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NF-κB proteins in viral oncogenesis and the regulation of viral transcription
seminars in
CANCER BIOLOGY, Vol 8, 1997: pp 121–129
The role of Rel/NF-kB proteins in viral oncogenesis and the regulation of viral transcription George Mosialos
Rel/NF-kB is a ubiquitous transcription factor that consists of multiple polypeptide subunits, and is subject to complex regulatory mechanisms that involve protein–protein interactions, phosphorylation, ubiquitination, proteolytic degradation, and nucleocytoplasmic translocation. The sophisticated control of Rel/NF-kB activity is not surprising since this transcription factor is involved in a wide array of cellular responses to extracellular cues, associated with growth, development, apoptosis, and pathogen invasion. Thus, it is not unexpected that this versatile cellular homeostatic switch would be affected by a variety of viral pathogens, which have evolved mechanisms to utilize various aspects of Rel/NF-kB activity to facilitate their replication, cell survival and possibly evasion of immune responses. This review will cover the molecular mechanisms that are utilized by mammalian oncogenic viruses to affect the activity of Rel/ NF-kB transcription factors and the role of Rel/NF-kB in the regulation of viral gene expression and replication.
A causative role for Tax in cellular transformation is supported by a number of experimental findings. First, the tax gene of HTLV-I is always present in infected tumor cells, occasionally in the absence of other regions of the viral genome. Second, Tax expression can immortalize human T cells, a phenotype that is likely to predispose these cells to tumorigenic genetic alterations.2 Third, expression of Tax in rodent fibroblasts results in oncogenic transformation as manifested by loss of contact inhibition, anchorageindependent growth and tumorigenicity in nude mice.3,4 Tax is a 40 kDa polypeptide that shows predominantly nuclear but, to some extent, also cytoplasmic localization. Tax appears to deregulate cellular growth control mechanisms primarily by affecting transcription regulation, and the activities of several transcription factors, including cyclic-AMP responsive element binding protein (CREB), serum response factor (SRF) and NF-kB, are influenced by Tax.1 However, Tax does not have intrinsic DNA-binding activity and therefore it must utilize cellular factors as docking vehicles to gain access to specific enhancer and promoter elements. Tax does contain transcription activation elements, and it can activate transcription when brought to DNA by fusion to a heterologous DNA-binding domain.5,6 Tax has a pronounced stimulatory effect on the activity of Rel/NF-kB, which is characterized by an increase in the nuclear accumulation of certain transactivating complexes. These complexes include p50 or p52 heterodimers with RelA and/or c-Rel. There are two phases in Tax-mediated generation of nuclear Rel complexes.7 In the early phase Tax promotes the nuclear accumulation of p50/RelA complexes which is then followed by the appearance of p52/c-Rel dimers. The increase in nuclear localization of p50/RelA following Tax expression is not due to an increase in protein levels of these subunits. However, the late response to Tax expression is accompanied by an elevation in mRNA and protein levels of p52 and c-Rel, which is most likely due to the
Key words: Epstein–Barr virus / malignant transformation / NF-kB / signal transduction / Tax protein ©1997 Academic Press Ltd
Activation of NF-kB by the HTLV-I transforming protein Tax Human T-cell leukemia virus type I (HTLV-I) is a human retrovirus that is associated with the development of adult T-cell leukemia (ATL) and a neurological disorder known as HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP).1 The critical player in HTLV-I-mediated T-cell transformation is the Tax protein, which is encoded by the pX region of the viral genome. From the Infectious Disease Division, Brigham & Women’s Hospital, Channing Labs, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA ©1997 Academic Press Ltd 1044-579X/97/020121 + 09$25.00/0/se970063
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G. Mosialos presence of kB-responsive elements in their promoters. Therefore, it appears that Tax activates Rel/ NF-kB through both cytoplasmic and nuclear events. The molecular mechanisms of Tax-mediated NF-kBactivation can be classified as those involving NF-kB/ Rel-inhibitory molecules (IkB-α, IkB-β, IkB-γ, p105, p100) and those that are associated with transactivating nuclear forms of Rel/NF-kB factors (RelA, c-Rel) (see Figure 1). The initial effect of Tax on nuclear translocation of p50/RelA is mediated by phosphorylation and degradation of IkB-α.7,8 IkB-α is responsible for retention of the p50/RelA complex in the cytoplasm and it is the primary target of transient NF-kB-activating signaling mechanisms (see Whiteside and Isra¨el, this issue). After stimulation, the initial drop in IkB-α protein levels is counterbalanced by a rapid increase in its mRNA levels since the IkB-α promoter is activated by NF-kB. Although the steady-state levels of IkB-α in Tax-expressing or HTLV-I-infected cells are not reduced as compared to cells not expressing Tax, the turnover rate of IkB-α is substantially increased, and thus, a significant portion of cytoplasmic p50/RelA complexes may escape into the nucleus during these rapid cycles of IkB-α degradation and resynthesis.7,9 The early NF-kB-activating response to Tax expression has features in common with the signaling mechanism of phorbol esters in promoting phosphorylation and degradation of IkB-α. However, the kinetics of IkB-α degradation following Tax expression are much slower than those achieved by phorbol esters.7 At present, the mechanism of Tax-mediated IkB-α phosphorylation is unclear. It is conceivable that Tax directly or indirectly activates a protein kinase cascade that leads to IkB-α phosphorylation and degradation. One report has identified a low abundance complex containing both Tax and IkB-α, suggesting that the association of Tax with IkB-α destabilizes IkB-α and promotes nuclear localization of transactivating NF-kB subunits.10 Along these lines, Lindholm et al11 have shown that Tax-induced activation of NF-kB is sensitive to the protein kinase C (PKC) inhibitor calphostin C and that Tax expression leads to PKC autophosphorylation and membrane translocation. Although these data implicate PKC in a Tax pathway that leads to NF-kB activation, the overall contribution of PKC to NF-kB activation by Tax is a matter of debate since other reports have failed to detect an effect of PKC inhibitors on Tax-mediated activation of NF-kB.8 It is possible that a particular isoform of PKC plays a role in Tax-mediated activation of NF-kB in certain cell lines, but it is likely that other protein
kinases are also involved. For example, it will be interesting to test the possible involvement of Tax in MEKK1 kinase family activation, which has recently been linked to IkB-α phosphorylation and degradation.12 Tax expression also targets IkB-β for degradation.13,14 IkB-β is a potent inhibitor of complexes containing RelA or c-Rel, but unlike IkB-α, the IkB-β promoter is not responsive to NF-kB activation. As a result, the levels of IkB-β are dramatically reduced or eliminated in Tax-expressing cell lines, perhaps accounting for the appearance of c-Rel-containing nuclear complexes in the late phase of cellular response to Tax. Tax also binds to the p105 and p100 NF-kB precursors that are known to act as cytoplasmic anchors and inhibitors of NF-kB activity.15,19 Genetic analysis of Tax indicates that its association with p105 correlates with its ability to activate NF-kB-responsive promoters and suggests that Tax overrides the inhibitory role of p105 on transactivating Rel/NF-kB subunits. In fact, Tax overexpression can liberate RelA and c-Rel from cytoplasmic complexes with p105 and allow them to be transported to the nucleus. Tax may facilitate p105 degradation by linking it to the 20S proteasome subunit.20 The outcome of Tax binding to p100 is a matter of debate as some investigators have shown that Tax can overcome the inhibitory effect of p100 on Rel subunits,17,18 whereas others have failed to detect any substantial effect of Tax on the IkB activity of p100.16,19,21 It is conceivable that the binding of Tax to p105 and possibly p100 changes their conformation or occludes their Rel binding sites so that they can no longer bind to Rel proteins. There is no evidence that Tax directly accelerates the proteolytic processing of p105 and p100 to their mature p50 and p52 subunits. Tax also binds to the ankyrin repeats of IkB-γ and blocks its ability to retain RelA in the cytoplasm.22 However, the significance of this effect on the pathogenesis of HTLV-I-associated human diseases is questionable since IkB-γ has been detected only in mouse cells. The predominantly nuclear localization of Tax argues for a specific role in the nucleus. Moreover, several mutations in Tax that compromise its nuclear localization also reduce its transactivating potential towards NF-kB-dependent and -independent promotes.23 Tax can associate with RelA and c-Rel through the Rel homology domain (RHD).24 These interactions appear to be functionally significant since Tax can cooperate with RelA or c-Rel for the activation of NF-kB-responsive elements in F9 embryonic carcinoma cells. These experiments indicate that 122
Viruses and activation of NF-kB the cell-cycle inhibitor p16INK4a, indicating that Tax is capable of influencing cellular growth properties at multiple levels.28
the binding of Tax to Rel subunits in the nucleus potentiates their transactivating potential and highlight an additional level of NF-kB regulation by Tax. The activation of Rel/NF-kB factors by Tax results in the upregulation of several cellular proteins that are known to have growth promoting effects.1 These include cytokines and growth factors (such as interleukin-6, tumor necrosis factor α and β, granulocytemacrophage colony stimulating factor, granulocyte colony stimulating factor), cytokine receptors (such as interleukin-2 receptor α), and proto-oncogenes (such as c-Myc). Cytokines and cytokine receptors are likely to participate in autocrine or paracrine routes of cellular activation that predispose infected cells to subsequent tumorigenic alterations. The role of Tax in the development of human T-cell leukemias is likely to be critical in the early stages of malignancy, whereas Tax may be dispensable in established ATL. This is supported by the fact that the levels of Tax protein are often low or undetectable in HTLV-I-infected ATL cells. In addition, the long latency for the development of this malignancy indicates that factors in addition to Tax contribute to malignant transformation. Nevertheless, in an elegant study where viral replication was separated from Tax activity it was demonstrated that Tax is absolutely essential for growth transformation of primary human T-lymphocytes by HTLV in vitro.25 In contrast, Tax appears to be dispensable for the long-term growth of transformed rodent fibroblast tumor cell lines that were generated by Tax expression in transgenic mice.26 Interestingly, elevated levels of NF-kB activity in these Tax-generated tumors were absolutely necessary for the continued growth of the tumor cells both in vitro and in vivo.26 On the other hand, mutational analysis of Tax has generated controversial results on the importance of NF-kB activation for Tax-mediated rodent fibroblast transformation.4,27 In-vitro transformation of rodent fibroblasts has provided invaluable information on the transforming properties of a number of human oncoproteins. However, it will be essential to perform similar genetic analyses of Tax function in human T-cell transformation in vitro in order to understand better the molecular events that underlie the development of HTLV-I-associated human malignancies. Overall it appears that activation of NF-kB by Tax is essential for upregulation of cellular activation and proliferation markers that are likely to set the stage for the disruption of cell-cycle checkpoints and the development of malignancies. Interestingly, it has recently been shown that Tax can target and inactivate
The role of NF-kB activation in B-lymphocyte growth transformation by Epstein–Barr virus Epstein–Barr virus (EBV) is a human herpes virus that causes infectious mononucleosis, a self-limiting lymphoproliferative disease.29 EBV is also associated with a number of human malignancies, such as nasopharyngeal carcinoma, African Burkitt’s lymphoma, Hodgkin’s disease, AIDS-associated central nervous system lymphomas and lymphoproliferative syndromes developed in organ transplant recipients.29 EBV targets primarily two cell types, epithelial cells and B lymphocytes. Genetic analysis of the EBV genome has identified six latent gene products that have an essential role in the process of B-lymphocyte transformation.30 For the purpose of this review, I will focus primarily on the function of latent membrane protein 1 (LMP1) which plays a critical role in B-cell growth transformation by EBV.31 LMP1 is an integral membrane protein that consists of a 24-amino-acid N-terminal cytoplasmic domain, six transmembrane domains connected by short reverse turns and a 200-amino acid cytoplasmic C terminus (CCT, see Figure 2A). LMP1 is expressed in most EBV-associated nasopharyngeal carcinomas and Hodgkin’s lymphomas.29 In rodent fibroblast cell lines LMP1 causes anchorage- and growth factorindependent growth, loss of contact inhibition and nude mouse tumorigenicity.32,33 In epithelial cells in culture or in transgenic mice LMP1 expression prevents differentiation.34-36 LMP1 induces the expression of many activation markers (CD23, CD30, CD39, CD40) and cell adhesion molecules (LFA1, ICAM1, LFA3) that are also upregulated in EBVtransformed B lymphocytes.37 LMP1 also upregulates the expression of anti-apoptotic factors, such as A20 and Bcl-2.38,39 Many of the cellular phenotypic changes caused by LMP1 are mediated at the transcriptional level. NF-kB is one of the cellular transcription factors that is activated by LMP1. The LMP1 domains that mediate NF-kB activation were characterized using standard molecular genetic approaches.40,41 Two NF-kB-activating domains were mapped to the LMP1 CCT. One domain (CTAR1 for carboxyl-terminal activating region 1, see Figure 2A) is located within the membrane proximal 45 amino acids of the LMP1 123
G. Mosialos CCT (amino acids 187–231) and accounts for approximately 20 to 30% of the LMP1-induced NF-kB activation. The second NF-kB-activating domain (CTAR2, see Figure 2A) mediates approximately 70 to
80% of the activating effect and is located within the last 55 amino acids of the LMP1 CCT (amino acids 332–386). Recombinant EBV genetic analysis has demonstrated that CTAR1 plays an essential role in
Figure 1. Molecular mechanisms of Rel/NF-kB activation by HTLV-I Tax and hepatitis B HBx proteins. Tax targets Rel complexes both in the cytoplasm and in the nucleus. In the cytoplasm Tax activates signaling pathways that lead to IkB-α and IkB-β (IkB) phosphorylation (P) and degradation. Tax can associate in the cytoplasm with IkB molecules such as IkB-α, p105 and p100. These interactions may prevent the inhibitory activity of IkB molecules on Rel-containing complexes, which are then freed to enter the nucleus and activate NF-kB -responsive (kB) promoters (PRO). Tax may act as a linker between proteasome subunits (20SP) 20 and certain IkB molecules, thus facilitating their degradation. In the nucleus Tax can associate with Rel subunits (Rel) such as RelA and c-Rel and potentiate their transactivating properties. The hepatitis B virus protein HBx can target IkB-α for degradation through signaling pathways that lead to Ras and protein kinase C (PKC) activation.
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Viruses and activation of NF-kB formation by EBV.42 Second, a recombinant EBV with CTAR1 fused to the transmembrane domains of LMP1 (amino acids 1–231) can induce B-lymphocyte transformation in the absence of the rest of the LMP1 CCT; however, the transforming potential of this recombinant protein is weaker than full-length LMP1.43 The transmembrane domains of LMP1 are critical for B-lymphocyte transformation and NF-kB activation because they enable LMP1 to aggregate in tight plasma membrane patches.31 The functional importance of the LMP1 CCT for cell transformation prompted a yeast two-hybrid screen to identify cellular factors that participate in the LMP1 signal transduction process. This genetic approach as well as biochemical studies revealed that LMP1 associates through CTAR1 with members of the tumor necrosis factor receptor associated factors (TRAF).44-46 TRAF1, TRAF3 and to a lesser extent TRAF2 constitutively associate with the CTAR1 domain of LMP1 in vitro and in EBV-transformed B lymphocytes. LMP1 expression dramatically alters the subcellular distribution of TRAF proteins from diffuse cytoplasmic to plasma-membrane aggregates that colocalize with LMP1. Mutational analysis of the LMP1 TRAF-binding site and transient overexpression of wild-type and mutant TRAFs indicate that TRAF1 and TRAF2 mediate NF-kB activation by the LMP1 CTAR1 domain, whereas TRAF3 inhibits NF-kB-activation by CTAR1.45 It appears that liganddependent association of TRAF molecules with the cytoplasmic tail of TNF receptors forms the core of a signaling complex that associates subsequently with other effector molecules that propagate signaling (Figure 2B).47,48 On the other hand, LMP1 appears to function as a constitutively crosslinked and active TNF receptor that propagates ligand-independent signaling in part through TRAF proteins (Figure 2A). A candidate TRAF2-associated effector molecule was recently identified as the protein kinase NIK, which is a homologue of protein kinase MEKK.49 Overexpression of NIK activates NF-kB and dominant-negative mutants of NIK block TRAF2-mediated activation of NF-kB. The similar role of TRAF2 and TRAF3 in LMP1 and CD40 signaling and the common phenotypic and growth promoting effects of LMP1 and CD40 on B lymphocytes strongly suggest that LMP1 acts as a constitutively active CD40 molecule (Figure 2). None of the known TRAFs binds directly to CTAR2 and the mechanism of NF-kB activation by this particular domain of LMP1 is unclear. LMP1 activates NF-kB in B-lymphocytes by inducing a rapid turnover of IkB-α.50 LMP1 expression in both epithelial cells
primary B-lymphocyte growth transformation in vitro. First, deletion of a core functional sequence of CTAR1 (amino acids 185–211) abrogates B-lymphocyte trans-
Figure 2. NF-kB activation by the EBV transforming protein LMP1 (A) and CD40 (B). LMP1 consists of a short 24-amino acid cytoplasmic N terminus (N), six transmembrane domains (vertical cylinders), and a 200-amino acid cytoplasmic C terminus (CCT). The LMP1 CCT contains two NF-kB -activating subdomains (CTAR1 and CTAR2). The membrane proximal NF-kB -activating domain (CTAR1) accounts for approximately 30% of NF-kB activation and interacts with TRAF1, TRAF2 and TRAF3. The second NF-kB -activating domain (CTAR2) accounts for 70% of the total NF-kB activation mediated by LMP1 and engages signaling pathways that are unclear at present. TRAF1 and TRAF2 form heterodimers and mediate NF-kB activation from CTAR1. TRAF3 inhibits NF-kB activation from CTAR1 possibly by competing with TRAF1 and TRAF2 for binding to this domain. Plasma membrane aggregation of TRAF1 and TRAF2 by LMP1 probably recruits effector molecules (X) that eventually lead to the activation of an IkB-α kinase, IkB-α degradation and nuclear translocation of NF-kB.
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G. Mosialos ing AP-1, AP-2, c/EBP, NF-kB and RNA polymerase III transcription factors. In this section, I will focus on the mechanism of NF-kB activation by HBx. HBx shows both cytoplasmic and nuclear localization. There is evidence that activation of NF-kB by HBx requires a cytoplasmic signaling event since exclusive nuclear localization of HBx compromises its ability to activate NF-kB.56 It has been shown that HBx activates the Ras pathway by increasing the abundance of the GTP-bound form of Ras57 (Figure 1). Activation of Ras is implicated in HBx-mediated NF-kB activation since a dominant-negative mutant of Ras has an inhibitory role in this process, at least in certain cell lines.58 Another pathway that has been associated with HBx-mediated activation of NF-kB involves PKC since inhibition or downregulation of PKC can prevent NF-kB activation by HBx59 (Figure 1). It should be noted, however, that Ras and PKC may not be universally involved in NF-kB activation by HBx since their involvement could not be demonstrated in other cell lines, albeit under somewhat different experimental conditions.60 HBx expression induces degradation of IkB-α and targets p105 for proteolytic processing.58 The molecular mechanisms behind these processes are unclear at present although the ability of HBx to activate the MAP kinase kinase (MEKK-1) may be linked to IkB-α degradation.57,12 These events lead to increased nuclear localization of RelA and c-Rel complexes with p50 and p52 and transactivation of NF-kB-responsive promoters.58 There is no evidence for direct association of HBx with Rel/NF-kB proteins. The lack of a detailed genetic analysis of the functional properties of HBx does not allow an assessment of the role of NF-kB activation in the tumorigenic potential of HBx. However, it is likely that induction of NF-kB-responsive genes by HBx contributes to the establishment of an activated cellular phenotype that can be the prelude in tumor development. HBx, like Tax and LMP1, is likely to play a role in tumor promotion rather than being sufficient for the establishment of the malignant phenotype given the long latency that precedes the development of hepatocellular carcinoma in cases of chronic HBV infection.
and B lymphocytes induces nuclear accumulation of RelA complexes that also contain p50 and/or p52.50,51 Interestingly c-Rel has not been implicated in LMP1mediated NF-kB activation. The critical role of the LMP1 CTAR1 domain in B-lymphocyte transformation suggests that NF-kB activation through TRAF molecules is an essential signaling event for the immortalization process. NF-kB activation by LMP1 is not sufficient for B-cell transformation since an EBV mutant that lacks a functional LMP1 CTAR1 domain is non-transforming but still capable for potent NF-kB activation through CTAR2.42 However, it is possible that CTAR1 and CTAR2 activate different subsets of NF-kB-responsive genes due to different effects on neighboring promoter elements. NF-kB is clearly important for certain growth-phenotypic changes mediated by LMP1 since it is involved in the upregulation of certain LMP1induced genes such as A20 and ICAM1.39,40 LMP1mediated induction of growth factor receptors and antiapoptotic factors is likely to increase the proliferative capacity of EBV-infected cells and thus predispose them to tumorigenic events. In this respect, LMP1 is similar to HTLV-I Tax in having a significant role in the early stages of tumor development although it may be dispensable in well-established malignancies.52 EBNA2 is a second EBV latent protein that can activate NF-kB. EBNA2 is essential for EBV-mediated B-cell growth transformation, but unlike LMP1, it is a nuclear protein and affects transcription directly through a potent transactivating domain in its C terminus. EBNA2 transactivates the human immunodeficiency virus (HIV) long terminal repeat through the induction of NF-kB activity.53 However, the molecular mechanism through which EBNA2 mediates activation of NF-kB is currently unknown.
Activation of NF-kB by the hepatitis B virus protein HBx HBx is a 17 kDa polypeptide encoded by mammalian hepatitis B viruses (HBV). Chronic HBV infection is associated with the development of hepatocellular carcinoma and HBx appears to be involved in the process of tumorigenesis. High levels of HBx expression in transgenic mice lead to a dramatic increase in the incidence of liver tumors and HBx can induce cellular transformation in certain assays.54,55 HBx has a broad effect on the cellular transcription machinery by activating a number of transcription factors includ-
The role of NF-kB/Rel proteins in viral gene expression and replication In the previous sections, emphasis was placed on the effects of viral activation of NF-kB on cellular gene 126
Viruses and activation of NF-kB
Conclusions
expression and relevant phenotypic changes. However, NF-kB also plays a crucial role in the control of viral gene expression and replication. NF-kB has been implicated in the control of gene expression of retroviruses (HIV),61 adenoviruses,62 papova viruses (JC virus)63 and herpes viruses (HSV-1, CMV).64,65 Two broadly-studied examples of the involvement of NF-kB in the regulation of viral gene expression are the retrovirus human immunodeficiency virus type 1 (HIV-1) and the herpes virus cytomegalovirus (CMV). Replication of HIV-1 requires the generation of genomic RNA from viral DNA that is integrated into the host genome as the provirus.66 The HIV proviral enhancer located in the U3 region of the long terminal repeat (LTR) contains two tandem NF-kB binding sites,66 and NF-kB plays an important role in transcription regulation of the HIV-1 LTR.61 Other transcription factors, such as Sp1, have also been implicated in the HIV-LTR transactivation. Interestingly it has been demonstrated that Rel/NF-kB proteins interact and cooperate with Sp1 for optimal transactivation of the HIV-1 LTR.67 Although the NF-kB sites in the HIV-1 LTR may not be absolutely required for virus replication in T lymphocytes, they clearly modulate replication efficiency and offer alternative routes of gene expression that can complement defects in other transcription factors.68,69 The presence of functional NF-kB sites in the HIV-1 enhancer renders the virus responsive to the plethora of extracellular stimuli that target NF-kB, including cytokines, bacterial lipopolysacharides, oxidative stress and other viruses that activate NF-kB. All of these agents can affect the outcome of HIV-1 infection and progression to AIDS, and it appears that Rel/ NF-kB proteins control an important regulatory switch in this process. Human cytomegalovirus (HCMV) has developed mechanisms for NF-kB activation and utilizes NF-kB for the expression of its immediate early (IE) genes. Activation of NF-kB by HCMV occurs in two phases.70 The early phase does not depend on protein synthesis and appears to be mediated simply by virus absorption onto the host cell. The second phase involves the upregulation of p105 and RelA promoters and an increase in the levels of nuclear p50/RelA complex. The CMV IE promoter is responsive to NF-kB and the IE gene product IE1 has been implicated in the induction of NF-kB activity.64,70 Therefore, an NF-kBdependent autoregulatory loop has been developed that is critically important for the initiation of a productive HCMV infection.
A number of mammalian oncogenic viruses have targeted transcription factor NF-kB for activation through mechanisms that involve cytoplasmic inactivation of inhibitory IkB molecules and also potentiation of the transactivating properties of nuclear Rel subunits. NF-kB was clearly a sensible choice because of its involvement in the induction of growth factors, cytokines, cytokine receptors, cell adhesion molecules and proto-oncogenes, all of which can promote active engagement of the cell cycle and possible extend he life span of the host cell. NF-kB has also recently been shown to play an active role in counteracting apoptotic signals and promoting cell survival (see Sonenshein, this issue). This property is particularly beneficial for a viral pathogen since apoptotic cell death is often triggered by infection-associated cellular stress and immune responses. Finally, the ability of NF-kB to respond to a broad spectrum of extracellular signals has been used cleverly by certain viruses to provide a sensor for the environment that regulates viral gene expression and replication.
Acknowledgements I am grateful to Dr Elliott Kieff and the members of his laboratory for invaluable help and support. My research has been supported by the Leukemia Society of America and the National Cancer Institute.
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proteins bound to the NF-kB binding site and activates transcription. Oncogene 9:3099-3105 Ross TM, Pettiford SM, Green PL (1996) The tax gene of human T-cell leukemia virus type 2 is essential for transformation of human T lymphocytes. J Virol 70:5194-5202 Kitajima I, Shinohara T, Bilakovics J, Brown DA, Xu X, Nerenberg M (1992) Ablation of transplanted HTLV-I Taxtransformed tumors in mice by antisense inhibition of NF-kB. Science 258:1792-1795 Yamaoka S, Inoue H, Sakurai M, Sugiyama T, Hazama M, Yamada T, Hatanaka M (1996) Constitutive activation of NF-kB is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. EMBO J 15:873-887 Low KG, Dorner LF, Fernando DB, Grossman J, Jeang KT, Comb MJ (1997) Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4α. J Virol 71:1956-1962 Rickinson AB, Kieff E (1996) Epstein-Barr virus, in Virology (Fields BN, Knipe DM, Howley PM, eds) pp 2397–2446. Raven Press, New York Kieff E (1996) Epstein-Barr virus and its replication, in Virology (Fields BN, Knipe DM, Howley PM, eds) pp 2343–2496. Raven Press, New York Kaye KM, Izumi KM, Kieff E (1993) Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc Natl Acad Sci USA 90:9150-9154 Wang D, Liebowitz D, Kieff E (1985) An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43:831-840 Baichwal VR, Sugden B (1988) Transformation of Balb/3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene 4:67-74 Dawson CW, Rickinson AB, Young LS (1990) Epstein-Barr virus latent membrane protein inhibits human epithelial cell differentiation. Nature 344:777-780 Fahraeus R, Rymo L, Rhim JS, Klein G (1990) Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus. Nature 345:447-449 Wilson JB, Weinberg W, Johnson R, Yuspa S, Levine AJ (1990) Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell 61:1315-1327 Wang F, Gregory C, Sample C, Rowe M, Liebowitz D, Murray R, Rickinson A, Kieff E (1990) Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J. Virol 64:2309-2318 Henderson S, Rowe M, Gregory C, Croom-Carter D, Wang F, Longnecker R, Kieff E, Rickinson A (1991) Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65:1107-1115 Laherty CD, Hu HM, Opipari AW, Wang F, Dixit VM (1992) The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kB. J Biol Chem 267:24157-24160 Huen DS, Henderson SA, Croom-Carter D, Rowe M (1995) The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-kB and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene 10:549-560 Mitchell T, Sugden B (1995) Stimulation of NF-kB-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J Virol 69:2968-2976 Izumi KM, Kaye KM, Kieff ED (1997) The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation. Proc Natl Acad Sci USA 94:1447-1452
Viruses and activation of NF-kB 43. Kaye KM, Izumi KM, Mosialos G, Kieff E (1995) The EpsteinBarr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues. J Virol 69:675-683 44. Mosialos G, Birkenbach M, Yalamanchili R, VanArsdale T, Ware C, Kieff E (1995) The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80:389-399 45. Devergne O, Hatzivassiliou E, Izumi KM, Kaye KM, Kleijnen MF, Kieff E, Mosialos G (1996) Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kB activation. Mol Cell Biol 16:7098-7108 46. Kaye KM, Devergne O, Harada JN, Izumi KM, Yalamanchili R, Kieff E, Mosialos G (1996) Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kB activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein. Proc Natl Acad Sci USA 93:11085-11090 47. Shu HB, Takeuchi M, Goeddel DV (1996) The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc Natl Acad Sci USA 93:13973-13978 48. VanArsdale TL, VanArsdale SL, Force WR, Walter BN, Mosialos G, Kieff E, Reed JC, Ware CF (1997) Lymphotoxin-β receptor signaling complex: the role of TRAF3 recruitment in cell death and activation of nuclear factor kB. Proc Natl Acad Sci USA 94:2460-2465 49. Malinin NL, Boldin MP, Kovalenko AV, Wallach D (1997) MAP3K-related kinase involved in NF-kB induction by TNF, CD95 and IL-1. Nature 385:540-544 50. Herrero JA, Mathew P, Paya CV (1995) LMP-1 activates NF-kB by targeting the inhibitory molecule IkBα. J Virol 69:2168-2174 51. Paine E, Scheinman RI, Baldwin AS Jr, Raab-Traub N (1995) Expression of LMP1 in epithelial cells leads to the activation of a select subset of NF-kB/Rel family proteins. J Virol 69:4572-4576 52. Pathmanathan R, Prasad U, Sadler R, Flynn K, Raab-Traub N (1995) Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med 333:693-698 53. Scala G, Quinto I, Ruocco MR, Mallardo M, Ambrosino C, Squitieri B, Tassone P, Venuta S (1993) Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1. J Virol 67:2853-2861 54. Shirakata Y, Kawada M, Fujiki Y, Sano H, Oda M, Yaginuma K, Kobayashi M, Koike K (1989) The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells. Jpn J Cancer Res 80:617-621 55. Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T, Kurokawa K (1994) High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19:810-819 56. Doria M, Klein N, Lucito R, Schneider RJ (1995) The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of
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Ras and nuclear activator of transcription factors. EMBO J 14:4747-4757 Benn J, Schneider RJ (1994) Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci USA 91:10350-10354 Su F, Schneider RJ (1996) Hepatitis B virus HBx protein activates transcription factor NF-kB by acting on multiple cytoplasmic inhibitors of rel-related proteins. J Virol 70:4558-4566 Kekule AS, Lauer U, Weiss L, Luber B, Hofschneider PH (1993) Hepatitis B virus transactivator HBx uses a tumor promoter signalling pathway. Nature 361:742-745 Chirillo P, Falco M, Puri PL, Artini M, Balsano C, Levrero M, Natoli G (1996) Hepatitis B virus pX activates NF-kB-dependent transcription through a Raf-independent pathway. J Virol 70:641-646 Nabel D, Baltimore D (1987) An inducible transcription factor activates expression of a human immunodeficiency virus. Nature 326:711-713 Williams JL, Garcia J, Harrich D, Pearson L, Wu F, Gaynor R (1990) Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kB binding motifs. EMBO J 9:4435-4442 Mayreddy RP, Safak M, Razmara M, Zoltick P, Khalili K (1996) Transcription of the JC virus archetype late genome: importance of the kB and the 23-base-pair motifs in late promoter activity in glial cells. J Virol 70:2387-2393 Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES (1989) NF-kB activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J 8:4251-4258 Rong BL, Libermann TA, Kogawa K, Ghosh S, Cao LX, PavanLangston D, Dunkel EC (1992) HSV-1-inducible proteins bind to NF-kB-like sites in the HSV-1 genome. Virology 189:750-756 Graves BJ, Rabson AB (1997) Retrovirus Gene Expression: Transcription and RNA Processing, in Retroviruses (Coffin JM, Hughes SH, Varmus HE, eds). Cold Spring Harbor Laboratory Press, in press Perkins ND, Edwards NL, Duckett CS, Agranoff AB, Schmid RM, Nabel GJ (1993) A cooperative interaction between NF-kB and Sp1 is required for HIV-1 enhancer activation. EMBO J 12:3551-3558 Leonard J, Parrott C, Buckler-White AJ, Turner W, Ross EK, Martin MA, Rabson AB (1989) The NF-kB binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J Virol 63:4919-4924 Ross EK, Buckler-White WJ, Rabson AB, Englund G, Martin MA (1991) Contribution of NF-kB and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. J Virol 65:4350-4358 Yurochko AD, Kowalik TF, Huong SM, Huang ES (1995) Human cytomegalovirus upregulates NF-kB activity by transactivating the NF-kB p105/p50 and p65 promoters. J Virol 69:5391-5400
CANCER BIOLOGY, Vol 8, 1997: pp 121–129
The role of Rel/NF-kB proteins in viral oncogenesis and the regulation of viral transcription George Mosialos
Rel/NF-kB is a ubiquitous transcription factor that consists of multiple polypeptide subunits, and is subject to complex regulatory mechanisms that involve protein–protein interactions, phosphorylation, ubiquitination, proteolytic degradation, and nucleocytoplasmic translocation. The sophisticated control of Rel/NF-kB activity is not surprising since this transcription factor is involved in a wide array of cellular responses to extracellular cues, associated with growth, development, apoptosis, and pathogen invasion. Thus, it is not unexpected that this versatile cellular homeostatic switch would be affected by a variety of viral pathogens, which have evolved mechanisms to utilize various aspects of Rel/NF-kB activity to facilitate their replication, cell survival and possibly evasion of immune responses. This review will cover the molecular mechanisms that are utilized by mammalian oncogenic viruses to affect the activity of Rel/ NF-kB transcription factors and the role of Rel/NF-kB in the regulation of viral gene expression and replication.
A causative role for Tax in cellular transformation is supported by a number of experimental findings. First, the tax gene of HTLV-I is always present in infected tumor cells, occasionally in the absence of other regions of the viral genome. Second, Tax expression can immortalize human T cells, a phenotype that is likely to predispose these cells to tumorigenic genetic alterations.2 Third, expression of Tax in rodent fibroblasts results in oncogenic transformation as manifested by loss of contact inhibition, anchorageindependent growth and tumorigenicity in nude mice.3,4 Tax is a 40 kDa polypeptide that shows predominantly nuclear but, to some extent, also cytoplasmic localization. Tax appears to deregulate cellular growth control mechanisms primarily by affecting transcription regulation, and the activities of several transcription factors, including cyclic-AMP responsive element binding protein (CREB), serum response factor (SRF) and NF-kB, are influenced by Tax.1 However, Tax does not have intrinsic DNA-binding activity and therefore it must utilize cellular factors as docking vehicles to gain access to specific enhancer and promoter elements. Tax does contain transcription activation elements, and it can activate transcription when brought to DNA by fusion to a heterologous DNA-binding domain.5,6 Tax has a pronounced stimulatory effect on the activity of Rel/NF-kB, which is characterized by an increase in the nuclear accumulation of certain transactivating complexes. These complexes include p50 or p52 heterodimers with RelA and/or c-Rel. There are two phases in Tax-mediated generation of nuclear Rel complexes.7 In the early phase Tax promotes the nuclear accumulation of p50/RelA complexes which is then followed by the appearance of p52/c-Rel dimers. The increase in nuclear localization of p50/RelA following Tax expression is not due to an increase in protein levels of these subunits. However, the late response to Tax expression is accompanied by an elevation in mRNA and protein levels of p52 and c-Rel, which is most likely due to the
Key words: Epstein–Barr virus / malignant transformation / NF-kB / signal transduction / Tax protein ©1997 Academic Press Ltd
Activation of NF-kB by the HTLV-I transforming protein Tax Human T-cell leukemia virus type I (HTLV-I) is a human retrovirus that is associated with the development of adult T-cell leukemia (ATL) and a neurological disorder known as HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP).1 The critical player in HTLV-I-mediated T-cell transformation is the Tax protein, which is encoded by the pX region of the viral genome. From the Infectious Disease Division, Brigham & Women’s Hospital, Channing Labs, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA ©1997 Academic Press Ltd 1044-579X/97/020121 + 09$25.00/0/se970063
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G. Mosialos presence of kB-responsive elements in their promoters. Therefore, it appears that Tax activates Rel/ NF-kB through both cytoplasmic and nuclear events. The molecular mechanisms of Tax-mediated NF-kBactivation can be classified as those involving NF-kB/ Rel-inhibitory molecules (IkB-α, IkB-β, IkB-γ, p105, p100) and those that are associated with transactivating nuclear forms of Rel/NF-kB factors (RelA, c-Rel) (see Figure 1). The initial effect of Tax on nuclear translocation of p50/RelA is mediated by phosphorylation and degradation of IkB-α.7,8 IkB-α is responsible for retention of the p50/RelA complex in the cytoplasm and it is the primary target of transient NF-kB-activating signaling mechanisms (see Whiteside and Isra¨el, this issue). After stimulation, the initial drop in IkB-α protein levels is counterbalanced by a rapid increase in its mRNA levels since the IkB-α promoter is activated by NF-kB. Although the steady-state levels of IkB-α in Tax-expressing or HTLV-I-infected cells are not reduced as compared to cells not expressing Tax, the turnover rate of IkB-α is substantially increased, and thus, a significant portion of cytoplasmic p50/RelA complexes may escape into the nucleus during these rapid cycles of IkB-α degradation and resynthesis.7,9 The early NF-kB-activating response to Tax expression has features in common with the signaling mechanism of phorbol esters in promoting phosphorylation and degradation of IkB-α. However, the kinetics of IkB-α degradation following Tax expression are much slower than those achieved by phorbol esters.7 At present, the mechanism of Tax-mediated IkB-α phosphorylation is unclear. It is conceivable that Tax directly or indirectly activates a protein kinase cascade that leads to IkB-α phosphorylation and degradation. One report has identified a low abundance complex containing both Tax and IkB-α, suggesting that the association of Tax with IkB-α destabilizes IkB-α and promotes nuclear localization of transactivating NF-kB subunits.10 Along these lines, Lindholm et al11 have shown that Tax-induced activation of NF-kB is sensitive to the protein kinase C (PKC) inhibitor calphostin C and that Tax expression leads to PKC autophosphorylation and membrane translocation. Although these data implicate PKC in a Tax pathway that leads to NF-kB activation, the overall contribution of PKC to NF-kB activation by Tax is a matter of debate since other reports have failed to detect an effect of PKC inhibitors on Tax-mediated activation of NF-kB.8 It is possible that a particular isoform of PKC plays a role in Tax-mediated activation of NF-kB in certain cell lines, but it is likely that other protein
kinases are also involved. For example, it will be interesting to test the possible involvement of Tax in MEKK1 kinase family activation, which has recently been linked to IkB-α phosphorylation and degradation.12 Tax expression also targets IkB-β for degradation.13,14 IkB-β is a potent inhibitor of complexes containing RelA or c-Rel, but unlike IkB-α, the IkB-β promoter is not responsive to NF-kB activation. As a result, the levels of IkB-β are dramatically reduced or eliminated in Tax-expressing cell lines, perhaps accounting for the appearance of c-Rel-containing nuclear complexes in the late phase of cellular response to Tax. Tax also binds to the p105 and p100 NF-kB precursors that are known to act as cytoplasmic anchors and inhibitors of NF-kB activity.15,19 Genetic analysis of Tax indicates that its association with p105 correlates with its ability to activate NF-kB-responsive promoters and suggests that Tax overrides the inhibitory role of p105 on transactivating Rel/NF-kB subunits. In fact, Tax overexpression can liberate RelA and c-Rel from cytoplasmic complexes with p105 and allow them to be transported to the nucleus. Tax may facilitate p105 degradation by linking it to the 20S proteasome subunit.20 The outcome of Tax binding to p100 is a matter of debate as some investigators have shown that Tax can overcome the inhibitory effect of p100 on Rel subunits,17,18 whereas others have failed to detect any substantial effect of Tax on the IkB activity of p100.16,19,21 It is conceivable that the binding of Tax to p105 and possibly p100 changes their conformation or occludes their Rel binding sites so that they can no longer bind to Rel proteins. There is no evidence that Tax directly accelerates the proteolytic processing of p105 and p100 to their mature p50 and p52 subunits. Tax also binds to the ankyrin repeats of IkB-γ and blocks its ability to retain RelA in the cytoplasm.22 However, the significance of this effect on the pathogenesis of HTLV-I-associated human diseases is questionable since IkB-γ has been detected only in mouse cells. The predominantly nuclear localization of Tax argues for a specific role in the nucleus. Moreover, several mutations in Tax that compromise its nuclear localization also reduce its transactivating potential towards NF-kB-dependent and -independent promotes.23 Tax can associate with RelA and c-Rel through the Rel homology domain (RHD).24 These interactions appear to be functionally significant since Tax can cooperate with RelA or c-Rel for the activation of NF-kB-responsive elements in F9 embryonic carcinoma cells. These experiments indicate that 122
Viruses and activation of NF-kB the cell-cycle inhibitor p16INK4a, indicating that Tax is capable of influencing cellular growth properties at multiple levels.28
the binding of Tax to Rel subunits in the nucleus potentiates their transactivating potential and highlight an additional level of NF-kB regulation by Tax. The activation of Rel/NF-kB factors by Tax results in the upregulation of several cellular proteins that are known to have growth promoting effects.1 These include cytokines and growth factors (such as interleukin-6, tumor necrosis factor α and β, granulocytemacrophage colony stimulating factor, granulocyte colony stimulating factor), cytokine receptors (such as interleukin-2 receptor α), and proto-oncogenes (such as c-Myc). Cytokines and cytokine receptors are likely to participate in autocrine or paracrine routes of cellular activation that predispose infected cells to subsequent tumorigenic alterations. The role of Tax in the development of human T-cell leukemias is likely to be critical in the early stages of malignancy, whereas Tax may be dispensable in established ATL. This is supported by the fact that the levels of Tax protein are often low or undetectable in HTLV-I-infected ATL cells. In addition, the long latency for the development of this malignancy indicates that factors in addition to Tax contribute to malignant transformation. Nevertheless, in an elegant study where viral replication was separated from Tax activity it was demonstrated that Tax is absolutely essential for growth transformation of primary human T-lymphocytes by HTLV in vitro.25 In contrast, Tax appears to be dispensable for the long-term growth of transformed rodent fibroblast tumor cell lines that were generated by Tax expression in transgenic mice.26 Interestingly, elevated levels of NF-kB activity in these Tax-generated tumors were absolutely necessary for the continued growth of the tumor cells both in vitro and in vivo.26 On the other hand, mutational analysis of Tax has generated controversial results on the importance of NF-kB activation for Tax-mediated rodent fibroblast transformation.4,27 In-vitro transformation of rodent fibroblasts has provided invaluable information on the transforming properties of a number of human oncoproteins. However, it will be essential to perform similar genetic analyses of Tax function in human T-cell transformation in vitro in order to understand better the molecular events that underlie the development of HTLV-I-associated human malignancies. Overall it appears that activation of NF-kB by Tax is essential for upregulation of cellular activation and proliferation markers that are likely to set the stage for the disruption of cell-cycle checkpoints and the development of malignancies. Interestingly, it has recently been shown that Tax can target and inactivate
The role of NF-kB activation in B-lymphocyte growth transformation by Epstein–Barr virus Epstein–Barr virus (EBV) is a human herpes virus that causes infectious mononucleosis, a self-limiting lymphoproliferative disease.29 EBV is also associated with a number of human malignancies, such as nasopharyngeal carcinoma, African Burkitt’s lymphoma, Hodgkin’s disease, AIDS-associated central nervous system lymphomas and lymphoproliferative syndromes developed in organ transplant recipients.29 EBV targets primarily two cell types, epithelial cells and B lymphocytes. Genetic analysis of the EBV genome has identified six latent gene products that have an essential role in the process of B-lymphocyte transformation.30 For the purpose of this review, I will focus primarily on the function of latent membrane protein 1 (LMP1) which plays a critical role in B-cell growth transformation by EBV.31 LMP1 is an integral membrane protein that consists of a 24-amino-acid N-terminal cytoplasmic domain, six transmembrane domains connected by short reverse turns and a 200-amino acid cytoplasmic C terminus (CCT, see Figure 2A). LMP1 is expressed in most EBV-associated nasopharyngeal carcinomas and Hodgkin’s lymphomas.29 In rodent fibroblast cell lines LMP1 causes anchorage- and growth factorindependent growth, loss of contact inhibition and nude mouse tumorigenicity.32,33 In epithelial cells in culture or in transgenic mice LMP1 expression prevents differentiation.34-36 LMP1 induces the expression of many activation markers (CD23, CD30, CD39, CD40) and cell adhesion molecules (LFA1, ICAM1, LFA3) that are also upregulated in EBVtransformed B lymphocytes.37 LMP1 also upregulates the expression of anti-apoptotic factors, such as A20 and Bcl-2.38,39 Many of the cellular phenotypic changes caused by LMP1 are mediated at the transcriptional level. NF-kB is one of the cellular transcription factors that is activated by LMP1. The LMP1 domains that mediate NF-kB activation were characterized using standard molecular genetic approaches.40,41 Two NF-kB-activating domains were mapped to the LMP1 CCT. One domain (CTAR1 for carboxyl-terminal activating region 1, see Figure 2A) is located within the membrane proximal 45 amino acids of the LMP1 123
G. Mosialos CCT (amino acids 187–231) and accounts for approximately 20 to 30% of the LMP1-induced NF-kB activation. The second NF-kB-activating domain (CTAR2, see Figure 2A) mediates approximately 70 to
80% of the activating effect and is located within the last 55 amino acids of the LMP1 CCT (amino acids 332–386). Recombinant EBV genetic analysis has demonstrated that CTAR1 plays an essential role in
Figure 1. Molecular mechanisms of Rel/NF-kB activation by HTLV-I Tax and hepatitis B HBx proteins. Tax targets Rel complexes both in the cytoplasm and in the nucleus. In the cytoplasm Tax activates signaling pathways that lead to IkB-α and IkB-β (IkB) phosphorylation (P) and degradation. Tax can associate in the cytoplasm with IkB molecules such as IkB-α, p105 and p100. These interactions may prevent the inhibitory activity of IkB molecules on Rel-containing complexes, which are then freed to enter the nucleus and activate NF-kB -responsive (kB) promoters (PRO). Tax may act as a linker between proteasome subunits (20SP) 20 and certain IkB molecules, thus facilitating their degradation. In the nucleus Tax can associate with Rel subunits (Rel) such as RelA and c-Rel and potentiate their transactivating properties. The hepatitis B virus protein HBx can target IkB-α for degradation through signaling pathways that lead to Ras and protein kinase C (PKC) activation.
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Viruses and activation of NF-kB formation by EBV.42 Second, a recombinant EBV with CTAR1 fused to the transmembrane domains of LMP1 (amino acids 1–231) can induce B-lymphocyte transformation in the absence of the rest of the LMP1 CCT; however, the transforming potential of this recombinant protein is weaker than full-length LMP1.43 The transmembrane domains of LMP1 are critical for B-lymphocyte transformation and NF-kB activation because they enable LMP1 to aggregate in tight plasma membrane patches.31 The functional importance of the LMP1 CCT for cell transformation prompted a yeast two-hybrid screen to identify cellular factors that participate in the LMP1 signal transduction process. This genetic approach as well as biochemical studies revealed that LMP1 associates through CTAR1 with members of the tumor necrosis factor receptor associated factors (TRAF).44-46 TRAF1, TRAF3 and to a lesser extent TRAF2 constitutively associate with the CTAR1 domain of LMP1 in vitro and in EBV-transformed B lymphocytes. LMP1 expression dramatically alters the subcellular distribution of TRAF proteins from diffuse cytoplasmic to plasma-membrane aggregates that colocalize with LMP1. Mutational analysis of the LMP1 TRAF-binding site and transient overexpression of wild-type and mutant TRAFs indicate that TRAF1 and TRAF2 mediate NF-kB activation by the LMP1 CTAR1 domain, whereas TRAF3 inhibits NF-kB-activation by CTAR1.45 It appears that liganddependent association of TRAF molecules with the cytoplasmic tail of TNF receptors forms the core of a signaling complex that associates subsequently with other effector molecules that propagate signaling (Figure 2B).47,48 On the other hand, LMP1 appears to function as a constitutively crosslinked and active TNF receptor that propagates ligand-independent signaling in part through TRAF proteins (Figure 2A). A candidate TRAF2-associated effector molecule was recently identified as the protein kinase NIK, which is a homologue of protein kinase MEKK.49 Overexpression of NIK activates NF-kB and dominant-negative mutants of NIK block TRAF2-mediated activation of NF-kB. The similar role of TRAF2 and TRAF3 in LMP1 and CD40 signaling and the common phenotypic and growth promoting effects of LMP1 and CD40 on B lymphocytes strongly suggest that LMP1 acts as a constitutively active CD40 molecule (Figure 2). None of the known TRAFs binds directly to CTAR2 and the mechanism of NF-kB activation by this particular domain of LMP1 is unclear. LMP1 activates NF-kB in B-lymphocytes by inducing a rapid turnover of IkB-α.50 LMP1 expression in both epithelial cells
primary B-lymphocyte growth transformation in vitro. First, deletion of a core functional sequence of CTAR1 (amino acids 185–211) abrogates B-lymphocyte trans-
Figure 2. NF-kB activation by the EBV transforming protein LMP1 (A) and CD40 (B). LMP1 consists of a short 24-amino acid cytoplasmic N terminus (N), six transmembrane domains (vertical cylinders), and a 200-amino acid cytoplasmic C terminus (CCT). The LMP1 CCT contains two NF-kB -activating subdomains (CTAR1 and CTAR2). The membrane proximal NF-kB -activating domain (CTAR1) accounts for approximately 30% of NF-kB activation and interacts with TRAF1, TRAF2 and TRAF3. The second NF-kB -activating domain (CTAR2) accounts for 70% of the total NF-kB activation mediated by LMP1 and engages signaling pathways that are unclear at present. TRAF1 and TRAF2 form heterodimers and mediate NF-kB activation from CTAR1. TRAF3 inhibits NF-kB activation from CTAR1 possibly by competing with TRAF1 and TRAF2 for binding to this domain. Plasma membrane aggregation of TRAF1 and TRAF2 by LMP1 probably recruits effector molecules (X) that eventually lead to the activation of an IkB-α kinase, IkB-α degradation and nuclear translocation of NF-kB.
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G. Mosialos ing AP-1, AP-2, c/EBP, NF-kB and RNA polymerase III transcription factors. In this section, I will focus on the mechanism of NF-kB activation by HBx. HBx shows both cytoplasmic and nuclear localization. There is evidence that activation of NF-kB by HBx requires a cytoplasmic signaling event since exclusive nuclear localization of HBx compromises its ability to activate NF-kB.56 It has been shown that HBx activates the Ras pathway by increasing the abundance of the GTP-bound form of Ras57 (Figure 1). Activation of Ras is implicated in HBx-mediated NF-kB activation since a dominant-negative mutant of Ras has an inhibitory role in this process, at least in certain cell lines.58 Another pathway that has been associated with HBx-mediated activation of NF-kB involves PKC since inhibition or downregulation of PKC can prevent NF-kB activation by HBx59 (Figure 1). It should be noted, however, that Ras and PKC may not be universally involved in NF-kB activation by HBx since their involvement could not be demonstrated in other cell lines, albeit under somewhat different experimental conditions.60 HBx expression induces degradation of IkB-α and targets p105 for proteolytic processing.58 The molecular mechanisms behind these processes are unclear at present although the ability of HBx to activate the MAP kinase kinase (MEKK-1) may be linked to IkB-α degradation.57,12 These events lead to increased nuclear localization of RelA and c-Rel complexes with p50 and p52 and transactivation of NF-kB-responsive promoters.58 There is no evidence for direct association of HBx with Rel/NF-kB proteins. The lack of a detailed genetic analysis of the functional properties of HBx does not allow an assessment of the role of NF-kB activation in the tumorigenic potential of HBx. However, it is likely that induction of NF-kB-responsive genes by HBx contributes to the establishment of an activated cellular phenotype that can be the prelude in tumor development. HBx, like Tax and LMP1, is likely to play a role in tumor promotion rather than being sufficient for the establishment of the malignant phenotype given the long latency that precedes the development of hepatocellular carcinoma in cases of chronic HBV infection.
and B lymphocytes induces nuclear accumulation of RelA complexes that also contain p50 and/or p52.50,51 Interestingly c-Rel has not been implicated in LMP1mediated NF-kB activation. The critical role of the LMP1 CTAR1 domain in B-lymphocyte transformation suggests that NF-kB activation through TRAF molecules is an essential signaling event for the immortalization process. NF-kB activation by LMP1 is not sufficient for B-cell transformation since an EBV mutant that lacks a functional LMP1 CTAR1 domain is non-transforming but still capable for potent NF-kB activation through CTAR2.42 However, it is possible that CTAR1 and CTAR2 activate different subsets of NF-kB-responsive genes due to different effects on neighboring promoter elements. NF-kB is clearly important for certain growth-phenotypic changes mediated by LMP1 since it is involved in the upregulation of certain LMP1induced genes such as A20 and ICAM1.39,40 LMP1mediated induction of growth factor receptors and antiapoptotic factors is likely to increase the proliferative capacity of EBV-infected cells and thus predispose them to tumorigenic events. In this respect, LMP1 is similar to HTLV-I Tax in having a significant role in the early stages of tumor development although it may be dispensable in well-established malignancies.52 EBNA2 is a second EBV latent protein that can activate NF-kB. EBNA2 is essential for EBV-mediated B-cell growth transformation, but unlike LMP1, it is a nuclear protein and affects transcription directly through a potent transactivating domain in its C terminus. EBNA2 transactivates the human immunodeficiency virus (HIV) long terminal repeat through the induction of NF-kB activity.53 However, the molecular mechanism through which EBNA2 mediates activation of NF-kB is currently unknown.
Activation of NF-kB by the hepatitis B virus protein HBx HBx is a 17 kDa polypeptide encoded by mammalian hepatitis B viruses (HBV). Chronic HBV infection is associated with the development of hepatocellular carcinoma and HBx appears to be involved in the process of tumorigenesis. High levels of HBx expression in transgenic mice lead to a dramatic increase in the incidence of liver tumors and HBx can induce cellular transformation in certain assays.54,55 HBx has a broad effect on the cellular transcription machinery by activating a number of transcription factors includ-
The role of NF-kB/Rel proteins in viral gene expression and replication In the previous sections, emphasis was placed on the effects of viral activation of NF-kB on cellular gene 126
Viruses and activation of NF-kB
Conclusions
expression and relevant phenotypic changes. However, NF-kB also plays a crucial role in the control of viral gene expression and replication. NF-kB has been implicated in the control of gene expression of retroviruses (HIV),61 adenoviruses,62 papova viruses (JC virus)63 and herpes viruses (HSV-1, CMV).64,65 Two broadly-studied examples of the involvement of NF-kB in the regulation of viral gene expression are the retrovirus human immunodeficiency virus type 1 (HIV-1) and the herpes virus cytomegalovirus (CMV). Replication of HIV-1 requires the generation of genomic RNA from viral DNA that is integrated into the host genome as the provirus.66 The HIV proviral enhancer located in the U3 region of the long terminal repeat (LTR) contains two tandem NF-kB binding sites,66 and NF-kB plays an important role in transcription regulation of the HIV-1 LTR.61 Other transcription factors, such as Sp1, have also been implicated in the HIV-LTR transactivation. Interestingly it has been demonstrated that Rel/NF-kB proteins interact and cooperate with Sp1 for optimal transactivation of the HIV-1 LTR.67 Although the NF-kB sites in the HIV-1 LTR may not be absolutely required for virus replication in T lymphocytes, they clearly modulate replication efficiency and offer alternative routes of gene expression that can complement defects in other transcription factors.68,69 The presence of functional NF-kB sites in the HIV-1 enhancer renders the virus responsive to the plethora of extracellular stimuli that target NF-kB, including cytokines, bacterial lipopolysacharides, oxidative stress and other viruses that activate NF-kB. All of these agents can affect the outcome of HIV-1 infection and progression to AIDS, and it appears that Rel/ NF-kB proteins control an important regulatory switch in this process. Human cytomegalovirus (HCMV) has developed mechanisms for NF-kB activation and utilizes NF-kB for the expression of its immediate early (IE) genes. Activation of NF-kB by HCMV occurs in two phases.70 The early phase does not depend on protein synthesis and appears to be mediated simply by virus absorption onto the host cell. The second phase involves the upregulation of p105 and RelA promoters and an increase in the levels of nuclear p50/RelA complex. The CMV IE promoter is responsive to NF-kB and the IE gene product IE1 has been implicated in the induction of NF-kB activity.64,70 Therefore, an NF-kBdependent autoregulatory loop has been developed that is critically important for the initiation of a productive HCMV infection.
A number of mammalian oncogenic viruses have targeted transcription factor NF-kB for activation through mechanisms that involve cytoplasmic inactivation of inhibitory IkB molecules and also potentiation of the transactivating properties of nuclear Rel subunits. NF-kB was clearly a sensible choice because of its involvement in the induction of growth factors, cytokines, cytokine receptors, cell adhesion molecules and proto-oncogenes, all of which can promote active engagement of the cell cycle and possible extend he life span of the host cell. NF-kB has also recently been shown to play an active role in counteracting apoptotic signals and promoting cell survival (see Sonenshein, this issue). This property is particularly beneficial for a viral pathogen since apoptotic cell death is often triggered by infection-associated cellular stress and immune responses. Finally, the ability of NF-kB to respond to a broad spectrum of extracellular signals has been used cleverly by certain viruses to provide a sensor for the environment that regulates viral gene expression and replication.
Acknowledgements I am grateful to Dr Elliott Kieff and the members of his laboratory for invaluable help and support. My research has been supported by the Leukemia Society of America and the National Cancer Institute.
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