POS1120 A RETROSPECTIVE COHORT STUDY OF EBV INFECTION STATUS AND SYSTEMIC LUPUS ERYTHEMATOSUS
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Abstract:
Background:
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease caused by immune dysregulation and characterized by the production of a large number of autoantibodies leading to multiple organ involvement. Despite considerable diagnostic and therapeutic advances in recent decades, SLE remains associated with premature death. However, the exact pathogenesis has not been fully elucidated, and specific treatments are lacking. The development of SLE results from an interaction between genetic and environmental factors. Viral infection is a widely reported environmental trigger of SLE. For example, the Epstein-Barr virus (EBV) is most closely associated with SLE, but the exact mechanisms involved remain unclear. EBV is responsible for two forms of infection: lytic and latent. The lytic phase of infection is mediated by proteins encoded by BZLF1 and BRLF1, which produce a large number of viral particles that express the early antigen (EA), viral capsid antigen (VCA), and membrane antigen (MA). Furthermore, Epstein-Barr nuclear antigen (EBNA) is expressed during latent infection; EBNA1 plays an important role in latency, and EBNA2 is a viral protein essential for B-cell growth transformation.Objectives:
Systemic lupus erythematosus (SLE)and the Epstein-Barr virus (EBV)are very closely related, but the specific role of EBV in the development of SLE remains unclear.This study estimated the impact of EBV infection status on clinical manifestations and disease remission in patients with SLE.Methods:
A retrospective study was performed using electronic health records of patients with SLE. The SLE Disease Activity Index (SLEDAI-2K)was used to assess disease activity, with mild activity as SLEDAI-2K ≤ 6, moderate activity as SLEDAI-2K 7 -12, and severe activity as SLEDAI-2K > 12. VCA-IgM-positive or EA-IgM-positive or EBV-DNA copies ≥50 IU/ml were defined as lytic infection group, EBNA-IgG or VCA-IgG-positive and negative for both VCA-IgM and EA-IgM, EBV-DNA copies <50 IU/ml were defined as latent infection group. The end piont was defined as a decrease in SLEDAI-2K score of ≥ one grade from baseline or a decrease in SLEDAI-2K score of ≥4 points from baseline. Cochran Armitage trend test and multivariable logistic regression were further used to analyze the factors influencing EBV proliferation infection.The association of different EBV infection status with treatment response and disease remission in patients with SLE was assessed using propensity score weighting and multivariable Cox regression models.Results:
The cohort included 217 patients with SLE, 75 in the EBV lytic infection group and 142 in the EBV latent infection group. The SLEDAI-2K score was higher in the proliferative infection group (10.00 (6.25, 16.00) vs. 8.00 (5.00, 10.00), Z=3.96, P<0.001). The proportion of vasculitis (14.7% vs. 2.8%, X2 = 10.71, P = 0.001), serositis (30.7% vs. 13.4%, X2 = 9.40, P = 0.002), and hematological involvement (52.0% vs. 36.6%, X2 = 4.77, P = 0.029) were higher in the lytic infection group than in the latent infection group. A higher proportion of the lytic infection group required methylprednisolone pulse therapy than the latent infection group (17.3% vs. 7.0%, X2=5.49, P=0.019). The results of propensity score-weighted Cox regression implied a statistically significant difference in the effect of EBV lytic infection on disease remission in SLE relative to latent infection (HR 0.30, 95% CI 0.19-0.49, P < 0.001), which was consistent with the results of Cox proportional-hazards regression models analysis (HR 0.16, 95% CI 0.11-0.25, P < 0.001).Conclusion:
Patients with SLE with lytic EBV infection have higher disease activity than those with latent EBV infection, and those with lytic EBV infection are more likely to have hematologic involvement and manifestations of serositis and vasculitis, and take longer to achieve remission than those with latent infection.REFERENCES:
NIL.Acknowledgements:
NIL.Disclosure of Interests:
None declared.Keywords:
Lytic cycle
BZLF1
Epstein–Barr virus infection
104 Background: Several tumor types commonly contain the Epstein Barr virus (EBV) genome, including lymphomas and nasopharyngeal carcinoma. EBV-associated tumors contain latent EBV. We and others have shown that expression of the viral immediate-early proteins, BZLF1 or BRLF1, is sufficient to induce lytic EBV replication in vitro. Induction of lytic replication using the BZLF1 or BRLF1 gene products has potential as a tumor-specific therapeutic approach, since it results in death of the host cell. Methods: Replication-defective adenovirus vectors containing the LacZ gene, the BZLF1 gene, or the BRLF1 gene were used to infect the EBV-positive Burkitt lymphoma line, Jijoye. Expression of the BZLF1 and BRLF1 genes was monitored by FACS analysis using BZLF1 and BRLF1 specific antibodies. Induction of lytic EBV infection was examined using an antibody specific to the EBV BMRF1 protein (an early viral protein which is expressed in lytic, but not latent, viral infection). Adenovirus-infected cells were treated with or without the antiviral drug ganciclovir at a dosage known to inhibit lytic EBV replication (10 uM). Results: The adenovirus vectors induced reasonably efficient infection of Jijoye cells in vitro using a MOI of 50 (15-40% of cells infected in various experiments). Expression of either the BZLF1 or BRLF1 proteins induced lytic EBV infection in Jijoye cells. Although the BZLF1 (or BRLF1) protein was expressed in only a minority of the Jijoye cells, greater than 90 percent of cells were killed, while the LacZ control vector had little effect. Furthermore, the combination of BZLF1 (or BRLF1) with ganciclovir resulted in equal (or greater) cell killing, suggesting that full lytic replication (with concomitant release of infectious EBV) is not required for inhibition of tumor cell growth. Conclusions: Adenovirus-based delivery of either the EBV BZLF1 or BRLF1 gene products in an EBV-positive Burkitt lymphoma line induced lytic EBV infection and resulted in death of the tumor cell. Since many more tumor cells were killed than the number expressing the EBV IE gene products, induction of lytic EBV infection in only a portion of latently infected tumor cells may result in killing of surrounding tumor cells through an as yet unknown "bystander" effect. The finding that tumor cell killing is at least as efficient in the presence of the antiviral drug ganciclovir suggests that these vectors could be used in patients (with concomitant ganciclovir administration) without fear of releasing infectious EBV. Phosphorylation of ganciclovir by the EBV thymidine kinase (tk) (which is induced during lytic replication) may result in synergistic cell killing using the BZLF1/ganciclovir combination. We are currently testing the BZLF1 and BRLFI adenovirus vectors (with and without ganciclovir) in a SCID mouse model for EBV-associated lymphomas.
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Objective To test the hypothesis that Epstein-Barr virus (EBV) infection of cells within the synovial membrane contributes to the pathogenesis of rheumatoid arthritis (RA). Methods Biopsy samples of synovial membrane from 37 patients with RA and from 51 patients with other joint diseases were studied for evidence of EBV infection using in situ hybridization specific for the EBV-encoded RNAs (EBERs). Latent membrane protein 1 (LMP1) and the lytic-cycle BZLF1 protein were detected by immunohistochemistry. Results Rare EBER-positive B lymphocytes were detected in 7 RA biopsy samples. EBV was not detectable in any other cells. Expression of the LMP1 and BZLF1 proteins of EBV was not observed in any of the samples. No EBV infection was detected in synovial membranes from patients with other joint diseases. Conclusion Our data indicate that EBV infection is not directly involved in the pathogenesis of RA. Any contribution of EBV to the pathogenic process leading to RA is likely to be indirect.
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Lytic cycle
Epstein–Barr virus infection
Gammaherpesvirinae
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Lytic cycle
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Gammaherpesvirinae
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EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1 , related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1’s initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV’s epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host’s mode of methylating DNA for its own epigenetic regulation.
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Although the Epstein-Barr virus (EBV) is associated with malignant and nonmalignant diseases, its lytic replication is predominantly associated with nonmalignant diseases such as acute infectious mononucleosis (IM) or chronic active EBV infection. Lytic replication is also associated with type B EBV more than with type A EBV. Sustained lytic replication, however, is not compatible with tumor growth. We investigated whether control of an EBV lytic regulatory gene, BZLF1, differed in these diseases.Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequence analyses were used to characterize the promoter sequence of BZLF1 (Zp) in 52 tumors (34 non-Hodgkin's lymphomas, 13 post-transplant lymphoproliferative disease samples, and five nasopharyngeal carcinomas), and in peripheral blood lymphocytes from seven patients with chronic active EBV, six with IM, and 40 healthy, EBV-seropositive individuals. All sequences were compared with the prototype EBV strain B95.8 sequence. All statistical tests were two-sided.Three polymorphic Zp sequences were detected. Among the malignant samples, sequence Zp-P, associated with 84% of type A EBV, was identical to that of EBV strain B95.8, whereas a second sequence (Zp-V3), associated exclusively with type B EBV (P<.001), contained three base substitutions. Among the nonmalignant samples, a distinct polymorphism, Zp-V4, containing the substitutions detected in Zp-V3 and an additional base change, was identified in all samples from chronic active EBV, IM, and healthy individuals, but in none of the malignant samples (P<.001). Zp-V4 was independent of the EBV type.Polymorphisms in the regulatory sequences of BZLF1 are differentially distributed among malignant and nonmalignant cells and may identify EBV subtypes with various lytic activities, including those not associated with malignancies.
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ABSTRACT Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a “superlytic” (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an “abortive” lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.
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The Epstein‐Barr Virus (EBV) is a member of the herpes virus family and causes infectious mononucleosis. Epstein‐Barr Virus was the first virus discovered to cause cancer in humans. After infection with EBV, the virus maintains a lifelong dormant infection within the host. The virus's life cycle consists of two phases, the latent and the lytic phase. The latent phase allows the virus to lie dormant within the host without presenting any symptoms, while during the lytic phase the virus reproduces and spreads among cells. The virus switches between the latent and lytic phases in response to environmental stimuli, including some pharmaceuticals. We investigated the response of the virus to atypical antipsychotic drugs. Antipsychotic drugs are used to treat conditions such as schizophrenia and bipolar disorder. Atypical antipsychotics, also known as second generation antipsychotics, have a different chemical structure and are generally more effective than the typical (first generation) antipsychotics. The effects of varying concentrations of the drugs on the reactivation of EBV into the lytic cycle were tested. The degree of viral reactivation was measured by expression of the viral BZLF1 gene, a regulatory gene expressed during reactivation into the Epstein‐Barr Virus lytic cycle. Quantitative polymerase chain reaction (qPCR) monitored BZLF1 gene expression. Expression of the BZLF1 gene and viral reactivation was found to be inhibited. Understanding the conditions and cellular pathways that inhibit the lytic phase of the virus will help to better understand the virus's life cycle in order to develop treatments for cancers caused by Epstein‐Barr Virus. Support or Funding Information This work was funded by the UWL College of Science and Health, UWL Faculty Research Grants to KLG, and Undergraduate Research and Creativity grants to AGA. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signalling after BCR cross-linking to inhibit activation of lytic EBV, and ectopically expressed LMP2B negatively regulates LMP2A. Here, it is demonstrated that silencing of LMP2B in EBV-harbouring Burkitt's lymphoma Akata cells results in reduced expression of EBV immediate-early lytic BZLF1 gene mRNA and late lytic gp350/220 protein upon BCR cross-linking. Similarly, reduction of lytic EBV activation was observed in Akata cells overexpressing LMP2A. In contrast, silencing of LMP2A expression resulted in higher lytic EBV mRNA and protein expression in BCR cross-linked Akata cells. These observations indicate a role for LMP2B distinct from that of LMP2A in regulation of lytic EBV activation in the host cell and support the hypothesis that LMP2B exhibits a negative-regulatory effect on the ability of LMP2A to maintain EBV latency by preventing the switch to lytic replication.
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Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.
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Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins involved in several cellular events as well as in processes that characterize the infective cycle of some viruses. In the present study, we investigated the role of poly(ADP-ribosylation) on Epstein-Barr Virus (EBV) lytic cycle activation.Inhibition of PARP-1 by 3-aminobenzamide (3-ABA) during EBV induction, diminished cell damage and apoptosis in the non-productive Raji cell line while markedly reducing the release of viral particles in the productive Jijoye cells. Furthermore, incubation with 3-ABA up-regulated the levels of LMP1 and EBNA2 latent viral proteins. At the same time, it slightly affected the expression of the immediate early BZLF1 gene, but largely down-regulated the levels of the early BFRF1 protein. The modulation of the expression of both latent and lytic EBV genes appeared to be post-transcriptionally regulated.Taken together the data indicate that PARP-1 plays a role in the progression of EBV lytic cycle and therefore, PARP inhibitors might represent suitable pharmacological adjuncts to control viral spread in EBV productive infection.
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