RESUMO
Overexpression of the cellular oncogene c-Myc frequently occurs during induction of leukemias and lymphomas in many species. Retroviruses have enhanced our understanding of the role of c-Myc in such tumors. Leukemias and lymphomas induced by retroviruses activate c-Myc by: (1) use of virally specified proteins that increase c-Myc transcription, (2) transduction and modification of c-Myc to generate a virally encoded form of the gene, v-Myc, and (3) proviral integration in or near c-Myc. Proviral integrations elevate transcription by insertion of retroviral enhancers found in the long terminal repeat (LTR). Studies of the LTR enhancer elements from these retroviruses have revealed the importance of these elements for c-Mycactivation in several cell types. Retroviruses also have been used to identify genes that collaborate with c-Myc during development and progression of leukemias and lymphomas. In these experiments, animals that are transgenic for c-Mycoverexpression (often in combination with the overexpression or deletion of known proto-oncogenes) have been infected with retroviruses that then insertionally activate novel co-operating cellular genes. The retrovirus then acts as a molecular 'tag' for cloning of these genes. This review covers several aspects of c-Myc involvement in retrovirally induced leukemias and lymphomas.
Assuntos
Leucemia/virologia , Linfoma/virologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Retroviridae/genética , Animais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/genética , Leucemia/metabolismo , Linfoma/genética , Linfoma/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas c-myc/genética , Sequências Repetidas Terminais , Integração ViralRESUMO
Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.
Assuntos
Apresentação de Antígeno/imunologia , Toxinas Bacterianas , Enterotoxinas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Superantígenos/metabolismo , Linfócitos T/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/classificação , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular , Enterotoxinas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-Ab cells (which expresses I-Ab that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-Ab-binding peptides. Here we investigate the contribution of I-Ab-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-Ab-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-Ab. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Toxinas Bacterianas , Enterotoxinas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Peptídeos/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Animais , Mapeamento de Epitopos , Hibridomas , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Nucleopoliedrovírus/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a DNA/genética , Mutagênese Sítio-Dirigida , Fosfoproteínas/genética , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Nucleopoliedrovírus/metabolismo , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Linhagem Celular , Núcleo Celular/virologia , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fosfoproteínas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/citologiaRESUMO
The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.
Assuntos
Baculoviridae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Animais , Baculoviridae/genética , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Immunoblotting , Imuno-Histoquímica , Insetos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Mapeamento por RestriçãoRESUMO
CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.