Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.

Treatment-dependent loss of polyfunctional CD8+ T-cell responses in HIV-infected kidney transplant recipients is associated with herpesvirus reactivation.

Antiretroviral-therapy has dramatically changed the course of HIV infection and HIV-infected (HIV(+)) individuals are becoming more frequently eligible for solid-organ transplantation. However, only scarce data are available on how immunosuppressive (IS) strategies relate to transplantation outcome and immune function. We determined the impact of transplantation and immune-depleting treatment on CD4+ T-cell counts, HIV-, EBV-, and Cytomegalovirus (CMV)-viral loads and virus-specific T-cell immunity in a 1-year prospective cohort of 27 HIV(+) kidney transplant recipients. While the results show an increasing breadth and magnitude of the herpesvirus-specific cytotoxic T-cell (CTL) response over-time, they also revealed a significant depletion of polyfunctional virus-specific CTL in individuals receiving thymoglobulin as a lymphocyte-depleting treatment. The disappearance of polyfunctional CTL was accompanied by virologic EBV-reactivation events, directly linking the absence of specific polyfunctional CTL to viral reactivation. The data provide first insights into the immune-reserve in HIV+ infected transplant recipients and highlight new immunological effects of thymoglobulin treatment. Long-term studies will be needed to assess the clinical risk associated with thymoglobulin treatment, in particular with regards to EBV-associated lymphoproliferative diseases.

Cloning of glucocorticoid-regulated genes in C6/ST1 rat glioma phenotypic reversion

The C6 rat glioma cell line is responsive to glucocorticoid hormones. C6 variants that are hyper-responsive (ST1) and resistant (P7) to hormone treatment have been derived previously. Glucocorticoid treatment of ST1 cells leads to complete reversion of the transformed phenotype and loss of tumorigenic potential. Production of C type retrovirus particles is also induced by glucocorticoids in ST1 cells. Cloning of the genes regulated by glucocorticoids in this cell system was used here as a strategy to uncover the gene products involved in the transformed-to-normal phenotypic change. Construction of a cDNA library from glucocorticoid-treated ST1 cells and screening by differential hybridization resulted in the isolation of three cellular sequences that code for rat metallothioneins (C27 and C41) and α1-acid glycoprotein (C36). Northern blot analysis revealed that expression of these genes was dramatically induced by hydrocortisone in ST1 but not in P7 cells. Viral genomic RNA was used to isolate and characterize retrovirus-related sequences that could also be responsible for the phenotypic reversion phenomenon.

Prevalence and risk factors for polyomavirus reactivation in solid-organ transplantation

Background. Polyomavirus reactivation is common in solid-organ transplant recipients who are given immunosuppressive medications as standard treatment of care. Previous studies have shown that polyomavirus infection can lead to allograft failure in as many as 45% of the affected patients. Hypothesis. Ubiquitous polyomaviruses when reactivated by post-transplant immunosuppressive medications may lead to impaired renal function and possibly lower survival prospects. Study Overview. Secondary analysis of data was conducted on a prospective longitudinal study of subjects who were at least 18 years of age and were recipients of liver and/or kidney transplant at Mayo Clinic Scottsdale, Arizona. Methods. DNA extractions of blinded urine and blood specimens of transplant patients collected at Mayo Clinic during routine transplant patient visits were performed at Baylor College of Medicine using Qiagen kits. Virologic assays included testing DNA samples for specific polyomavirus sequences using QPCR technology. De-identified demographic and clinical patient data were merged with laboratory data and statistical analysis was performed using Stata10. Results. 76 patients enrolled in the study were followed for 3.9 years post transplantation. The prevalence of BK virus and JC virus urinary excretion was 30% and 28%. Significant association was observed between JC virus excretion and kidney as the transplanted organ (P = 0.039, Pearson Chi-square test). The median urinary JCV viral loads were two logs higher than those of BKV. Patients that excreted both BKV and JCV appeared to have the worst renal function with a mean creatinine clearance value of 71.6 millimeters per minute. A survival disadvantage was observed for dual shedders of BKV and JCV, log-rank statistics, p = 0.09; 2/5 dual-shedders expired during the study period. Liver transplant and male sex were determined to be potential risk factors for JC virus activation in renal and liver transplant recipients. All patients tested negative for SV40 and no association was observed between polyomavirus excretion and type of immunosuppressive medication (tacrolimus, mycophenolate mofetil, cyclosporine and sirolimus). Conclusions. Polyomavirus reactivation was common after solid-organ transplantation and may be associated with impaired renal function. Male sex and JCV infection may be potential risk factors for viral reactivation; findings should be confirmed in larger studies.^

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

Rôle de PP2A dans l'activation constitutive de MEK1/2 de cellules MDCK transformées par le virus du sarcome de Moloney

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of Sa˜o Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

Epstein–Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter

The Epstein–Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs). Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF. P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography. EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300. In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression. Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect. In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity. Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.

Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication

CD4+ T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.

Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Purα

JC virus is activated to replicate in glial cells of many AIDS patients with neurological disorders. In human glial cells, the human immunodeficiency virus 1 (HIV-1) Tat protein activates the major late promoter of JC virus through a Tat-responsive DNA element, termed upTAR, which is a recognition site for cellular Purα, a sequence-specific single-stranded DNA binding protein implicated in cell cycle control of DNA replication and transcription. Tat interacts with two leucine-rich repeats in Purα to form a complex that can be immunoprecipitated from cell extracts. Tat enhances the ability of purified glutathione S-transferase-Purα (GST-Purα) to bind the upTAR element. Tat acts synergistically with Purα, in a cell-cycle-dependent manner, to activate transcription at an upTAR element placed upstream of a heterologous promoter. Since Purα is ubiquitously expressed in human cells and since PUR elements are located near many promoters and origins of replication, the Tat-Purα interaction may be implicated in effects of HIV-1 throughout the full range of HIV-1-infected cells.

Trans-activation by human immunodeficiency virus Tat protein requires the C-terminal domain of RNA polymerase II.

Human immunodeficiency virus (HIV)-encoded trans-activator (Tat) acts through the trans-activation response element RNA stem-loop to increase greatly the processivity of RNA polymerase II. Without Tat, transcription originating from the HIV promoter is attenuated. In this study, we demonstrate that transcriptional activation by Tat in vivo and in vitro requires the C-terminal domain (CTD) of RNA polymerase II. In contrast, the CTD is not required for basal transcription and for the formation of short, attenuated transcripts. Thus, trans-activation by Tat resembles enhancer-dependent activation of transcription. These results suggest that effects of Tat on the processivity of RNA polymerase II require proteins that are associated with the CTD and may result in the phosphorylation of the CTD.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus.

Infection of cells with picornaviruses, such as poliovirus and encephalomyocarditis virus (EMCV), causes a shutoff of host protein synthesis. The molecular mechanism of the shutoff has been partly elucidated for poliovirus but not for EMCV. Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure to which the multisubunit translation initiation factor eIF4F binds to promote ribosome binding. Picornaviruses use a mechanism for the translation of their RNA that is independent of the cap structure. Poliovirus infection engenders the cleavage of the eIF4G (formerly p220) component of eIF4F and renders this complex inactive for cap-dependent translation. In contrast, EMCV infection does not result in eIF4G cleavage. Here, we report that both EMCV and poliovirus activate a translational repressor, 4E-BP1, that inhibits cap-dependent translation by binding to the cap-binding subunit eIF4E. Binding of eIF4E occurs only to the underphosphorylated form of 4E-BP1, and this interaction is highly regulated in cells. We show that 4E-BP1 becomes dephosphorylated upon infection with both EMCV and poliovirus. Dephosphorylation of 4E-BP1 temporally coincides with the shutoff of protein synthesis by EMCV but lags behind the shutoff and eIF4G cleavage in poliovirus-infected cells. Dephosphorylation of 4E-BP1 by specifically inhibiting cap-dependent translation may be the major cause of the shutoff phenomenon in EMCV-infected cells.