway by downregulating the expression 597  

of Ku80 in CREF cells, suggesting that C-NHEJ is inhibited (Ducu et al., 2011; 598  

Majone & Jeang, 2012), although in Tax immortalised WT4, WT4B and WT4I and 599  

HTLV-1 infected cell lines HR is inhibited and NHEJ is induced (Baydoun et al., 600  

2012). Regarding Ku70, the expression of Tax might increase the nuclear levels 601  

of deacetylated Ku70. Nuclear, deacetylated but not acetylated, Ku70 has been 602  

shown to interact with nuclear matrix-associated protein scaffold/matrix-603  

associated region-binding protein 1 (SMAR1) (Chaudhary et al., 2014). 604  

Phosphorylation of the Ku70/SMAR1 at Ser-370 complex by ATM facilitates the 605  

recruitment of Ku70 to sites of DNA damage that might also recruit DNA-PK, 606  

leading to DNA repair (Chaudhary et al., 2014). Tax might therefore induce the 607  

formation of the Ku70/SMAR1 complex and recruit DNA-PK independent of Ku80 608  

despite the downregulation of Ku80 expression. Wip1 however might antagonise 609  

the formation of K70/SMAR1 by inhibiting ATM dependent phosphorylation of 610  

SMAR1 thus increasing Ku70 and subsequently promoting NHEJ (Fig.4A). 611  

Since the expression of Tax however does not interfere with the activation of 612  

 

 

ATR pathway and the induction of ATR by various agents including Cisplatin, 613  

Camptothecin, Hydroxyurea, and MMS induces the degradation of 614  

phosphorylated Chk-1 by chaperone mediated autophagy (Park et al., 2015), the 615  

failure of Tax to interfere with the ATR pathway might indicate that Tax interferes 616  

specifically with (macro-) autophagy (Supplemental Fig.2) 617  

Tax and Micronuclei 618  

One characteristic of cancer cells is the presence of abnormalities in the 619  

karyotype, both numerical and structural caused by erroneous DNA repair 620  

leading to deletions, duplications, inversions, and chromosome translocation. 621  

Two sequential mechanisms have been proposed to induce the rearrangement of 622  

chromosomal loci in a single event, chromothripsis (“chromosome shattering”) 623  

followed by chromoanasynthesis (“chromosome reconstitution”), which is 624  

preceded by the formation of micronuclei (reviewed in (Holland & Cleveland, 625  

2012)). Micronuclei are believed to be formed by mitotic errors in which lagging 626  

chromosomes fail to segregate properly and consequently contained in a 627  

micronucleus (MN) (Thompson & Compton, 2011). Owing to the decreased 628  

density of nuclear pore complexes within the MN membrane, DNA replication 629  

within the MN takes place in G2 phase of the cell cycle instead of S phase and is 630  

not completing at the beginning of M phase (Crasta et al., 2012). Consequently 631  

entry into M phase leads to chromothripsis –shattering or pulverization of 632  

chromosomes- and subsequent repair by NHEJ in the G1 phase succeeding M 633  

phase (chromoanasynthesis) with the MN not being disassembled during mitosis 634  

 

 

(reviewed in (Holland & Cleveland, 2012)). In the subsequent S phase, the highly 635  

rearranged chromosome then replicates and is distributed among the daughter 636  

cells. 637  

MEF derived from GFP-LC3 transgenic mice carrying a mutation of pH222P in 638  

the Lamin A gene (LmnaH222P-/H222P-) exhibit a higher frequency of MN that are 639  

positive for Lamin B, γH2AX, DNA, Rab7, LC3, Atg16L, Atg9, Atg7, or LAMP2, 640  

indicating that MN are autophagic substrates (Park et al., 2009). Treatment of 641  

LmnaH222P-/H222P- MEF with either Wortmannin or 3-MA not only decreased cell 642  

viability but also increased the frequency of MN (Germain et al., 2011) similar to 643  

U2OS cells treated with Hydroxyurea or Cytochalasin D followed by Bafilomycin-644  

A or NH4Cl treatment (Rello-Varona et al., 2012). Interestingly, only GFP-LC3+-645  

p62/SQSTM-1+ micronuclei exhibited signs of envelope degradation and γH2AX 646  

positive DNA damage foci, indicating that the induction of autophagic degradation 647  

of MN is associated with the presence of DNA damage (Rello-Varona et al., 648  

2012) and inhibition of the NHEJ with concomitant inhibition of autophagy has 649  

been associated with the accumulation of DNA lesions in Atg7-/- MEF (Liu et al., 650  

2015). 651  

The expression of HTLV-1 Tax in Cos, HeLa, and CREF cells has been shown to 652  

induce the formation of MN (Ducu et al., 2011; Majone et al., 1993; Semmes et 653  

al., 1996) and of nucleocytoplasmic bridges containing free DNA ends (Ducu et 654  

al., 2011), indicating that the MN indeed contain damaged DNA. Interestingly, 655  

Ku80 has been reported to localise to intranuclear bodies positive for Tax (Ducu 656  

 

 

et al., 2011), suggesting that these bodies might represent MN that are repaired 657  

via the NHEJ pathway. In this model, Tax would increase NHEJ despite the 658  

downregulation of Ku80 by relocalising both nuclear Ku70 and Ku80 to MN in an 659  

ATM dependent or independent pathway (Fig.4A and B) resulting in rearranged 660  

chromosomes that might be facilitated by an increase in nuclear –deacetylated- 661  

Ku70 in a Wip-1 dependent manner. Due to impaired nuclear import of repair 662  

factors into MN (Hoffelder et al., 2004), non-repaired DNA contained in MN 663  

and/or nucleocytoplasmic bridges induces the formation of MN that are 664  

subsequently degraded via autophagy. Prolonged Tax expression however might 665  

inhibit the degradation of MN via autophagy inhibition as discussed above, 666  

leading to apoptosis and/or chromosome aberrations. 667  

Conclusion 668  

In conclusion, Tax mediated stabilization of Mcl-1 might not only prevent OIS 669  

associated decrease in cell proliferation, but also apoptosis as well as the 670  

induction of mitophagy via decreasing mitochondrial localized Beclin-1, ER 671  

and/or lipid-raft based formation of the autophagosome however might be 672  

activated by Tax induced expression of Bcl-3 via hyperactivation of NF-κB as well 673  

as the recruitment of Beclin-1 to lipid rafts, downregulating p53 dependent 674  

apoptosis, increasing the degradation of caspase-8 via p62/SQSTM-1, as well as 675  

inducing the expression and stabilisation of cFLIP (inhibiting the DISC complex 676  

and/or inhibiting the necrosome) in addition to at least a partial inhibition of the 677  

autophagic flux by sequestering Beclin-1. Inhibiting autophagic flux might also 678  

 

 

prevent the secretion of inflammatory cytokines, which is not only autophagy 679  

dependent (Kraya et al., 2015) but might also be dependent on the activation of 680  

caspase-3 (Sirois et al., 2012), and contributing to the proliferation of HTLV-1 681  

infected and Tax expressing cells by preventing senescence. Deacetylation of 682  

Ku70 might contribute to preventing apoptosis by sequestering Bax in cytosolic 683  

Ku70/Bax complexes as well as stabilising cFLIP. 684  

Deacetylated Ku70 has also been demonstrated to localise to the nucleus where 685  

it binds sites of DNA damage by forming a complex with Ku80, thus recruiting 686  

DNA-PK (Chaudhary et al., 2014; Meng et al., 2015). Increasing the amount of 687  

deacetylated Ku70 in the cytoplasm might therefore also increase the nuclear 688  

import of Ku70 and interfering with the DNA damage response, in particular the 689  

NHEJ pathway (Fell & Schild-Poulter, 2012) which is also induced by inhibiting 690  

autophagy (Liu et al., 2015) concomitant with an inhibition of the homologous 691  

recombination pathway (HR) due to inhibiting the Rad6 dependent degradation of 692  

heterochromatin protein HP1 by inhibiting autophagy (Chen et al., 2015b). The 693  

NHEJ pathway however is also regulated by Protein Phosphatase (PP2A) via 694  

dephosphorylating DNA-PK, thus increasing the activity of DNA-PK (Chan & 695  

Lees-Miller, 1996), and Tax has been shown to form a complex with PP2A and 696  

IKKγ thereby inhibiting PP2A (Fu et al., 2003; Hong et al., 2007). It might 697  

therefore possible that the inhibition of PP2A might also inactivate DNA-PK 698  

despite the formation of Ku70/Ku80 heterodimers thus favoring a pathway that 699  

involves the formation of ATM dependent complex of Ku70 with SMAR1 who 700  

 

 

itself is subject to inhibition by Wip-1. If the expression of Tax also destabilizes 701  

NBS1 –and prevents the formation of the MRN complex- by decreased ATM 702  

dependent phosphorylation of NBS1 and/or decreased p300/CBP dependent 703  

acetylation (Jang et al., 2010) thus contributing to the attenuation of ATM, ATR, 704  

and PARP1 dependent DDR pathways (and impairment in DNA end-resection) 705  

also remains to be investigated. In addition, destabilizing NBS1 could also 706  

prevent MRE11 and ATM dependent phosphorylation of Bid (Stracker et al., 707  

2007), thus preventing Tax induced apoptosis. 708  

The expression of Tax might also induce the ubiquitination of Sirtuin-1 (SIRT1) by 709  

MDM2 and thus induce the relocalisation of SIRT1 to the cytoplasm as described 710  

for irradiated DU 145 and HeLa cells treated with Etoposide (Peng et al., 2015). 711  

Tax induced DNA damage therefore might prevent the deacetylation of NBS1 712  

and thus activating instead of attenuating the activation of ATM, ATR, and PARP-713  

1 (whilst attenuating the DDR by sequestering DNA repair factors into pseudo-714  

repair foci). Cytoplasmic SIRT1 has been hypothesized to facilitate the 715  

acetylation of pro-apoptotic substrates such as Bax and caspase and induce 716  

apoptosis (Jin et al., 2007). Tax might therefore induce both the acetylation and 717  

deacetylation of proapoptotic substrates thus explaining the ability of Tax to not 718  

only transform infected cells but also inducing apoptosis. 719  

Interaction of Tax with p300/CBP in the cytoplasm, in particular in the perinuclear 720  

region, might prevent the acetylation of cytoplasmic LC3-I and thus the 721  

translocation of LC3-I into the nucleus, similar to HEK-293T cells transfected with 722  

 

 

p300 RNAi (Huang et al., 2015), thus preventing the induction of autophagy as a 723  

result of amino-acid starvation (Fig.5A). The increase of cytoplasmic LC3-I might 724  

then facilitate the formation of autophagosomes at lipid rafts and the ER as well 725  

as facilitating microautophagy and mitophagy. The sequestration of p300/CBP by 726  

Tax inside the nucleus might contribute to the export of nuclear deacetylated LC3 727  

in a SIRT1 independent manner and thus the formation of autophagosomes at 728  

lipid rafts (Fig.5B), although a contribution of nuclear p300/CBP to the 729  

accumulation of deacetylated LC3 has not been demonstrated. However, in HeLa 730  

cells constitutively expressing Tax and transfected with GFP-LC3, GFP-LC3 is 731  

absent from the nucleus and located in the perinuclear region (Ren et al., 2015). 732  

Further research however is needed to analyze the interplay between Tax and 733  

the regulation of both autophagy and the DNA damage response. 734  

735  

736  

737  

738  

739  

740  

741  

742  

743  

744  

 

 

References 745  

Abou-­‐Kandil,  A.,  Chamias,  R.,  Huleihel,  M.,  Godbey,  W.  T.  &  Aboud,  M.  (2011).  746  Role  of  caspase  9  in  activation  of  HTLV-­‐1  LTR  expression  by  DNA  damaging  747  agents.  Cell  cycle  10,  3337-­‐3345.  748  

Alexander,  A.,  Cai,   S.  L.,  Kim,   J.,  Nanez,  A.,   Sahin,  M.,  MacLean,  K.  H.,   Inoki,  K.,  749  Guan,  K.  L.,  Shen,  J.,  Person,  M.  D.,  Kusewitt,  D.,  Mills,  G.  B.,  Kastan,  M.  B.  750  &  Walker,   C.   L.   (2010).  ATM  signals   to  TSC2   in   the   cytoplasm   to   regulate  751  mTORC1  in  response  to  ROS.  Proceedings  of  the  National  Academy  of  Sciences  752  of  the  United  States  of  America  107,  4153-­‐4158.  753  

Andersen,  J.  L.  &  Kornbluth,  S.  (2012).  Mcl-­‐1  rescues  a  glitch  in  the  matrix.  Nature  754  cell  biology  14,  563-­‐565.  755  

Avesani,  F.,  Romanelli,  M.  G.,  Turci,  M.,  Di  Gennaro,  G.,  Sampaio,  C.,  Bidoia,  C.,  756  Bertazzoni,   U.   &   Bex,   F.   (2010).   Association   of   HTLV   Tax   proteins   with  757  TAK1-­‐binding   protein   2   and   RelA   in   calreticulin-­‐containing   cytoplasmic  758  structures  participates  in  Tax-­‐mediated  NF-­‐kappaB  activation.  Virology  408,  759  39-­‐48.  760  

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1355  

1356  

1357  

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1366  

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1368  

 

 

Figure legends 1369  

Figure 1: Domains of HTLV-1 Tax. 1370   1371  Figure 2: 1372   1373  

(A) Induction of autophagosomes. The formation of autophagosomes is 1374  

induced by drugs such as Rapamycin, starvation, or by endogenous 1375  

factors such as AMPK that inhibit mTORC1 leading to the activation of 1376  

the ULK1/2 complex. Subsequent binding and phosphorylation of FIP22, 1377  

ATG 13 and ATG101 leads to the recruitment of Beclin-1 and VPS34, 1378  

forming complexes with ATG14L, AMBRA1 or UVRAG. These complexes 1379  

recruit ATG9 and WIPI among other proteins inducing the formation of the 1380  

nascent autophagosome. 1381  

(B) The formation of autophagosomes is induced by drugs such as Rapa-1382  

mycin, starvation, or by endogenous factors such as AMPK that inhibit 1383  

mTORC1 leading to the activation of the ULK1/2 complex. Subsequent 1384  

binding and phosphorylation of FIP220, ATG 13 and ATG101 leads to the 1385  

recruitment of Beclin-1 and VPS34, forming complexes with ATG14L, 1386  

AMBRA1 or UVRAG. These complexes recruit ATG9 and WIPI among 1387  

other proteins inducing the formation of the nascent autophagosome. 1388  

(C) Tax induces the formation of autophagosomes by forming a complex with 1389  

Beclin-1, Bif-1 and the IKK complex on lipid rafts. Alternatively, ATM 1390  

mediated activation of AMPK and/ Bcl-3 might bypass this requirement. 1391  

The recruitment of Beclin-1 and Bif-1 to lipid rafts by Tax might also inhibit 1392  

 

 

the fusion of the mature autophagosome with the lysosome and activate a 1393  

complex consisting of Rubicon and VPS34, inhibiting the fusion of the 1394  

autophagosome with the lysosome. 1395  

(D) HTLV-1 Tax increases the expression of cFLIP, thus inhibiting TRAIL/ 1396  

CD95L induced signalling pathways by inhibiting the activation of caspase- 1397  

8. cFLIP might also be stabilised by Tax via the accumulation of deace- 1398  

tylated Ku70 by inhibiting p300/CBP and/or P/CAF dependent acetylation 1399  

of Ku70. 1400  

1401  Figure 3: 1402  

(A) The DNA damage response is initiated by binding of sensor proteins to 1403  

sites of DNA damage and transduced via ATR/ATM/DNA-PK mediated 1404  

signalling. Potential outcomes are the activation of checkpoints, DNA 1405  

repair, or senescence. Cells with unrepaired DNA lesions might either 1406  

undergo apoptosis, necrosis, and autophagy related apoptosis/autosis. 1407  

Alternatively, cells might survive by PARP-1 and TSC-2 induced 1408  

autophagy. 1409  

(B) HTLV-1 Tax and the DNA damage response and the induction of 1410  

autophagosome formation. DNA damage is induced by the induction of 1411  

ROS and hyperreplication of cellular DNA due to increased expression of 1412  

PCNA. The DNA damage response is attenuated by Tax at various 1413  

stages, mainly by the increased expression of Wip-1 but also by 1414  

downregulating Ku70. Autophagosome formation however might be 1415  

 

 

induced due to activation of ATM and AMPK, whereas autophagic flux is 1416  

inhibited. 1417  

(C) In addition to HR mediated repair, tax might also interfere with both A- and 1418  

C-NHEJ mediated repair of DNA damage 1419  

Figure 4: 1420  

(A) Deacetylated Ku70 might translocate into the nucleus where it binds 1421  

SMAR-1 which following binding Ku70 is phosphorylated by ATM, thus 1422  

inducing the recruitment of downstream repair factors. The induced 1423  

expression of WIP-1 however might prevent the phosphorylation of 1424  

SMAR-1, thus dissociating the Ku70/SMAR-1 complex and inhibiting DNA 1425  

repair. 1426  

(B) Nuclear Ku70 might also associate with Ku80 in micronuclei, inducing C-1427  

NHEJ, which might be inhibited by Wip-1, and leading to the accumulation 1428  

of micronuclei. 1429  

(C) Summary of the pathways governing the induction of autophagosomes in 1430  

response to the induction of DNA damage by Tax and potential points of 1431  

attenuation by Tax. 1432  

Figure 5: 1433  

Interaction of HTLV-1 with cytoplasmic and/or nuclear p300/CBP might prevent 1434  

the acetylation of LC3-I and facilitating the localization of LC3-I in the cytoplasm, 1435  

inducing the formation of autophagosomes. 1436  

 

 

(A) Acetylated LC3-I is imported into the nucleus and upon starvation 1437  

deacetylated by SIRT1. Deacetylated LC3-I is exported from the nucleus 1438  

in a DOR dependent manner and binds ATG7 (modified from (Huang et 1439  

al., 2015). 1440  

(B) HTLV-1 might sequester p300/CBP in the perinuclear region and thus 1441  

prevent nuclear import of LC3-I, facilitating the formation of 1442  

autophagosomes independent of external stimuli, whereas sequestration 1443  

of nuclear p300 facilitates the export of deacetylated LC3-I independent of 1444  

SIRT1 1445  

1446  

1447  

1448  

1449  

1450  

1451  

1452  

1453  

1454  

1455  

1456  

1457  

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1460  

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1465  

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Figure 1 1474  

1475  

 

 

1476  

1477  

1478  

Figure 2 1479  

 

 

1480  

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Figure 3 1483  

1484  

 

 

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1488  

Figure 4 1489  

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1497  

1498  

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1500  

1501  

Figure 5 1502  

 

 

Supplemental Figure legends 1503  

Figure 1: 1504  

Inhibition of autophagy and the regulation of apoptosis 1505  

(A) Inhibition of autophagy upregulates Bik, inducing caspase 1506  

dependent apoptosis. 1507  

(B) and (C) Inhibition of autophagic flux by HTLV-1 Tax might 1508  

upregulate Bik and thus activating caspase-3/-9 and thus HTLV-1 1509  

LTR dependent expression of viral genes whilst apoptosis is 1510  

inhibited due to the stabilization of mitochondrial Mcl-1. Increasing 1511  

the expression of cFLIP and subsequent inhibition of autophago- 1512  

some maturation might favor apoptosis by excessive expression of 1513  

Bik. 1514  

Figure 2: 1515  

HTLV-1 Tax and degradation of phosphorylated Chk-1 1516  

The induction of ROS by HTLV-1 Tax due to hyperreplication of DNA 1517  

induces the ATR pathway of the DDR and might promote the degra- 1518  

dation of phosphorylated Chk-1 via chaperone mediated autophagy (II) 1519  

but not macroautophagy (I). 1520  

1521  

1522  

1523  

1524  

 

 

1525  

1526  

1527  

1528  

A

B

 

 

1529  

1530  

1531  

1532  

1533  

1534  

Supplemental Figure 1 1535  

1536  

1537  

C  

 

 

1538  

1539  

1540  

1541  

Supplemental Figure 2 1542  

1543  

1544