Virology tidbits

Thursday, 5 February 2015

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.

Sunday, 1 February 2015

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^Kip1levels 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^Kip1induced 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.