Deoxyuridine Triphosphate Nucleotidohydrolase Research Articles

Molecular Characterization of UL50 (dUTPase) Gene of Bovine Herpes Virus 1.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells.

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.

Examination of control asymptomatic cohorts reveals heightened anti-EBV and HHV-6 A/B dUTPase antibodies in the aging populations.

Members of the human Herpesviridae are found in high prevalence in the human virome. While these viruses are known to cause numerous disease pathologies in symptomatic individuals little is known concerning the role that these viruses may have in modulating the host immune system in asymptomatic “healthy” individuals, especially during the aging process. Examination of three cohorts of “healthy asymptomatic” individuals (n = 255) for the presence of antibodies against the herpesviruses deoxyuridine triphosphate nucleotidohydrolase (dUTPase) as a marker for lytic/abortive‐lytic replication demonstrated that all cohorts exhibited differential anti‐herpesvirus dUTPase antibodies positivity frequencies ranging from 40.4% to 84% with some individuals in these cohorts expressing antibodies to the dUTPases of multiple herpesviruses (17.2%–56%). Furthermore, our results demonstrate that there was a statistically significant difference in anti‐human herpesvirus 6 A and 6B (HHV‐6 A/B) dUTPase antibodies in Cohort 3 (age = 66.2 ± 15.02 years) versus Cohort 1 (age 46.88 ± 8.61 years), suggesting that reactivation of HHV‐6 A/B is not attenuated by aging. It is well established/documented that herpesvirus dUTPases induce immune dysfunction, as such it is of critical importance that additional studies be performed to determine how these viral proteins alter immune responses in asymptomatic individuals.

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.

Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition.

We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response–88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.

Mitochondrial DUT-M potentiates RLR-mediated antiviral signaling by enhancing VISA and TRAF2 association

Upon recognition of intracytoplasmic viral RNA, activated RIG-I is recruited to the mitochondrion-located adaptor protein VISA (also known as MAVS, CARDIF, and IPS-1). VISA then acts as a central signaling platform for linking RIG-I and downstream signaling components, such as TRAF2, 5, and 6, TBK1, and IKK, leading to activation of the kinases TBK1 and IKK. These activated kinases further phosphorylate the transcription factors IRF3/7 and NF-κB, leading to the induction of downstream antiviral genes. Here, we report a mitochondrial isoform, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), DUT-M, as a positive regulator in RLR-VISA-mediated antiviral signaling. DUT-M interacts with VISA and RIG-I to facilitate the assembly of the VISA-TRAF2 complex and to augment the polyubiquitination of TRAF2, leading to potentiated activation of IRF3 dimerization and phosphorylation of P65, and enhanced VISA-mediated innate immune response. RLR-VISA-mediated IRF3 dimerization and P65 phosphorylation, were inhibited in DUT-knockdown and DUT-deficient 293 cells. Thus, DUT-M is a positive regulator of the RIG-I-VISA-mediated innate immune response to RNA viruses.

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

PurposeNeuroinflammation 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. MethodsWith 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. FindingsEBV 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. ImplicationsThe 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.

Asymmetric biodegradation of the nerve agents Sarin and VX by human dUTPase: chemometrics, molecular docking and hybrid QM/MM calculations

Organophosphorus compounds (OP) nerve agents are among the most toxic chemical substances known. Their toxicity is due to their ability to bind to acetylcholinesterase. Currently, some enzymes, such as phosphotriesterase, human serum paraoxonase 1 and diisopropyl fluorophosphatase, capable of degrading OP, have been characterized. Regarding the importance of bioremediation methods for detoxication of OP, this work aims to study the interaction modes between the human human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and Sarin and VX, considering their Rp and Sp enantiomers, to evaluate the asymmetric catalysis of those compounds. In previous work, this enzyme has shown good potential to degrade phosphotriesters, and based on this characteristic, we have applied the human dUTPase to the OP degradation. Molecular docking, chemometrics and mixed quantum and molecular mechanics calculations have been employed, showing a good interaction between dUTPase and OP. Two possible reaction mechanisms were tested, and according to our theoretical results, the catalytic degradation of OP by dUTPase can take place via both mechanisms, beyond being stereoselective, that is, dUTPase cleaves one enantiomer preferentially in relation to other. Chemometric techniques provided excellent assistance for performing this theoretical investigation. The dUTPase study shows importance by the fact of it being a human enzyme.Communicated by Ramaswamy H. Sarma

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

EBV-dUTPase Modulates Host Immune Responses Potentially Altering the Tumor Microenvironment in EBV-associated Malignancies

While the use of long-term antiretroviral therapy (ART) has improved immune function and reduced the incidence of some malignancies in human immunodeficiency virus (HIV-1) infected patients, this is not the case for non-Hodgkin’s lymphoma, an AIDS-defining cancer, and Hodgkin lymphoma, a non-AIDS defining cancer. Epstein-Barr virus (EBV) is an independent factor that adversely affects risk and/or survival among patients with Diffuse Large B cell Lymphoma (DLBCL) or classical Hodgkin lymphoma in immunocompetent patients as well as in HIV-1 infected individuals on ART. While cells infected with EBV in EBV-associated malignancies generally express one type of the latency programs, a small number of cells in these tumors are expressing EBV genes associated with lytic replication of the virus, which suggests that products from lytic or abortive-lytic replication may contribute to lymphomagenesis. However, the potential roles of these proteins in lymphomagenesis remain unclear. In this study we demonstrate that the EBV deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an early protein produced during abortive/lytic EBV replication, is expressed in nasopharyngeal carcinoma (NPC) tumors in a mouse model of NPC, an EBV associated malignancy. Furthermore, screening of sera from 4 cohorts of patients diagnosed with AIDS, DLBCL, NPC and breast cancer (BC) by ELISA revealed an increase in IgG antibodies to the EBV-dUTPase in a subset of patients with AIDS, DLBCL and NPC relative to the controls. Finally, using an in vitro model of EBV infection we demonstrate that the EBV-dUTPase also inhibits T-cell function thus, allowing the proliferation of EBV immortalized B-cells. Altogether, our data support the premise that the EBV-dUTPase can modulate host immune responses and potentially alter the tumor microenvironment by promoting the survival/proliferation of immortalized B-cells, which may lead to lymphomagenesis. Key Words: Epstein-Barr virus, dUTPase, lymphomagenesis

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.

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 15days 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.

Evidence for the Role of Epstein Barr Virus Infections in the Pathogenesis of Acute Coronary Events

BackgroundThe role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events.Methodology/Principal FindingsBlood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6±2.6 pg/mL in patients with AMI vs. 3.2±2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304±116 pg/mL in AMI vs. 265±86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p = 0.008; 369±183 pg/mL in AMI and positive for dUTPase vs. 249±70 pg/mL in SA negative for dUTPase antibody).Conclusions/SignificanceThese clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.

Antibody to Epstein-Barr Virus Deoxyuridine Triphosphate Nucleotidohydrolase and Deoxyribonucleotide Polymerase in a Chronic Fatigue Syndrome Subset

BackgroundA defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar “clinical” conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbach's alpha.MethodsAntibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13–16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS.ResultsAntibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls.ConclusionsThere is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.

Inhibition of dUTPase Induces Synthetic Lethality with Thymidylate Synthase–Targeted Therapies in Non–Small Cell Lung Cancer

Chemotherapies that target thymidylate synthase (TS) continue to see considerable clinical expansion in non-small cell lung cancer (NSCLC). One drawback to TS-targeted therapies is drug resistance and subsequent treatment failure. Novel therapeutic and biomarker-driven strategies are urgently needed. The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is reported to protect tumor cells from aberrant misincorporation of uracil during TS inhibition. The goal of this study was to investigate the expression and significance of dUTPase in mediating response to TS-targeted agents in NSCLC. The expression of dUTPase in NSCLC cell lines and clinical specimens was measured by quantitative real-time reverse transcriptase PCR and immunohistochemistry. Using a validated RNA interference approach, dUTPase was effectively silenced in a panel of NSCLC cell lines and response to the fluoropyrimidine fluorodeoxyuridine (FUdR) and the antifolate pemetrexed was analyzed using growth inhibition and clonogenic assays. Apoptosis was analyzed by flow cytometry. Significant variation in the quantity and cellular expression of dUTPase was observed, including clear evidence of overexpression in NSCLC cell line models and tumor specimens at the mRNA and protein level. RNA interference-mediated silencing of dUTPase significantly sensitized NSCLC cells to growth inhibition induced by FUdR and pemetrexed. This sensitization was accompanied by a significant expansion of intracellular dUTP pools and significant decreases in NSCLC cell viability evaluated by clonogenicity and apoptotic analyses. Together, these results strongly suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition in NSCLC and that inhibition of dUTPase is a mechanism-based therapeutic approach to significantly enhance the efficacy of TS-targeted chemotherapeutic agents.

Pharmacokinetic and Metabolism Determinants of Fluoropyrimidines and Oxaliplatin Activity in Treatment of Colorectal Patients

Fluoropyrimidines and oxaliplatin continued to be the mainstay of therapeutic regimens in the treatment of colorectal cancer (CRC). For this reason, pharmacokinetic and metabolism of these drugs were analyzed and the identification of accurate and validated predictive, prognostic and toxicity markers became necessary to develop an effective therapy adapted to the patient's molecular profile, while minimizing life-threatening toxicities. In this review, we discuss literature data, defining predictive and prognostic markers actually identified in the treatment of CRC. We analyzed predictive markers of fluoropyrimidines effectiveness, principally for 5-Fluorouracil (5- FU) and also for oral fluoropyrimidines, as thymidylate Synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), methylenetetrahydrofolate reductase (MTHFR), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), microsatellite instability. DPD represent the more studied 5-FU toxicity marker, followed by TS and OPRT. Oxaliplatin effectiveness is principally regulated by nucleotide excision repair (NER) pathway, including excision repair cross-complementation group 1 (ERCC1), X-ray cross-complementing group 1 (XRCC1) and xeroderma pigmentosum group D (XDP). The major oxaliplatin toxicity marker is represented by glutathione S-transferase (GST). All these results are based principally on retrospective studies. The future challenge became to validate molecular markers and their association with clinical outcomes in prospective trials, refining technologic platforms and bioinformatics to accommodate the complexity of the multifaceted molecular map that may determine outcome, and determining CRC patients most likely to benefit from therapeutic interventions tailored specifically for them.

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.

Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.