EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies.

There is increasing evidence that put into question the classical dogma that the Epstein-Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a third state has now been described, known as the abortive-lytic phase, which is characterized by the expression of some immediate early (IE) and early (E) genes, but no new virus progeny is produced. While the function of these IE and E gene products is not well understood, several recent studies support the concept they may contribute to tumor promotion by altering the tumor microenvironment (TME). The mechanisms by which these viral gene products may contribute to tumorigenesis remain unclear; however, it has been proposed that some of them promote cellular growth, immune evasion, and/or inhibit apoptosis. One of these EBV early gene products is the deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by BLLF3, which not only contributes to the establishment of latency through the production of activin A and IL-21, but it may also alter the TME, thus promoting oncogenesis.

EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.

Viral dUTPases: Modulators of Innate Immunity.

Most free-living organisms encode for a deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23). dUTPases represent a family of metalloenzymes that catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, preventing dUTP from being incorporated into DNA by DNA polymerases, maintaining a low dUTP/dTTP pool ratio and providing a necessary precursor for dTTP biosynthesis. Thus, dUTPases are involved in maintaining genomic integrity by preventing the uracilation of DNA. Many DNA-containing viruses, which infect mammals also encode for a dUTPase. This review will summarize studies demonstrating that, in addition to their classical enzymatic activity, some dUTPases possess novel functions that modulate the host innate immune response.

Tissue specific signature of HHV-6 infection in ME/CFS.

First exposure to various human herpesviruses (HHVs) including HHV-6, HCMV and EBV does not cause a life-threatening disease. In fact, most individuals are frequently unaware of their first exposure to such pathogens. These herpesviruses acquire lifelong latency in the human body where they show minimal genomic activity required for their survival. We hypothesized that it is not the latency itself but a timely, regionally restricted viral reactivation in a sub-set of host cells that plays a key role in disease development. HHV-6 (HHV-6A and HHV-6B) and HHV-7 are unique HHVs that acquire latency by integration of the viral genome into sub-telomeric region of human chromosomes. HHV-6 reactivation has been linked to Alzheimer's Disease, Chronic Fatigue Syndrome, and many other diseases. However, lack of viral activity in commonly tested biological materials including blood or serum strongly suggests tissue specific localization of active HHV-6 genome. Here in this paper, we attempted to analyze active HHV-6 transcripts in postmortem tissue biopsies from a small cohort of ME/CFS patients and matched controls by fluorescence in situ hybridization using a probe against HHV-6 microRNA (miRNA), miR-aU14. Our results show abundant viral miRNA in various regions of the human brain and associated neuronal tissues including the spinal cord that is only detected in ME/CFS patients and not in controls. Our findings provide evidence of tissue-specific active HHV-6 and EBV infection in ME/CFS, which along with recent work demonstrating a possible relationship between herpesvirus infection and ME/CFS, provide grounds for renewed discussion on the role of herpesviruses in ME/CFS.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: The Human Herpesviruses Are Back!

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) or Systemic Exertion Intolerance Disease (SEID) is a chronic multisystem illness of unconfirmed etiology. There are currently no biomarkers and/or signatures available to assist in the diagnosis of the syndrome and while numerous mechanisms have been hypothesized to explain the pathology of ME/CFS, the triggers and/or drivers remain unknown. Initial studies suggested a potential role of the human herpesviruses especially Epstein-Barr virus (EBV) in the disease process but inconsistent and conflicting data led to the erroneous suggestion that these viruses had no role in the syndrome. New studies using more advanced approaches have now demonstrated that specific proteins encoded by EBV could contribute to the immune and neurological abnormalities exhibited by a subgroup of patients with ME/CFS. Elucidating the role of these herpesvirus proteins in ME/CFS may lead to the identification of specific biomarkers and the development of novel therapeutics.

Epstein-Barr Virus dUTPase Induces Neuroinflammatory Mediators: Implications for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

PURPOSE: Neuroinflammation is a common feature in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), affecting 85%-90% of all patients, yet the underlying mechanism or mechanisms responsible for the initiation and/or promotion of this process is largely unknown. Multiple reports, however, have suggested a role for Epstein-Barr virus (EBV), in particular, in ME/CFS, but its potential role, if any, in the neuroinflammatory process has not been addressed. In support of this premise, studies by our group have found that the EBV protein deoxyuridine triphosphate nucleotidohydrolase (dUTPase) induces anxiety and sickness behaviors in female mice. We also found that a small subset of patients with ME/CFS exhibited prolonged and significantly elevated neutralizing antibodies against EBV dUTPase protein in serum, which inversely correlated with ME/CFS symptoms. A larger ME/CFS case-control cohort study further confirmed that a significant percentage of patients with ME/CFS (30.91%-52.7%) were simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or human dUTPase. Altogether, these findings suggest that EBV dUTPase protein may be involved in the neuroinflammatory process observed in ME/CFS. Thus, the aim of the present study was to determine whether the EBV dUTPase protein could contribute to neuroinflammation by altering the expression of genes involved with maintaining blood-brain barrier (BBB) integrity and/or modulating synaptic plasticity. METHODS: With the use of human immortalized astrocytes, microglia, and cerebral microvascular endothelial cells, we conducted time-course (0-24 h) experiments with EBV dUTPase protein (10 µg/mL) to determine what effect(s) it may have on the expression of genes involved with BBB permeability, astrocytes and microglia cell function, tryptophan metabolism, and synaptic plasticity by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In parallel, in vivo studies were conducted in female C57Bl/6 mice. Mice were injected by the intraperitoneal route with EBV dUTPase protein (10 µg) or vehicle daily for 5 days, and the brains were collected and processed for further qRT-PCR analysis of the in vivo effect of the dUTPase on the dopamine/serotonin and γ-aminobutyric acid/glutamate pathways, which are important for brain function, using RT2 Profiler PCR Arrays. FINDINGS: EBV dUTPase protein altered the expression in vitro (12 of 15 genes and 32 of 1000 proteins examined) and in vivo (34 of 84 genes examined) of targets with central roles in BBB integrity/function, fatigue, pain synapse structure, and function, as well as tryptophan, dopamine, and serotonin metabolism. IMPLICATIONS: The data suggest that in a subset of patients with ME/CFS, the EBV dUTPase could initiate a neuroinflammatory reaction, which contributes to the fatigue, excessive pain, and cognitive impairments observed in these patients.

EBV Positive Diffuse Large B Cell Lymphoma and Chronic Lymphocytic Leukemia Patients Exhibit Increased Anti-dUTPase Antibodies.

The Epstein-Barr virus (EBV), which is a ubiquitous γ-herpesvirus, establishes a latent infection in more than 90% of the global adult population. EBV-associated malignancies have increased by 14.6% over the last 20 years, and account for approximately 1.5% of all cancers worldwide and 1.8% of all cancer deaths. However, the potential involvement/contribution of lytic proteins to the pathophysiology of EBV-associated cancers is not well understood. We have previously demonstrated that the EBV-deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate and adaptive immune responses by engaging the Toll-Like Receptor 2 (TLR2), which leads to the modulation of downstream genes involved in oncogenesis, chronic inflammation, and in effector T-cell function. Furthermore, examination of serum samples from diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia patients revealed the presence of increased levels of anti-dUTPase antibodies in both cohorts compared to controls with the highest levels (3.67-fold increase) observed in DLBCL female cases and the lowest (2.12-fold increase) in DLBCL males. Using computer-generated algorithms, dUTPase amino acid sequence alignments, and functional studies of BLLF3 mutants, we identified a putative amino acid motif involved with TLR2 interaction. These findings suggest that the EBV-dUTPase: TLR2 interaction is a potential molecular target that could be used for developing novel therapeutics (small molecules/vaccines).

Myalgic encephalomyelitis/chronic fatigue syndrome and gulf war illness patients exhibit increased humoral responses to the herpesviruses-encoded dUTPase: Implications in disease pathophysiology.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), and varicella-zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91-52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV-6 and human dUTPases than controls (P = 0.0053 and P = 0.0036, respectively), while the ME/CFS cohort had higher anti-EBV-dUTPase antibodies than in both GWI patients (P = 0.0008) and controls (P < 0.0001) as well as significantly higher anti-human dUTPase antibodies than in controls (P = 0.0241). These results suggest that screening of patients' sera for the presence of various combinations of anti-dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment.

Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease.

The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person's lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein-Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer.

Chronic Physical Stress Does Not Interact with Epstein-Barr Virus (EBV)-Encoded Dutpase to Alter the Sickness Response.

Most adult humans have been infected with Epstein-Barr virus (EBV), which is thought to contribute to the development of chronic fatigue syndrome. Stress is known to influence the immune system and can exacerbate the sickness response. Although a role for psychological stress in the sickness response, particularly in combination with EBV-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) has been established, and the role of physical stressors in these interactions remains unspecified. In this study, we seek to determine the interaction of chronic physical (swim) stress and EBV-encoded dUTPase injection. We hypothesize that a chronic physical stressor will exacerbate the sickness response following EBV-encoded dUTPase injection. To test this hypothesis mice receive daily injections of EBV-encoded dUTPase or vehicle and are subjected to 15 min of swim stress each day for 14 days or left unmanipulated. On the final evening of injections mice undergo behavioral testing. EBV-encoded dUTPase injection alone produces some sickness behaviors. The physical swimming stress does not alter the sickness response.

Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity.

We have previously shown that Epstein-Barr virus (EBV)-encoded dUTPase can modulate innate immune responses through the activation of TLR2 and NF-κB signaling. However, whether this novel immune function of the dUTPase is specific for EBV or a common property of the Herpesviridae family is not known. In this study, we demonstrate that the purified viral dUTPases encoded by herpes simplex virus type 2 (HSV-2), human herpesvirus-6A (HHV-6A), human herpesvirus-8 (HHV-8) and varicella-zoster virus (VZV) differentially activate NF-κB through ligation of TLR2/TLR1 heterodimers. Furthermore, activation of NF-κB by the viral dUTPases was inhibited by anti-TLR2 blocking antibodies (Abs) and the over-expression of dominant-negative constructs of TLR2, lacking the TIR domain, and MyD88 in human embryonic kidney 293 cells expressing TLR2/TLR1. In addition, treatment of human dendritic cells and PBMCs with the herpesviruses-encoded dUTPases from HSV-2, HHV-6A, HHV-8, and VZV resulted in the secretion of the inflammatory cytokines IL-1ß, IL-6, IL-8, IL-12, TNF-α, IL-10, and IFN-γ. Interestingly, blocking experiments revealed that the anti-TLR2 Ab significantly reduced the secretion of cytokines by the various herpesviruses-encoded dUTPases (p < 0.05). To our knowledge, this is the first report demonstrating that a non-structural protein encoded by herpesviruses HHV-6A, HHV-8, VZV and to a lesser extent HSV-2 is a pathogen-associated molecular pattern. Our results reveal a novel function of the virus-encoded dUTPases, which may be important to the pathophysiology of diseases caused by these viruses. More importantly, this study demonstrates that the immunomodulatory functions of dUTPases are a common property of the Herpesviridae family and thus, the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by these herpesviruses.

Epstein-Barr virus (EBV)-encoded dUTPase and chronic restraint induce impaired learning and memory and sickness responses.

Most adult humans have been infected with Epstein-Barr virus (EBV) and carry the latent virus. The EBV genome codes for several proteins that form an early antigen complex important for viral replication; one of these proteins is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The EBV-encoded dUTPase can induce sickness responses in mice. Because stress can increase latent virus reactivation, we hypothesized that chronic restraint would exacerbate sickness behaviors elicited by EBV-encoded dUTPase. Male Swiss-Webster mice were injected daily for 15 days with either saline or EBV-encoded dUTPase. Additionally, half of the mice from each condition were either restrained for 3h daily or left undisturbed. Restraint stress impaired learning and memory in the passive avoidance chamber; impaired learning and memory was due to EBV-encoded dUTPase injected into restrained mice. EBV-encoded dUTPase induced sickness responses and restraint stress interacts with EBV-encoded dUTPase to exacerbate the sickness response. These data support a role for EBV-encoded dUTPase and restraint stress in altering the pathophysiology of EBV independent of viral replication.

Epstein-Barr virus encoded dUTPase containing exosomes modulate innate and adaptive immune responses in human dendritic cells and peripheral blood mononuclear cells.

We have recently demonstrated that Epstein-Barr virus (EBV)-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) modulates innate immunity in human primary monocyte-derived macrophages through toll-like receptor (TLR) 2 leading to NF-κB activation and the production of pro-inflammatory cytokines. Our previous depletion studies indicated that dendritic cells (DCs) may also be a target of the EBV-encoded dUTPase. However, the role of EBV-encoded dUTPase in DC activation/function and its potential contribution to the inflammatory cellular milieu characteristic of EBV-associated diseases remains poorly understood. In the present study, we demonstrate that EBV-encoded dUTPase significantly altered the expression of genes involved in oncogenesis, inflammation and viral defense mechanisms in human primary DCs by microarray analysis. Proteome array studies revealed that EBV-encoded dUTPase modulates DC immune responses by inducing the secretion of pro-inflammatory TH1/TH17 cytokines. More importantly, we demonstrate that EBV-encoded dUTPase is secreted in exosomes from chemically induced Raji cells at sufficient levels to induce NF-κB activation and cytokine secretion in primary DCs and peripheral blood mononuclear cells (PBMCs). Interestingly, the production of pro-inflammatory cytokines in DCs and PBMCs was TLR2-dependent. Together these findings suggest that the EBV-encoded dUTPase may act as an intercellular signaling molecule capable of modulating the cellular microenvironment and thus, it may be important in the pathophysiology of EBV related diseases.

Targeting IL-17 in psoriasis: from cutaneous immunobiology to clinical application.

Psoriasis vulgaris is a chronic, immune-mediated inflammatory skin disease associated with complex genetic susceptibility. Although the hallmark of psoriasis is characterized by cutaneous inflammation and keratinocyte hyperproliferation, recent studies show that the pathologic features observed in psoriasis arises as a result of innate and adaptive immune activation in genetically prone individuals. Studies focused on the microenvironment in the skin of psoriasis lesions have revealed novel cellular and cytokine abnormalities of the immune system. One pathway important is the role of the T(H)17/IL-17 dysregulation. The recent development of biologics that target the IL-17 cytokine pathway has confirmed the importance of T(H)17 and IL-17 homeostasis in the skin and yielded potent therapies in the treatment of psoriasis, and potentially other autoimmune diseases.

Protective effect of human endogenous retrovirus K dUTPase variants on psoriasis susceptibility.

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 × 10(-15), odds ratio =2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis.

A human endogenous retrovirus K dUTPase triggers a TH1, TH17 cytokine response: does it have a role in psoriasis?

Psoriasis is a chronic inflammatory immune disease of the skin characterized by a complex interplay between multiple risk genes and their interactions with environmental factors. Recent haplotype analyses have suggested that deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by a human endogenous retrovirus K (HERV-K) may be a candidate gene for the psoriasis susceptibility 1 locus. However, no functional studies have been conducted to determine the role of HERV-K dUTPase in psoriasis. For this purpose, we constructed an HERV-K dUTPase wild-type sequence, as well as specific mutations reflecting the genotype characteristic of high- and low-risk haplotypes, purified the recombinant proteins, and evaluated whether they could modulate innate and/or adaptive immune responses. In this study, we demonstrate that wild-type and mutant HERV-K dUTPase proteins induce the activation of NF-κB through Toll-like receptor 2, independent of enzymatic activity. Proteome array studies revealed that treatment of human primary cells with wild-type and mutant HERV-K dUTPase proteins triggered the secretion of T(H)1 and T(H)17 cytokines involved in the formation of psoriatic plaques, including IL-12p40, IL-23, IL-17, tumor necrosis factor-α, IL-8, and CCL20, in dendritic/Langerhans-like cells and to a lesser extent in keratinocytes. These data support HERV-K dUTPase as a potential contributor to psoriasis pathophysiology.

Bryostatin-1, a naturally occurring antineoplastic agent, acts as a Toll-like receptor 4 (TLR-4) ligand and induces unique cytokines and chemokines in dendritic cells.

Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11c(high)CD8α(-) CD11b(+)CD4(+) phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent.

The EBV-encoded dUTPase activates NF-kappa B through the TLR2 and MyD88-dependent signaling pathway.

The innate immune response plays a key role as the primary host defense against invading pathogens including viruses. We have previously shown that treatment of human monocyte-derived macrophages with EBV-encoded dUTPase induces the expression of proinflammatory cytokines through the activation of NF-kappaB. However, the receptor responsible for EBV-encoded dUTPase-mediated biological effects is not known. In this study, we demonstrate that the purified EBV-encoded dUTPase activates NF-kappaB in a dose-dependent manner through TLR2 and requires the recruitment of the adaptor molecule MyD88 but not CD14. Furthermore, activation of NF-kappaB was abrogated by anti-TLR2, anti-EBV-encoded dUTPase blocking Abs and the overexpression of a dominant negative construct of MyD88 in human embryonic kidney 293 cells expressing TLR2. In addition, treatment of human monocyte-derived macrophages with the anti-EBV-encoded dUTPase Ab 7D6 or the anti-TLR2 Ab blocked the production of IL-6 by the EBV-encoded dUTPase. To our knowledge, this is the first report demonstrating that a nonstructural protein encoded by EBV is a pathogen-associated molecular pattern and that it has immunomodulatory functions. Although additional studies are necessary to define the signaling pathways activated by the EBV-encoded dUTPase and to determine its role in modulating immune responses to EBV infection, our results suggest that the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by EBV.

A novel PAAD-containing protein that modulates NF-kappa B induction by cytokines tumor necrosis factor-alpha and interleukin-1beta.

PAAD domains are found in diverse proteins of unknown function and are structurally related to a superfamily of protein interaction modules that includes death domains, death effector domains, and Caspase activation and recruitment domains. Using bioinformatics strategies, cDNAs were identified that encode a novel protein of 110 kDa containing a PAAD domain followed by a putative nucleotide-binding (NACHT) domain and several leucine-rich repeat domains. This protein thus resembles Cryopyrin, a protein implicated in hereditary hyperinflammation syndromes, and was termed PAN2 for PAAD and NACHT-containing protein 2. When expressed in HEK293 cells, PAN2 suppressed NF-kappaB induction by the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), suggesting that this protein operates at a point of convergence in these two cytokine signaling pathways. This PAN2-mediated suppression of NF-kappaB was evident both in reporter gene assays that measured NF-kappaB transcriptional activity and electromobility shift assays that measured NF-kappaB DNA binding activity. PAN2 also suppressed NF-kappaB induction resulting from overexpression of several adapter proteins and protein kinases involved in the TNF or IL-1 receptor signal transduction, including TRAF2, TRAF6, RIP, IRAK2, and NF-kappaB-inducing kinase as well as the IkappaB kinases IKKalpha and IKKbeta. PAN2 also inhibited the cytokine-mediated activation of IKKalpha and IKKbeta as measured by in vitro kinase assays. Furthermore, PAN2 association with IKKalpha was demonstrated by co-immunoprecipitation assays, suggesting a direct effect on the IKK complex. These observations suggest a role for PAN2 in modulating NF-kappaB activity in cells, thus providing the insights into the potential functions of PAAD family proteins and their roles in controlling inflammatory responses.