Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA sequence analysis of the BamHI-C fragment of Marek's Disease Virus (MDV) reveals the presence of a 513 amino acid open reading frame (ORF). This ORF codes for a protein with an estimated M(r) of 58,901. Comparison of the amino acid sequence with those available in the Swiss-Prot database indicates extensive homology with a protein kinase (PK) of herpes simplex virus (HSV) and varicella-zoster virus (VZV). In Northern blot hybridization, a transcript of 2.0 kb was detected in MDV (GA strain) infected duck embryo fibroblasts (DEFs). A portion of the ORF was expressed in Escherichia coli as a trpE-fusion protein and used to generate antiserum in New Zealand rabbits. This antiserum specifically detected a protein of 60 kDa in MDV serotype 1, 2 and 3 infected DEFs or chicken embryo fibroblasts (CEFs) by Western blot analysis. This ORF codes for a functional PK.
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PMID:Identification and structural analysis of a MDV gene encoding a protein kinase. 1069 41

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.
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PMID:Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC. 1763 22

Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ''non-spreading phenotype'' of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.
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PMID:Comparative sequence analysis of a highly oncogenic but horizontal spread-defective clone of Marek's disease virus. 1772 13

Marek's disease (MD) is a highly contagious, lymphoproliferative disease of chickens caused by the cell-associated MD virus (MDV), a member of the alphaherpesvirus subfamily. In a previous study we showed that the absence of the serine/threonine protein kinase (pU(S)3) encoded in the MDV unique-short region resulted in accumulation of primarily enveloped virions in the perinuclear space and significant impairment of virus growth in vitro. It was also shown that pU(S)3 is involved in actin stress fiber breakdown [Schumacher, D., Tischer, B. K., Trapp, S., and Osterrieder, N. (2005). Here, we constructed a recombinant virus to test the importance of pU(S)3 kinase activity for MDV replication and its functions in actin rearrangement. Disruption of the kinase active site was achieved by substituting a lysine at position 220 with an alanine (K220A). Titers of a kinase-negative MDV mutant, 20U(S)3()K220A, were reduced when compared to parental virus similar to those of the U(S)3 deletion mutant. We were also able to demonstrate complete absence of phosphorylation of MDV-specific phosphoprotein pp38 in cells infected with the kinase-deficient virus, indicating that pp38 phosphorylation depends entirely on the kinase activity of pU(S)3. Enzymatically inactive pU(S)3()K220A was, however, still capable of mediating breakdown of the actin cytoskeleton in transfection studies, and this activity was indistinguishable from that of wild-type pU(S)3(). Furthermore, we demonstrated that pU(S)3 possesses anti-apoptotic activity, which is dependent on its kinase activity. Taken together, our results demonstrate that pU(S)3 and MDV-specific phosphoprotein pp38 represent a kinase-substrate pair and that growth impairment in the absence of pU(S)3 is caused by the absence of kinase activity. The unaltered disruption of F-actin by the K220A pU(S)3 mutant suggests that F-actin disassembly is unrelated to MDV growth restrictions in the absence of the unique-short protein kinase.
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PMID:Enzymatically inactive U(S)3 protein kinase of Marek's disease virus (MDV) is capable of depolymerizing F-actin but results in accumulation of virions in perinuclear invaginations and reduced virus growth. 1830 99

MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27(Kip1). Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27(Kip1) 3'-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27(Kip1) in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27(Kip1) levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27(Kip1) in the induction and progression of T-cell lymphomas.
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PMID:MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1. 1926 8

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.
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PMID:Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1. 2614 91

The Herpesviridae encode many conserved genes, including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture to various degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek's disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken to chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and Western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread and autophosphorylation of CHPK and that mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens, and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread.IMPORTANCE Marek's disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek's disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation but do not block interindividual spread from chicken to chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek's disease while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined that MDV CHPK is not required for cell-to-cell spread, for disease induction, and for oncogenicity. However, it is required for interindividual spread, and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.
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PMID:Expression of the Conserved Herpesvirus Protein Kinase (CHPK) of Marek's Disease Alphaherpesvirus in the Skin Reveals a Mechanistic Importance for CHPK during Interindividual Spread in Chickens. 3180 54

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, U_S3. The functions of U_S3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV U_S3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV U_S3 and identified chicken CREB (chCREB) as a substrate of MDV U_S3. We show that wild-type MDV U_S3, but not kinase-dead U_S3 (U_S3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using U_S3 deletion and U_S3 kinase-dead recombinant MDV, we identified U_S3-responsive MDV genes during infection and found that the majority of U_S3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some U_S3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by U_S3. Furthermore, biochemical studies demonstrated that MDV U_S3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a U_S3 substrate. MDV U_S3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. U_S3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV U_S3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.
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PMID:Role of Marek's Disease Virus (MDV)-Encoded U_S3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation. 3258 Oct 93


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