RESUMO
Epstein-Barr virus (EBV) recombinants which carry three different deletion mutations in the LMP2A cytoplasmic amino-terminal domain were constructed. The presence of each mutation, LMP2A delta 21-36, LMP2A delta 21-64, and LMP2A delta 21-85, in EBV-infected transformed lymphoblastoid cell lines was confirmed by PCR analysis and Southern blot hybridization. Confirmation of mutant LMP2A protein expression was by immunofluorescence and immunoblotting with a newly identified rat monoclonal antibody that recognizes each of the LMP2A deletion mutations. Lymphoblastoid cell lines infected with recombinant EBV DNAs containing the mutations were analyzed for loss of LMP2A's dominant-negative effect on surface immunoglobulin signal transduction by monitoring induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication following surface immunoglobulin cross-linking. Domains of LMP2A important for induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication were identified.
Assuntos
Linfócitos B/imunologia , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , Proteínas da Matriz Viral/fisiologia , Replicação Viral , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular Transformada , Primers do DNA , DNA Viral , Imunofluorescência , Vetores Genéticos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Dados de Sequência Molecular , Mutagênese , Proteínas Oncogênicas Virais/fisiologia , Fosfotirosina/análise , Reação em Cadeia da Polimerase , Ratos , Recombinação Genética , Deleção de Sequência , Proteínas da Matriz Viral/biossíntese , Proteínas da Matriz Viral/químicaRESUMO
The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.
Assuntos
Herpesvirus Humano 4/fisiologia , Proteínas Virais/fisiologia , Replicação Viral/genética , Linfócitos B/virologia , Sequência de Bases , Linhagem Celular , Transformação Celular Viral , Clonagem Molecular , DNA Viral , Deleção de Genes , Herpesvirus Humano 4/genética , Humanos , Dados de Sequência Molecular , Proteínas do Envelope Viral , Proteínas Virais/genéticaRESUMO
Feeding experiments with Actinoplanes sp. SN223/29 showed that 3-amino-5-hydroxy-[7-13C]benzoic acid is not incorporated into acarbose (I). The valienamine moiety of I is thus not derived in the same way, from the shikimate pathway, as the m-C7N units in the ansamycin, mitomycin and ansamitocin antibiotics. Feeding experiments with [U-13C3]-glycerol followed by analysis of I by multiple quantum NMR spectroscopy support this conclusion and point to formation of the valienamine moiety by cyclization of a heptulose phosphate which arises from a triose phosphate via successive transfer of two 2-carbon fragments by transketolase, as proposed by Pape and co-workers.