RESUMO
Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.
Assuntos
Herpesvirus Humano 3/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Sequência de Aminoácidos , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infected human ganglia and encodes a 278-amino-acid protein, IE63, with immediate-early kinetics. IE63 is expressed in the cytoplasm of neurons during VZV latency and in both the cytoplasm and the nucleus during productive infection; however, the mechanism(s) involved in IE63 nuclear import and retention has remained unclear. We constructed and identified a recombinant monoclonal antibody to detect a posttranslationally modified form of IE63. Analysis of a series of IE63 truncation and substitution mutants showed that amino acids 186 to 195 are required for antibody binding. Synthetic peptides corresponding to this region identified IE63 S186 as a target for casein kinase II phosphorylation. In addition, acidic charges supplied by E194 and E195 were required for antibody binding. Immunofluorescence analysis of VZV-infected MeWo cells using the recombinant monoclonal antibody detected IE63 exclusively in the nuclei of infected cells, indicating that casein kinase II phosphorylation of S186 occurs in the nucleus and possibly identifying an initial molecular event operative in VZV reactivation.
Assuntos
Caseína Quinase II/metabolismo , Herpesvirus Humano 3/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Serina/metabolismo , Proteínas do Envelope Viral/metabolismo , Anticorpos Antivirais/metabolismo , Linhagem Celular , Núcleo Celular/química , Humanos , Fosforilação , Ligação ProteicaRESUMO
Activated protein C (APC) resistance is a common risk factor for venous thromboembolism (VTE) attributed to various mechanisms, including factor V Leiden (FVL) polymorphism. FVL is considered responsible for up to 95% of APC resistance; however, other factor V polymorphisms and elevated factor VIII levels also have been implicated. We assessed whether additional mechanisms contribute to APC resistance in a blinded case-control study of 65 subjects by measuring APC resistance using 3 methods: 2 activated partial thromboplastin time-based methods with and without dilution in factor V-deficient plasma and 1 Russell viper venom-based assay (RVV). Without factor V-deficient plasma, 24 subjects were APC resistant; with factor V-deficient plasma, the assay identified fewer APC-resistant subjects, as did RVV. All assays detected the 7 heterozygous FVL subjects. Thirteen subjects had factor VIII levels above 150% (1.50). After excluding subjects with FVL or elevated factor VIII levels, 4 subjects still had APC resistance. VTE risk trended higher for subjects with APC resistance in the absence of FVL. Measurement of APC resistance without dilution in factor V-deficient plasma is needed to assess for potentially important thrombotic risk factors other than FVL.
Assuntos
Resistência à Proteína C Ativada/genética , Fator V/genética , Predisposição Genética para Doença , Trombose Venosa/etiologia , Resistência à Proteína C Ativada/sangue , Testes de Coagulação Sanguínea/métodos , Estudos de Casos e Controles , DNA/análise , Fator V/análise , Fator VIII/análise , Genoma Humano , Haplótipos , Humanos , Pessoa de Meia-Idade , Polimorfismo Genético , Fatores de Risco , Método Simples-Cego , Trombose Venosa/patologiaRESUMO
Five varicella zoster virus (VZV) genes are known to be transcribed in latently infected human ganglia. Transcripts from VZV gene 63, which encodes an immediate early (IE) protein, are the most prevalent and abundant. To obtain a reagent that might facilitate studies of the role of the IE63 protein in latency and reactivation, we selected an IE63-specific Fab fragment from a phage library and used it to prepare a recombinant mouse IgG1 antibody that detects IE63 and functions in Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assays.