Endoplasmic reticulum stress causes EBV lytic replication.

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Effects of storage and leukoreduction on lymphocytes and Epstein-Barr virus genomes in platelet concentrates.

BACKGROUND: Epstein-Barr virus (EBV) persists in infected B lymphocytes in blood donors. Lymphocytes are viable during platelet (PLT) storage. The effects of storage and leukoreduction on lymphocytes and EBV genomes are evaluated. STUDY DESIGN AND METHODS: Forty nonleukoreduced PLT concentrates were stored at 20 to 24°C for up to 7 days. EBV genomes in B cells were quantified on Days 1 and 5. Viable white blood cells (WBCs) and T and B cells were quantified in 10 of 40 units on Days 1, 3, 5, and 7 of storage. For the leukoreduction study, four pools of PLTs were leukoreduced within 24 hours of collection. B cells from before leukoreduction and all peripheral blood mononuclear cells from after leukoreduction were assayed for EBV. RESULTS: Viable WBCs and T cells were stable whereas viable B cells were reduced to 71% of the Day 1 level by Day 5. A total of 31 of 37 (83.8%) units were EBV positive. Although EBV genomes remained stable in most units, 12 of 37 units demonstrated a median of 5.1 (range 2- to 134)-fold increase in EBV genomes per 105 B cells on Day 5. For the leukoreduction study, EBV genomes were detected in four of four pools before leukoreduction with a median of 3.8 (range, 0.2-93.6) EBV genomes per 105 B cells. EBV genomes were not detected in any of the postleukoreduction specimens. CONCLUSIONS: Seventy percent of B lymphocytes are viable on Day 5 of PLT storage. Although the mean number of EBV genomes remained stable, a subset of units had increased EBV genomes during storage. Leukoreduction removed polymerase chain reaction-detectable EBV genomes from PLT pools.

Chronic high Epstein-Barr viral load carriage in pediatric liver transplant recipients.

Development of EBV disease and PTLD is usually accompanied by the detection of a high EBV load in peripheral blood. However, many children undergoing primary EBV infection following LTx will maintain chronically elevated EBV loads in the absence of clinical symptoms. To better understand this phenomenon, we retrospectively reviewed the records of children undergoing LTx at our center from 1997 to 2007 to identify chronic high EBV load carriers in this population. A CHL state was defined by the presence of a high load for >50% of samples for greater than or equal to six months following either asymptomatic or complete clinical resolution of EBV disease/PTLD. A total of 35 CHL carriers were identified. Pretransplant serologies were available for 29 of the 35; 22/29 (76%) were EBV negative prior to LTx; eight of these 22 developed their CHL state at the time of their primary EBV infection. Fourteen of the 35 had EBV disease (n = 7) or PTLD (n = 7) prior to development of the CHL state. Only one of 35 CHL carriers developed PTLD or lymphoma while they were a high load carrier. In all, 23/35 resolved their CHL state without apparent sequelae while 11 children continue to be asymptomatic high load carriers. These data provide important information about the outcome of chronic EBV high load carriage in pediatric liver transplant recipients.

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes.

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.

Calcium mobilization is responsible for Thapsigargin induced Epstein Barr virus lytic reactivation in in vitro immortalized lymphoblstoid cell lines.

The latent state is a critical component of all herpesvirus infections, and its regulation remains one of the most active areas of Epstein-Barr Virus (EBV) research. In particular, identifying environmental factors that trigger EBV reactivation into a virus-productive state has become a central goal in EBV latency research. Recently, a category of chemicals known as inducers of the endoplasmic reticulum unfolded protein response (UPR) have been shown to trigger EBV lytic reactivation in various established EBV-associated lymphoma cell lines. This has led to the recent belief that UPR is a universal cellular signaling pathway that directly triggers EBV lytic reactivation irrespective of cell type. We tested the potency of several widely used UPR inducers for EBV lytic reactivation on virus-immortalized primary lymphoblastoid cell lines (LCLs) in vitro. We found that, with the exception of Thapsigargin (Tg), UPR inducers did not trigger significant increases in BZLF1 transcripts or changes in the numbers of EBV genomic copies/cell in our panel of primary LCLs. Further investigation revealed that induction of lytic reactivation by Tg appeared to be due to its ability to trigger intracellular Ca2+ mobilization rather than its ability to induce UPR, based on our observations in which UPR induction alone was not sufficient to trigger the EBV lytic cycle in our LCLs. EBV immortalized LCLs have rarely been included in the majority of the lytic reactivation studies yet the characteristics of latent infection in LCLs should resemble those of proliferating B cells in clinically encountered lymphoproliferative diseases. Based on these observations, we propose an alternative mechanism of action for Tg in triggering EBV lytic reactivation in LCLs, and suggest that the proposed use of any chemical inducers of UPR for a purpose of oncolytic/lytic induction therapy needs to be fully evaluated pre-clinically in a panel of LCLs.

Response of elevated Epstein-Barr virus DNA levels to therapeutic changes in pediatric liver transplant patients: 56-month follow up and outcome.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a serious complications after liver transplantation. Epstein-Barr virus (EBV) load measured by quantitative competitive polymerase chain reaction (PCR) has been used as an early marker for the development of PTLD and a guide for initiating preemptive therapy. The aim of this study is to report the results of EBV DNA PCR screening in a transplant population and to examine the risk factors for developing elevated EBV DNA PCR and the effect of interventions for reducing EBV DNA levels. METHODS: Medical records of 44 children who underwent liver transplantation and EBV DNA PCR screening during a 3-year period were reviewed, and the patients were prospectively followed up for another 2 years. Eleven patients who developed elevated EBV DNA PCR levels, defined as >/=40 genomes/105 peripheral blood lymphocytes (PBL) in pretransplant EBV-seronegative patients and >/=200 genomes/105 PBL in pretransplant-seropositive patients, were treated. The initial intervention was reduction of immunosuppression and initiation of anti-viral therapy in all patients, with administration of cytomegalovirus immunoglobulin (CMV-IgG) in two patients. CMV-IgG was then given to five of the patients who did not respond to the initial intervention. RESULTS: The initial intervention resulted in the reduction of EBV DNA PCR levels to below threshold values in 4 of 11 patients. Five patients who did not respond to the initial interventions were subsequently given intravenous CMV-IgG. The EBV DNA PCR level fell in all five of these patients during the course of treatment with CMV-IgG, with a significant reduction (to threshold levels or by two dilutions) in four of the five patients. No episodes of graft rejection were observed. CONCLUSION: Eleven patients (25%) developed elevated EBV DNA PCR after liver transplantation. There were no identifiable risk factors for developing elevated EBV DNA PCR. A combination of reducing immunosuppression, adding antiviral agents, and initiating CMV-IgG resulted in a significant reduction of EBV DNA levels in nine (82%) patients during the follow-up period.

The effect of Epstein-Barr virus Latent Membrane Protein 2 expression on the kinetics of early B cell infection.

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein.

Cellular factors associated with latency and spontaneous Epstein-Barr virus reactivation in B-lymphoblastoid cell lines.

EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV.

Minimal protein domain requirements for the intracellular localization and self-aggregation of Epstein-Barr virus latent membrane protein 2.

The EBV Latent Membrane Protein 2 (LMP2) may have a role in the establishment and maintenance of in vivo latency. The gene is transcribed into two mRNAs that produce two LMP2 protein isoforms. The LMP2a protein isoform has 12 transmembrane segments (TMs) and an amino terminal cytoplasmic signaling domain (CSD) while the LMP2b isoform is identical but lacks the CSD. There has not been a consensus on the cellular membrane localization being sometimes ascribed to either a plasma membrane or an intracellular location [M. Rovedo, R. Longnecker, J. Virol. 81:89-94, 2007; D. Lynch, J. Zimmerman, D.T. Rowe, J. Gen. Virol. 83:1025-1035, 2002; C. Dawson, J. George, S. Blake, R. Longnecker, L.S. Young, Virology 289:192-207, 2001]. Fluorescent marker and epitope tagged LMP2b truncation mutants progressively removing TMs from the N and C termini were used to assess the localization and aggregation properties of LMP2b. wtLMP2b had an exclusively intracellular perinuclear localization, while all truncations of the protein resulted in localization to the cell surface. By epitope loop-tagging, all the truncated LMP2b proteins were verified to be in the predicted membrane orientation. In co-transfection experiments, the C-terminal region was implicated in the self-aggregation properties of LMP2b. Thus, an intact 12 TM domain was required for intracellular localization and protein-protein interaction while a C-terminal region was responsible for auto-aggregative properties.

Progress in the development of the DexAide right ventricular assist device.

The DexAide right ventricular assist device (RVAD) is an implantable centrifugal pump modified from the CorAide left ventricular assist device. As previously published, in vitro performance testing of the DexAide RVAD has met design criteria, and the nominal operating condition of 4 l/min and 20 mm Hg pressure rise was achieved at 2,000 rpm, with a power consumption of 1.9 watts. In vivo studies in 14 calves have demonstrated acceptable hemodynamic characteristics. The calf inflow cannula design is still evolving to minimize depositions on the cannula observed in most experiments. Fitting studies were performed in 5 cadavers and 2 patients to reconfigure the cannulae for use in humans. The design and development of external electronics have been completed for the stand-alone RVAD system, and verification tests are under way in preparation for preclinical tests. Work on the external electronics design for the biventricular assist system is ongoing. In conclusion, the initial in vitro and in vivo studies have demonstrated acceptable hemodynamic characteristics of the DexAide RVAD. The design and development of the external electronic components for the stand-alone RVAD system have been completed. The calf inflow cannula is being redesigned, and chronic in vivo tests are under way.

Epstein-Barr virus latent membrane protein 2B (LMP2B) co-localizes with LMP2A in perinuclear regions in transiently transfected cells.

Epstein-Barr virus is a human gammaherpesvirus that infects and establishes latency in B lymphocytes in vivo. The latent membrane protein 2 (LMP2) gene is expressed in latently infected B cells and encodes two protein isoforms, LMP2A and LMP2B, that are identical except for an additional N-terminal 119 aa cytoplasmic domain which is present in the LMP2A isoform. A panel of fusion proteins was constructed in which the fluorescent enhanced green fluorescent protein and DsRed protein domains were fused to the N- and C-termini of LMP2A and LMP2B. By fluorescence microscopy, LMP2B localized to perinuclear regions of both live and fixed transiently transfected cells. Co-localization was detected with markers for the endoplasmic reticulum and the trans-Golgi network. No evidence of co-localization of LMP2B with endosomes or surface expression was obtained. Transiently expressed LMP2B co-localized with transiently or constitutively expressed LMP2A. Confocal microscopy confirmed that LMP2A proteins localized to intracellular perinuclear compartments with markers for the trans-Golgi network. Only LMP2A proteins with C-terminal truncations were detected in the plasma membrane with extracellular loop1 epitope tags. These results indicate that the transmembrane domain of LMP2 proteins possess intracellular retention signals and suggest that LMP2A-mediated signalling effects are likely to be ectopic, originating from sites inside the cell close to the nucleus.