RESUMO
Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.
Assuntos
Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas da Matriz Viral/genética , Quinases da Família src/metabolismoRESUMO
Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membrane of B-lymphocytes and blocks B cell receptor (BCR) signaling in EBV-transformed B-lymphocytes in vitro. The LMP2A amino-terminal domain, which is essential for the LMP2A-mediated block of B cell signal transduction, contains eight tyrosine residues. Three of these tyrosine residues (Y74, Y85, and Y112) have been demonstrated to be essential for the LMP2A-mediated block on protein tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression after BCR activation. To investigate the importance of tyrosines at positions 60, 64, and 101 on B cell signaling, EBV recombinants were constructed containing a tyrosine-to-phenylalanine point mutation at amino acid 60, 64, or 101 within LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were not affected by any of the tyrosine point mutations after BCR activation. In addition, constitutive phosphorylation of LMP2A was unaffected by any of the tyrosine point mutations. These data indicate that tyrosines 60, 64, and 101 are not essential for the LMP2A-mediated block of B cell signal transduction in transformed cell lines.
Assuntos
Linfócitos B/metabolismo , Herpesvirus Humano 4/metabolismo , Transdução de Sinais , Tirosina/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Proteínas Virais , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Linfócitos B/virologia , Cálcio/metabolismo , Sinalização do Cálcio , Callithrix , Linhagem Celular , Linhagem Celular Transformada , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/genética , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , Fosforilação , Fosfotirosina/metabolismo , Mutação Puntual/genética , Agregação de Receptores , Receptores de Antígenos de Linfócitos B/metabolismo , Transativadores/metabolismo , Tirosina/genética , Proteínas da Matriz Viral/genéticaRESUMO
Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.
Assuntos
Herpesvirus Humano 4/fisiologia , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Virais , Latência Viral , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Primers do DNA , DNA Complementar , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Transativadores/genética , Células Tumorais Cultivadas , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B lymphocytes in vitro. The cytoplasmic amino-terminal domain of LMP2A contains an immunoreceptor tyrosine activation motif (ITAM). ITAMs consist of paired tyrosine and leucine residues and play a central role in signal transduction of the BCR and the T-cell receptor (TCR). To investigate the importance of the LMP2A ITAM, two EBV recombinants were constructed, each containing a tyrosine-to-phenylalanine point mutation at amino acid 74 or 85 within the ITAM of LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were no longer blocked in the LMP2A ITAM mutant LCLs following BCR cross-linking. In addition, the Syk protein tyrosine kinase (PTK) was unable to bind LMP2A in unstimulated LCLs infected with either of the LMP2A ITAM mutants. Analysis of Syk phosphorylation before and after BCR cross-linking in the LMP2A mutant ITAM LCLs compared with wild-type EBV LCLs indicates a specific role of the LMP2A ITAM on the LMP2A-mediated negative effect on the Syk PTK. These data indicate the importance of the LMP2A ITAM motif in the LMP2A-mediated block on BCR signal transduction and position the role of the Syk PTK as being central to the function of LMP2A.
Assuntos
Receptores de Antígenos de Linfócitos B/fisiologia , Proteínas da Matriz Viral/química , Sequência de Aminoácidos , Células Cultivadas , Precursores Enzimáticos/metabolismo , Imunofluorescência , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase Syk , Transfecção , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/fisiologiaRESUMO
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.