A new approach for analysis of polyprenols by a combination of thin-film chemical deposition and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: The polyprenols are involved in some essential biosynthetic pathways and serve as ubiquitous components of cellular membranes, so their fingerprinting in natural samples is of great interest. Previous studies indicate that due to the high hydrophobicity of polyprenols their direct analysis by mass spectrometry with soft ionization techniques may be difficult and require preliminary off-line derivatization. Hence, a method for rapid and sensitive screening of polyprenols is required. METHODS: A combination of thin-film chemical deposition and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for analysis of the polyprenol profile of Abies sibirica L. extract. Polyprenol-based monolayers were formed at the interphase of aqueous barium acetate solution, supplemented with 2,5-dihydroxybenzoic acid, and an n-hexane solution of polyprenols directly on a MALDI target plate. RESULTS: Peaks corresponding to [M - H + Ba]+ ions were observed in the MALDI-TOF mass spectra of polyprenols. A total of nine polyprenol homologues were identified with a polyprenol of 16 isoprene units dominating. The limit of detection was established at the level of 6 pg. Possible mechanisms of formation of [M - H + Ba]+ ions of polyprenols were discussed. CONCLUSIONS: The proposed approach can be suitable for high-throughput screening of polyprenols in biological samples of different origin due to easy sample preparation and high sensitivity.

B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition.

Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication.IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation.

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch.

An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1ß (IL-1ß). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the IL-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting the IL1R1 3' untranslated region (UTR) by EBV miR-BHRF1-2-5p was confirmed using 3'-UTR luciferase reporter assays and Western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently infected B cells altered steady-state cytokine levels and disrupted IL-1ß responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis.IMPORTANCE IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell.