HTLV-1 Tax, NF-κB and autophagy

Human T-Cell Leukaemia/Lymphoma Virus type-1 (HTLV-1) is a member of the deltaretrovirus family which also include Bovine Leukaemia Virus (BLV) and Simian T-Lymphotropic Viruses (STLVs) and was the first human retrovirus to be associated with the formation of tumours in humans, being the etiologic agent of Adult T-Cell Leukaemia/Lymphoma (ATL/L; commonly referred to as ATL) as well as a chronic neuroinflammatory disease, HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP).  Worldwide an estimated number of 10 to 20 million individuals are infected with HTLV-1, with a high prevalence in Japan and the Caribbean, out of which approx. 1-5%  develop ATL during their lifetime usually late in life following a period of persistence which can be up to 20 years. HTLV-1 infects a wide variety of cells, including CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, macrophages, and fibroblasts thus reflecting the ubiquitous expression pattern of its hypothesised receptors (glucose transporter 1, heparan sulfate and proteoglycans and neuropilin-1), although primarily CD4+ T lymphocytes are targeted.

HTLV-1 genome

The key protein involved in the malignant transformation of CD4+ T lymphocytes is the viral Transactivator from the X-gene region (Tax) protein, which activates the expression of viral proteins via the viral cyclic-AMP-responsive promoter located within the HTLV-1 LTR region by binding the cellular transcription factor CREB as well as recruiting the cellular Histone Acetyltransferases p300 and P/CAF via its N-terminus as well as TATA-box-bound TBP protein via the C-terminus, thus enabling  the initiation of transcriptional and RNA polymerase elongation. It should be noted that Tax however does not exhibit any DNA binding activity. 

As described below, the expression of Tax modulates the cell cycle, induces the DNA damage response, autophagy and other processes, with the activation of Nuclear Factor-kB (NF-κB) pathway being central. The prevailing model involves the binding of Tax to IKKγ/NEMO in the cytoplasm thereby activating  IKKα/IKKβ/IKKγ, inducing the phosphorylation (and proteasomal degradation) of IKKα and IKKβ and subsequent nuclear localisation of NF-κB. Following the nuclear translocation of both NF-κB/Rel family members, nuclear Tax associates with c-Rel, RelA, p50 and p52, thereby increasing their dimerisation which is necessary to bind to promoters, both viral and cellular, with p300/CBP and P/CAF being co-activators. Tax mediated nuclear translocation of members of the NF-κB/Rel family therefore facilities the expression of both viral and cellular genes, and indeed hyperactivation of NF-κB is required for Tax induced malignant transformation of cells both in vitro and in vivo. 

Tax and Autophagy: inhibition v. activation

Jurkat cells which have been infected with HTLV-1 by co-culturing them with a HTLV-1 transformed cell line, MT-2, exhibit an increase in NF-κB activity as measured by the luciferase activity following the transfection with a pNF-κB-luc reporter plasmid as expected, similar to a Tax-positive Jurkat-TaxP cell line stably expressing Tax. Upon examination of the changes in gene expression in the latter using Microarray analysis, approx. 48 genes exhibited a change in gene expression of at least twofold with 10 genes associated with oncogenesis, differentiation, proliferation, cell development being unregulated and 38 genes down regulated in the presence of Tax.  Interestingly one of the genes, Bcl-3, was upregulated not only in Jurkat-TaxP cells but also significantly in MT-2 cells, accompanied by nuclear translocation of Bcl-3 in Jurkat-TaxP. These results indicate that Tax not only induces the translocation of NF-κB but also the expression and nuclear translocation of Bcl-3, which is a negative inhibitor of NF-κB. Furthermore, Bcl-3 also binds Tax and inhibits Tax mediated transcriptional activation of the viral LTR independent of the ability of Tax to activate NF-κB. Also, IKK induced phosphorylation of AMPK and subsequent inactivation of mTOR induces the formation of autophagosomes thus explaining the inability of TaxM22 to induce the formation of autophagosomes since TaxM22 can not bind IKK.

Regarding autophagy, the overexpression of Bcl-3 in HeLa cells co-transfected with Tax induces the formation of autophagosomes following Rapamycin or Pifithrin-α treatment as well as in mock treated cells whereas under starvation the formation of autophagosomes decreased for unknown reasons. The formation of autophagosomes also increases in MT2 and MT4 cells treated with an inhibitor of NF-κB, Bay 11-7082, that inhibits IκBα phosphorylation, suggesting that the inhibition of the NF-κB signalling pathway is sufficient to induce autophagosome formation.  Interestingly the expression of a Tax mutant, TaxM22, incapable of inducing the activation of NF-κB also fails to induce the formation of autophagosome, indicating that inducing the expression of Bcl-3 via NF-κB activation is necessary for the induction of autophagy, whereas the expression of TaxM47 not only activates NF-κB but also increases the formation of autophagosomes similar to wt Tax.
In addition to Bcl-3, Tax mediated activation of the IKKβ kinase complex has been postulated to activate autophagy by recruiting a complex containing Beclin-1 and Bif-1 to lipid raft microdomains. In this model, Tax interacts with both complexes directly, thereby promoting the assembly of LC3-II positive autophagosomes.

Increasing the formation of autophagosomes has been shown to increase the replication of HTLV-1 in HEK-293T cells transfected  with a HTLV-1 molecular clone (K30) whilst the expression of Tax in HeLa and Jurkat cells as well as in other established T cell lines such as PTX4-1 or PL9-1 not only increases the formation of autophagosomes as measured by the increase in LC3-II but at the same time also inhibiting the fusion with the lysosome as indicated by the accumulation of Neutrophil Cytosolic Factor 4(NCF)/p40phox, which -as discussed for positive strand RNA viruses before - is a common principle in cells expressing viral proteins interfering with the autophagy pathway. Paradoxically, both in HEK-293T cells transfected with the HTLV-1 molecular clone as well as in MT1, MT2, and HUT102 cells the fusion of the autophagosome with the lysosome is not inhibited, suggesting that that in the context of the presence of other viral proteins autophagic flux is functional; it should be noted that in 50% of ATL patients, HTLV-1 infected T lymphocytes do not express Tax as a result of the accumulation of mutations which render Tax inactive.

 NF-ϰB  dependent and independent signalling pathways

Blocking the fusion step using Bafilomycin-A, Chloroquine or siLAMP2 however increases the production of infectious HTLV-1 by approx. 3-fold as measured by p19 ELISA, suggesting that autophagy is part of the antiviral response. Inhibiting autophagy by the application of PI3K class III inhibitors or transfection of siBeclin-1 as well as shTax indeed resulted in impaired proliferation of HTLV-1 infected SLB1 and MT2 cells as well as a decrease in LC3-II levels. One mechanism of Tax induced blocking of the fusion might be by sequestering Beclin-1 to lipid rafts. The expression of a Tax mutant not capable of binding Beclin-1 or overexpression of Beclin-1 therefore might allow the formation of the autolysosome. Unfortunately, such a mutant would also fail to prevent the induction of autophagosome formation. An alternative might be the overexpression of a Beclin-1 mutant unable to bind Tax providing that such a mutant does not prevent the formation of the phagophore.

Tax induced autophagy has also been implicated in the degradation of phosphorylated IKKβ. Therefore it might be possible that the formation of autophagosomes via direct recruitment of Beclin-1 and Bif-1 is necessary in order to activate NF-κB early infection, whereas later in infection the inactivation of NF-κB mediated signalling is necessary for persistence and that the formation of the autophagosome indeed is beneficial. Unfortunately, so far it has not been shown if the induction of autophagy by Bcl-3 is accompanied by an increase in autophagic flux as measured by the degradation of p62/SQSTM-1. It should be noted however that cytosolic -but not nuclear- HTLV-1 Tax interacts with HDAC-6 and prevents the formation of stress granules. Since the deacetylation of microtubuli by HDAC-6 has been shown to be required for the fusion of autophagosomes with lysosomes, the expression of Tax-1 (at least the  cytosolic form) might therefore increase the clearance of stress granules by autophagy induced via the recruitment of Beclin-1 to lipid rafts as well as facilitating the fusion of the autophagosome with the lysosome via interaction with HDAC-6. In the opinion of the author of these lines, the observed decrease in autophagy in cells infected with HTLV-1 might therefore be the result of the decrease of cytosolic Tax-1 -and maybe even nuclear Tax-1 during infection. This again would favour a "biphasic" model in which Tax induces autophagy early in infection whilst inhibiting autophagy at later stages. 

Tax and autophagy: promotion of the formation of autophagosomes via
NF-ϰB dependent and independent pathways

HTLV-1 Tax and KSHV vFLIP: similarities and differences

Kaposi’s Sarcoma associated Herpesvirus (KSHV) or Human Herpesvirus 8 is a γ-Herpesvirus associated with two lymphoproliferative diseases, body cavity-based B-cell lymphoma/primary effusion lymphoma and multicentric Castleman’s disease. As discussed previously, the viral vCyclin-D and vFLIP proteins have to the ability to transform infected cells by promoting cell cycle progression whilst inhibition the induction of oncogene induced senescence (OIS) by way of inhibiting the formation of mature autophagosomes, in addition to NF-κB activation, the latter being reminiscent of Tax mediated activation of NF-κB. Both proteins have been shown to bind the regulatory subunits of I-κB kinases (IKK), NF-κB B essential modulator/IKKγ as well as activating IKKα and IKKβ, thus causing the activation of both the classical and alternative NF-κB signalling pathways. Indeed, KSHV vFLIP mediated activation of NF-κB has been demonstrated to be crucial for the survival of B-cell lymphoma/primary effusion lymphoma cells and the expression of Tax in mice and cell lines has been demonstrated to be required for the oncogenic activity of Tax. Similar to vCyclin, the expression of Tax induces senescence, which in this case is induced by (hyperactivated) p65/RelA and mediated by two Cyclin Kinase Inhibitors (CKI), p21^CIP1/WAF1 (p21) and p27^Kip1 (p27). Interestingly, similar to Tax, the expression of KSHV vFLIP in HeLa cells induces an arrest of the cell cycle in G1 phase in the absence of senescence concomitant with a decrease in I-κBα and an increase in p100, suggesting that both the classical and alternative NF-κB signalling pathway are activated, as well as an increase in both p21 and p27 levels. Co-expression of KSHV vCyclin however can abrogate vFLIP induced cell cycle arrest as well as Tax induced senescence probably by sequestering both p21 and p27 without affecting the ability of vFLIP and Tax to activate NF-κB dependent signalling. In contrast to Tax expressing HeLa cells however, cells transduced vFLIP in the presence of vCyclin do not present binucleation nor increased formation of micronuclei, which are commonly present in Tax expressing cells. Since the increase in both p21 and p27 is induced by activation of p65/RelA, the expression of ΔN-I-Kα (which cannot be degraded) as well as the transfection with shRelA prevents vFLIP induced increase of p21 and p27 and thus presumably vFLIP induced cell cycle arrest akin to cells expressing Tax.

In conclusion, both KSHV vFLIP and HTLV-1 Tax increase p21^CIP1/WAF1 and p27^Kip1 levels via hyperactivation of RelA/ NF-κB; in the case of HTLV-1 Tax this induces senescence whereas in the case of KSHV vFLIP cells are arrested in G1 phase of the cell cycle. In the case of HTLV-1 infected cells, senescence is inhibited by the accumulation of mutations within the cellular genome leading to a deregulation of cellular G1 Cyclin dependent kinases and CKI, whereas in KSHV infected cells the expression of the viral Cyclin inhibits p21CIP1^/WAF1 and p27^Kip1 induced cell cycle arrest by sequestering these proteins. It remains to be seen if the ability of Tax to induce the formation of mature autophagosomes is related to the induction of senescence and if inhibiting autophagy by e.g. by expression of the Atg3 binding domain of vFLIP or specific inhibitors prevents Tax induced senescence. Indeed, Tax induced overexpression of Bcl-3 might not only promote cell survival but also inhibit Tax induced senescence by inhibiting NF-κB. It is therefore interesting that the expression of KSHV vFLIP does not induce senescence - despite the hyperactivation of NF-κB. Future experiments might shed some light of the complex interactions between these oncogenic proteins, senescence, autophagy, and NF-κB.

Sequestering of Beclin-1 and Bif-1 by Tax: inhibition of fusion of the autophagosome
with the lysosome?

Tax not only induces the hyperactivation of NF-κB via facilitating the degradation of the IKK complex but also via attenuating the ATM dependent DNA damage response by sequestering DNA repair factors which can be inhibited by Bcl-3. Constitutive activation of ATM results in an intranuclear activation of NEMO and translocation of the ATM/NEMO complex to the cytoplasm where in subsequent steps the IKK complex is degraded and the p65/p50 complex is activated. It remains to be seen to which extent this pathway is responsible for Tax induced autophagy, in particular early in infection prior to the establishment of persistently infected cells as well as in cell transformation.

The expression of Tax has also been linked to the prevention of TRAIL induced apoptosis in U251 astroglioma cells by inducing the expression of c-FLIP, the cellular equivalent of KSHV vFLIP, as well as inducing the formation of autophagosomes. In this model, the expression of c-FLIP via Tax prevents the cleavage of Beclin-1 by preventing the cleavage of Caspase-9/-3. Tax therefore not only promotes the formation of autophagosomes by sequestering Beclin-1 to lipid rafts and increasing the expression of Bcl-3 but also by preventing the activation of Caspase-3 via inducing c-FLIP expression. Interestingly, the expression of KSHV vFLIP also inhibits Caspase-3 cleavage, suggesting that the expression of KSHV vFLIP in Tax expressing cells depleted of cFLIP might increase the resistance of these cells to TRAIL induced apoptosis – hypothetically. 

Tax and autophagy: central role of NF-ϰB in inducing autophagy
and preventing apoptosis

One final question is if the expression of Tax also mediates the secretion of neuroinflammatory factors since autophagy has also been implicated in the secretion of IL1B (Interleukin 1-beta), CXCL8 (Chemokine (C-X-C motif) ligand 8), LIF (Leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (Dickkopf WNT signaling pathway inhibitor 3). In order to answer this question, not only is it necessary to characterise the secretome of Tax expressing cells, but also determine if Tax indeed increases autophagic flux. So far the data obtained from HeLa cells expressing Tax indicate that the fusion of mature autophagosomes with lysosomes is inhibited thus increasing the stability of Tax. Consequently, the induction of senescence in Tax expressing cells is independent of the induction of autophagy and thus different from cells expressing KSHV vCyclin. 

Further reading

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Kfoury Y, Nasr R, Journo C, Mahieux R, Pique C, & Bazarbachi A (2012). The multifaceted oncoprotein Tax: subcellular localization, posttranslational modifications, and NF-κB activation. Advances in cancer research, 113, 85-120 PMID: 22429853

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Morozov VA, & Weiss RA (1999). Two types of HTLV-1 particles are released from MT-2 cells. Virology, 255 (2), 279-84 PMID: 10069953

Wang J, Niu Z, Shi Y, Gao C, Wang X, Han J, Li J, Gao Z, Zhu X, Song X, Qin Z, & Wang H (2013). Bcl-3, induced by Tax and HTLV-1, inhibits NF-κB activation and promotes autophagy. Cellular signalling, 25 (12), 2797-804 PMID: 24044922 

Gao C, Wang X, Chen L, Wang JH, Gao ZT, & Wang H (2013). Knockdown of Bcl-3 inhibits cell growth and induces DNA damage in HTLV-1-infected cells. Asian Pacific journal of cancer prevention : APJCP, 14 (1), 405-8 PMID: 23534762 

Saito K, Saito M, Taniura N, Okuwa T, & Ohara Y (2010). Activation of the PI3K-Akt pathway by human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax increases Bcl3 expression, which is associated with enhanced growth of HTLV-1-infected T cells. Virology, 403 (2), 173-80 PMID: 20471052 

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