Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments.     

SNARE assembly and disassembly exhibit a pronounced hysteresis.

SNARE proteins are essential for intracellular membrane fusion of eukaryotes. Their assembly into stable four-helix bundles bridges membranes and may provide the energy for initiating membrane fusion. In vitro, assembly of soluble SNARE fragments is accompanied by major structural rearrangements that can be described as a folding reaction. The pathways and the thermodynamics of SNARE protein interactions, however, are not known. Here we report that assembly and dissociation of two distantly related SNARE complexes exhibit a marked hysteresis. The assembled and disassembled native states are separated by a kinetic barrier and cannot equilibrate on biologically relevant timescales. We suggest that the hysteresis is a hallmark of all SNARE complexes and that complex assembly and disassembly follow different pathways that may be independently controlled.     

Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation

To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-γ) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZV-specific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-γ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of ≥3 (BIMAS) or ≥20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide binding assay (ProImmune, Oxford, United Kingdom). HLA-A2 binding affinity was determined by the ability of the peptides to stabilize the HLA-peptide complex. Based on the binding affinity measurements, 34 high- to medium-affinity HLA-A2 binding peptides were selected and used to generate ProVE MHC pentamers (ProImmune, Oxford, United Kingdom). To enable screening of this large number of pentamers, the pentamers were divided into five pools, each containing six or seven pentamers. In the initial screening with pooled pentamers, four HLA-A2-positive patients were screened after a clinical diagnosis of VZV reactivation after TCD alloSCT. The presence of viral DNA in plasma at the time of clinical observations of VZV reactivation was confirmed by real-time PCR on plasma samples as previously described (7). After informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were cryopreserved and thawed and 0.5 × 10⁶ cells were incubated with pentamers at a concentration of 0.03 mg/ml for 10 min at room temperature in RPMI medium supplemented with 2% fetal bovine serum. After the cells were washed twice, 8 μl of FluoroTag-phycoerythrin (PE) was added for 20 min of incubation at 4°C and the cells were counterstained with CD4, CD40, and CD19-fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed on a FACScalibur fluorescence-activated cell sorter (FACS; Becton-Dickinson [BD], San Jose, CA). In one of four patients, pentamer pool 6, containing pentamers 61, 62, 64, 65, 66, and 67, was positive (0.06% of CD8 T cells); no other positive signals were observed. Staining with the individual pentamers revealed that pentamer 66, containing the epitope ALWALPHAA derived from the IE62 protein of VZV (IE62-ALW-A2) was responsible for the positive signal (0.06% of CD8 T cells, Fig. ​Fig.1B1B).Open in a separate windowFIG. 1.Screening with pentamers containing VZV-derived immunogenic epitopes. PBMCs of a patient after VZV reactivation following TCD alloSCT were incubated with pentamers and then stained with FluoroTag-PE to detect the pentamer-positive cells (A and B) and counterstained with CD4-, CD40-, and CD19-FITC. Pentamer staining of the CD4-, CD40-, and CD19-negative cells is shown. (A) PBMCs stained with pentamer 67 containing the epitope ALPHAAAAV, showing no specific staining. (B) PBMCs stained with pentamer 66 containing the epitope ALWALPHAA, showing specific staining. IE62-ALW-A2-specific T-cell clones were sorted into a single cell per well and expanded nonspecifically. The clones were stained with an irrelevant tetramer (C) and the IE62-ALW-A2 tetramer (D) in combination with CD8-FITC. Clones 1 and 2 were stained with a Vβ kit (BD) to demonstrate that clone 1 (E) and clone 2 (F) express different T-cell receptors. The results demonstrate that we isolated different T-cell clones that specifically stain with the IE62-ALW-A2 tetramer.To confirm the specificity of the IE62-ALW-A2-specific T cells, the pentamer-positive T cells were sorted into a single cell per well with a FACSDiva (BD) and expanded as previously described (5). The expanded T-cell clones were labeled specifically with the IE62-ALW-A2 PE-conjugated tetramer that was constructed as previously described (3) (Fig. ​(Fig.1D),1D), and Vβ analysis with the T-cell receptor Vβ repertoire kit (BD) showed that at least two different T-cell clones were isolated, demonstrating the oligoclonal origin of IE62-ALW-A2-positive T cells (Fig. 1E and F). To assess the cytolytic capacity of IE62-ALW-A2 T cells, chromium release assays were performed as described earlier (5). ⁵¹Cr-labeled Epstein-Barr virus (EBV) lymphoblastoid cell lines (LCLs) loaded with the IE62-ALW peptide were incubated with IE62-ALW-A2 T cells for 4 h. As demonstrated in Fig. ​Fig.2A,2A, HLA-A2-positive EBV LCLs loaded with the IE62-ALW-A2 peptide were lysed by both T-cell clones, whereas unloaded EBV LCLs were not lysed. To determine the avidity of the T-cell clones, the IE62-ALW-A2 peptide was titrated on EBV LCLs, and after 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells by standard enzyme-linked immunosorbent assay. Half-maximum IFN-γ production of the T-cell clones was observed when the stimulator cells were loaded with 10 ng/ml peptide, indicative of high-avidity T-cell clones (Fig. ​(Fig.2B).2B). To determine whether the T cells recognized cells endogenously expressing the IE-62-encoding gene, COS-A2 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) by using pcDNA vectors coding for different VZV genes, which were kindly provided by E. Wiertz (Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands). The transfected COS-A2 cells were used 24 h after transfection as stimulator cells in this assay. After 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells. IE62-ALW-A2 T-cell clones produced IFN-γ in response to COS-A2 cells endogenously expressing the IE62 protein, as well as COS-A2 cells pulsed with the IE62-ALW-A2 peptide. No IFN-γ was produced when the COS-A2 cells were transfected with the IE63-encoding gene of VZV or pulsed with an irrelevant peptide (Fig. ​(Fig.2C2C).Open in a separate windowFIG. 2.IE62-ALW-A2 T cells recognize IE62-ALW-A2 peptide-loaded target cells and target cells endogenously expressing IE62. (A) The cytolytic activity of IE62-ALW-A2-positive T-cell clones 1 and 2 was analyzed with the ⁵¹Cr release assay. T cells were incubated for 4 h with IE62-ALW-A2 peptide (pep)-loaded or unloaded, HLA-A2-positive EBV LCLs at an effector-to-target ratio of 10:1. (B) IE62-ALW-A2 T-cell clone 1 was stimulated with HLA-A2-positive EBV LCLs loaded with different concentrations of the IE62-ALW-A2 peptide. Release of IFN-γ (pg/ml) after 24 h of stimulation is shown. (C) IE62-ALW-A2 T-cell clones 1 and 2 were stimulated with HLA-A2-positive COS-A2 cells, left untreated, or loaded with the IE62-ALW-A2 peptide or with the IE4-ALR-B8 peptide as an irrelevant peptide or transfected with the IE63-encoding gene (COS-A2-IE63) or the IE62-encoding gene (COS-A2-IE62). Release of IFN-γ (picograms per milliliter) after 24 h of stimulation is shown.To determine whether IE62-ALW-A2-specific T cells were present in healthy individuals, cryopreserved PBMCs from 18 healthy, VZV-seropositive, HLA-A2-positive individuals were screened with the PE-conjugated VZV tetramer. PBMCs were labeled with tetramers for 15 min at 37°C in RPMI medium without phenol supplemented with 2% fetal bovine serum, washed, and analyzed with a FACScalibur. In 3 of these 18 serologically VZV-positive individuals, IE62-ALW-A2 tetramer-positive T cells could be detected (range, 0.01 to 0.02% of CD8 T cells). These data demonstrate that IE62-ALW-A2-specific T cells can be observed and that the frequency of these T cells is low under steady-state conditions in immunocompetent persons.To assess the frequency of IE62-ALW-A2-specific T cells in a cohort of patient who suffered from VZV reactivation following TCD alloSCT, 19 HLA-A2-positive patients after VZV reactivation following TCD alloSCT were screened by using the IE62-ALW-A2 tetramer. We screened these patients at a median of 47 days after the clinical diagnosis of VZV reactivation. In 8 of these 19 patients, IE62-ALW-A2-specific T cells could be directly detected ex vivo (mean, 0.04% [range, 0.01 to 0.11%] of CD8 T cells), indicating that this epitope is recognized in 42% of the HLA-A2-positive patients during VZV reactivation (Table ​(Table1).1). In VZV-seronegative patients (six screened), no IE62-ALW-A2 tetramer-positive cells could be detected.

TABLE 1.

Presence of IE62-ALW-A2-specific T cells in HLA-A2 patients after VZV reactivation following TCD alloSCT

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

A new method for class prediction based on signed-rank algorithms applied to Affymetrix<Superscript>®</Superscript> microarray experiments

Background

The huge amount of data generated by DNA chips is a powerful basis to classify various pathologies. However, constant evolution of microarray technology makes it difficult to mix data from different chip types for class prediction of limited sample populations. Affymetrix^® technology provides both a quantitative fluorescence signal and a decision (detection call: absent or present) based on signed-rank algorithms applied to several hybridization repeats of each gene, with a per-chip normalization. We developed a new prediction method for class belonging based on the detection call only from recent Affymetrix chip type. Biological data were obtained by hybridization on U133A, U133B and U133Plus 2.0 microarrays of purified normal B cells and cells from three independent groups of multiple myeloma (MM) patients.

Results

After a call-based data reduction step to filter out non class-discriminative probe sets, the gene list obtained was reduced to a predictor with correction for multiple testing by iterative deletion of probe sets that sequentially improve inter-class comparisons and their significance. The error rate of the method was determined using leave-one-out and 5-fold cross-validation. It was successfully applied to (i) determine a sex predictor with the normal donor group classifying gender with no error in all patient groups except for male MM samples with a Y chromosome deletion, (ii) predict the immunoglobulin light and heavy chains expressed by the malignant myeloma clones of the validation group and (iii) predict sex, light and heavy chain nature for every new patient. Finally, this method was shown powerful when compared to the popular classification method Prediction Analysis of Microarray (PAM).

Conclusion

This normalization-free method is routinely used for quality control and correction of collection errors in patient reports to clinicians. It can be easily extended to multiple class prediction suitable with clinical groups, and looks particularly promising through international cooperative projects like the "Microarray Quality Control project of US FDA" MAQC as a predictive classifier for diagnostic, prognostic and response to treatment. Finally, it can be used as a powerful tool to mine published data generated on Affymetrix systems and more generally classify samples with binary feature values.