Protein Kinase CK2 and Epstein–Barr Virus

Protein kinase CK2 is a pleiotropic protein kinase, which phosphorylates a number of cellular and viral proteins. Thereby, this kinase is implicated in the regulation of cellular signaling, controlling of cell proliferation, apoptosis, angiogenesis, immune response, migration and invasion. In general, viruses use host signaling mechanisms for the replication of their genome as well as for cell transformation leading to cancer. Therefore, it is not surprising that CK2 also plays a role in controlling viral infection and the generation of cancer cells. Epstein–Barr virus (EBV) lytically infects epithelial cells of the oropharynx and B cells. These latently infected B cells subsequently become resting memory B cells when passing the germinal center. Importantly, EBV is responsible for the generation of tumors such as Burkitt’s lymphoma. EBV was one of the first human viruses, which was connected to CK2 in the early nineties of the last century. The present review shows that protein kinase CK2 phosphorylates EBV encoded proteins as well as cellular proteins, which are implicated in the lytic and persistent infection and in EBV-induced neoplastic transformation. EBV-encoded and CK2-phosphorylated proteins together with CK2-phosphorylated cellular signaling proteins have the potential to provide efficient virus replication and cell transformation. Since there are powerful inhibitors known for CK2 kinase activity, CK2 might become an attractive target for the inhibition of EBV replication and cell transformation

Epstein–Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein–Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3′-untranslated region (3′UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3′UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3′UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40–50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication

microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19-26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processe

An estimate of the total number of true human miRNAs

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs

Modulation of intracellular calcium signaling by microRNA-34a-5p

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions

Epstein-Barr Virus EBER Transcripts Affect miRNA-Mediated Regulation of Specific Targets and Are Processed to Small RNA Species

The oncogenic Epstein-Barr virus (EBV) expresses 44 mature microRNAs and two non-coding EBER RNAs of 167 (EBER1) and 172 (EBER2) nt length. MiRNA profiling of NK/T cell lines and primary cells and Northern blotting of EBV-infected cell lines and primary tumors revealed processing of EBER1 to short 5′-derived RNAs of approximately 23, 52 and 70 nt (EBER123, EBER152, and EBER170) and of EBER2 to 3′ fragments. The biogenesis of these species is independent of Dicer, and EBER123 does not act like a miRNA OPEN ACCESS Non-Coding RNA 2015, 1 171 to target its complementary sequence. EBER1, EBER2 and EBER123 were bound by the lupus antigen (La), a nuclear and cytoplasmic protein that facilitates RNAi. Consistent with this, the EBERs affect regulation of interleukin 1alpha (IL1α) and RAC1 reporters harboring miR target sequences, targets of miR-142-3p. However, the EBERs have no effect upon another target of miR-142-3p, ADCY9, nor on TOMM22, a target of ebv-miR-BART16, indicative of selective modulation of gene expression by the EBERs

Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma

Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation

microRNA profiling in Epstein–Barr virus-associated B-cell lymphoma

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes

Protein Kinase CK2 and Epstein–Barr Virus

Protein kinase CK2 is a pleiotropic protein kinase, which phosphorylates a number of cellular and viral proteins. Thereby, this kinase is implicated in the regulation of cellular signaling, controlling of cell proliferation, apoptosis, angiogenesis, immune response, migration and invasion. In general, viruses use host signaling mechanisms for the replication of their genome as well as for cell transformation leading to cancer. Therefore, it is not surprising that CK2 also plays a role in controlling viral infection and the generation of cancer cells. Epstein–Barr virus (EBV) lytically infects epithelial cells of the oropharynx and B cells. These latently infected B cells subsequently become resting memory B cells when passing the germinal center. Importantly, EBV is responsible for the generation of tumors such as Burkitt’s lymphoma. EBV was one of the first human viruses, which was connected to CK2 in the early nineties of the last century. The present review shows that protein kinase CK2 phosphorylates EBV encoded proteins as well as cellular proteins, which are implicated in the lytic and persistent infection and in EBV-induced neoplastic transformation. EBV-encoded and CK2-phosphorylated proteins together with CK2-phosphorylated cellular signaling proteins have the potential to provide efficient virus replication and cell transformation. Since there are powerful inhibitors known for CK2 kinase activity, CK2 might become an attractive target for the inhibition of EBV replication and cell transformation