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J L Strominger

Publications and source records attributed to J L Strominger.

At least 109 records · Page 6Linked to original sources
PubMed1996

A simple reverse transcriptase PCR assay to distinguish EBNA1 gene transcripts associated with type I and II latency from those arising during induction of the viral lytic cycle.

In Epstein-Barr virus (EBV)-associated tumors that arise in immunocompetent individuals, the pattern of viral gene expression is very restricted compared with that of latently infected B cells in tissue culture. A hallmark of viral gene expression in these tumors is the exclusive expression of only one EBV-encoded nuclear antigen, EBNA1, which is driven from a promoter (Qp) that lies near the junction of the viral BamHI F and Q fragments. During induction of the lytic cycle, a viral promoter, Fp, which lies ca. 200 bp upstream of Qp, gives rise to transcripts which overlap with Qp-initiated EBNA1 gene transcripts. Distinguishing between latency-associated EBNA1 gene transcripts and those associated with the early phase of the viral lytic cycle is critical for correct identification of restricted viral latency. Here we describe a reverse transcriptase PCR protocol which employs a nested set of upstream primers from the BamHI Q region of the viral genome and readily distinguishes Fp-initiated transcripts from Qp-initiated transcripts. A single set of amplification conditions was used for the various PCR primer combinations, which allowed all reactions to be run simultaneously. An in vitro-generated transcript, diluted in RNA from an EBV-negative cell line, was used to demonstrate that the efficiencies of amplification with the different primer combinations were very similar. This protocol was used to demonstrate that EBNA1 gene transcription in two previously uncharacterized EBV-positive epithelial cell lines initiates from Qp. In addition, we assessed the site(s) of initiation of EBNA1 gene transcripts in cell lines exhibiting restricted viral latency. Contrary to the results of Nonkwelo et al. (J. Virol. 70:623-627, 1996), which indicated that EBNA1 gene transcription during restricted viral latency initiates at multiple sites downstream of Fp, we show here that nearly all EBNA1 transcripts start at the previously identified Qp transcription initiation site.

Cell Line↗
PubMed1996

Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction.

Induction of the Epstein-Barr virus lytic cycle is mediated through the immediate-early BZLF1 gene and the coordinately regulated BRLF1 gene. The BZLF1 gene product, Zta, transactivates its own promoter, as well as the promoters of a number of lytic genes, thereby initiating a cascade of viral gene expression. Previous work identified four related elements (ZIA, ZIB, ZIC, and ZID) and a cyclic AMP response element binding-AP-1 element (ZII) that are involved in the induction of the BZLF1 promoter (Zp) by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (E. Flemington and S. H. Speck, J. Virol. 64:1217-1226, 1990). Here we report a detailed characterization of TPA induction mediated by the ZI domains. Mutation of individual ZI domains within the context of the intact promoter significantly diminished TPA induction. Cloning of individual ZI domains upstream of a minimal promoter demonstrated that the ZIA, ZIC, and ZID domains, but not the ZIB domain, are TPA responsive. Furthermore, cloning of the ZII domain downstream of the ZI domains significantly augmented TPA induction. The critical regions within the ZIA and ZIC elements involved in binding of cellular factors were identified by using methylation interference and electrophoretic mobility shift analyses of ZI domain mutants. Four specific complexes were observed with the ZIA and ZID domains, all of which could be specifically competed for by either the ZIA or ZID domain. Methylation interference analyses of bound complexes revealed the presence of two overlapping binding sites for cellular factors in the ZIA domain, and functional studies provided evidence that both of these sites are involved in TPA induction. Functional analyses of the ZIC domain revealed that the 5' region of this domain is largely responsible for mediating TPA induction. Binding data correlated well with functional activity and revealed that the ZIC domain binds only a subset of the cellular factors that bind to the ZIA and ZID domains. Analysis of factor binding to the ZIB domain revealed only a single shifted complex, which correlated with the most slowly migrating complex observed with the ZIA and ZID domains. These data provide a direct demonstration of TPA induction mediated by the ZIA, ZIC, and ZID domains and also provide the first evidence that the ZI domains exhibit distinct functional characteristics.

Base Sequence↗
PubMed1995

Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

B-Lymphocytes↗
PubMed1995

Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region.

A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation.

Amino Acid Sequence↗
PubMed1995

Structural basis for major histocompatibility complex (MHC)-linked susceptibility to autoimmunity: charged residues of a single MHC binding pocket confer selective presentation of self-peptides in pemphigus vulgaris.

Human T-cell-mediated autoimmune diseases are genetically linked to particular alleles of MHC class II genes. Susceptibility to pemphigus vulgaris (PV), an autoimmune disease of the skin, is linked to a rare subtype of HLA-DR4 (DRB1*0402, 1 of 22 known DR4 subtypes). The PV-linked DR4 subtype differs from a rheumatoid arthritis-associated DR4 subtype (DRB1*0404) only at three residues (DR beta 67, 70, and 71). The disease is caused by autoantibodies against desmoglein 3 (DG), and T cells are thought to trigger the autoantibody production against this keratinocyte adhesion molecule. Based on the DRB1*0402 binding motif, seven candidate peptides of the DG autoantigen were identified. T cells from four PV patients with active disease responded to one of these DG peptides (residues 190-204); two patients also responded to DG-(206-220). T-cell clones specific for DG-(190-204) secreted high levels of interleukins 4 and 10, indicating that they may be important in triggering the production of DG-specific autoantibodies. The DG-(190-204) peptide was presented by the disease-linked DRB1*0402 molecule but not by other DR4 subtypes. Site-directed mutagenesis of DRB1*0402 demonstrated that selective presentation of DG-(190-204), which carries a positive charge at the P4 position, was due to the negatively charged residues of the P4 pocket (DR beta 70 and 71). DR beta 71 has a negative charge in DRB1*0402 but a positive charge in other DR4 subtypes, including the DR4 subtypes linked to rheumatoid arthritis. The charge of the P4 pocket in the DR4 peptide binding site therefore appears to be a critical determinant of MHC-linked susceptibility to PV and rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1995

Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors.

Antigens, Viral↗
PubMed1995

Human class II MHC molecule HLA-DR1: X-ray structure determined from three crystal forms.

The three-dimensional structure of the extracellular region of a 60 kDa class II major histocompatibility glycoprotein, HLA-DR1, was determined to 3.3 A by X-ray crystallography using three crystal forms, each containing two molecules per asymmetric unit. Phases were initially determined to 4.2 A using two crystal forms both containing DR1 from human lymphocytes complexed with a mixture of endogenous peptides. One of these crystal forms also contained a 28 kDa superantigen, Staphylococcus aureus enterotoxin B (SEB), bound to each DR1 molecule. Single-isomorphous replacement phasing followed by iterative two- and fourfold non-crystallographic real-space averaging between the two crystal forms resulted in 4.2 A resolution electron-density maps from which the paths of the polypeptides could be traced. Cryocrystallography and synchrotron radiation were then used to extend the resolution to 3.3 A for the two lymphocyte-derived crystal forms and for a third crystal form grown from DR1 produced in insect cells and complexed in vitro with a specific antigenic peptide. Iterative sixfold non-crystallographic real-space averaging resulted in an electron- density map into which 340 of 371 residues could be fit unambiguously. Crystal contacts and the existence of a parallel dimer of the DR1 alphabeta heterodimer in the three crystal forms are discussed.

Journal Article↗
PubMed1995

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.

Amino Acid Sequence↗
PubMed1995

Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II.

The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.

Amino Acid Sequence↗
PubMed1995

Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression.

The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.

Base Sequence↗
PubMed1995

Cooperativity between the J and S elements of class II major histocompatibility complex genes as enhancers in normal and class II-negative patient and mutant B cell lines.

The class II major histocompatibility complex genes all contain in their proximal promoters three cis-elements called S, X, and Y that are conserved in both sequence and position, and a fourth element, J, conserved in sequence but not in position. J, X, and Y and, to some extent, S, have been shown to be functionally important in regulation of expression of these genes. In the present study, a protein factor that binds cooperatively to the S plus J elements of the promoter of the class II major histocompatibility complex gene DPA has been detected. Moreover, functional cooperativity between S and J in activation of the enhancerless -40 interferon-beta (-40 IFN-beta) promoter has been demonstrated. Finally, the latter assay appears to subdivide complementation group A of class II negative human B cell lines that includes both mutants generated in vitro and cells from patients with the bare lymphocyte syndrome (type II). In three of these cell lines, the enhancerless -40 IFN-beta promoter containing the S plus J elements was functionally active, while in the others it was inactive.

B-Lymphocytes↗
PubMed1995

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.

Amino Acid Sequence↗
PubMed1995

Activation of transcription by binding of NF-E1 (YY1) to a newly identified element in the first exon of the human DR alpha gene.

A previously unrecognized element, located downstream of the start site of transcription in the first exon of the DR alpha gene, has been defined that enhances promoter activity up to eightfold in a position-dependent manner. Mutations in this DNA-binding site abolished binding of a nuclear factor in human B cell nuclear extract and decreased the activity of the DR alpha promoter to a basal level. Significant sequence homology of this element was found in the DNA of the DR beta, DP alpha and -beta, and DQ alpha genes, always located downstream of the transcriptional start site. The nuclear factor binds to the DR alpha and DP alpha element but not to the element in the DQ alpha gene. It was identified as NF-E1 (YY1). This protein, previously identified by its binding to the Ig kappa 3' enhancer and the Ig heavy chain mu E1 site, thus also appears to be quite important in the regulation of MHC class II gene expression.

B-Lymphocytes↗
PubMed1995

Cyclosporin A and FK506 block induction of the Epstein-Barr virus lytic cycle by anti-immunoglobulin.

The Epstein-Barr virus (EBV) BZLF1 gene is expressed early upon induction of the viral lytic cycle and its protein product is unique in its ability to disrupt viral latency in some latently infected cell lines. Anti-immunoglobulin (anti-Ig) treatment of the Burkitt's lymphoma cell line Akata, which bears surface IgG, has previously been shown to synchronously induce transcription of the BZLF1 gene (K. Takada and Y. Ono, 1989, J. Virol. 63, 445-449). We have previously shown that anti-Ig induction of Akata cells activates expression of the tumor necrosis factor alpha (TNF-alpha) gene via a calcineurin-dependent mechanism (Goldfeld et al., 1992, Proc. Natl. Acad. Sci. USA 89, 12198-12201). Here, we report that anti-Ig induction of the EBV lytic cycle in Akata cells can be blocked by the immunosuppressants cyclosporin A and FK506. Furthermore, we demonstrate that synergistic induction by phorbol ester and calcium ionophore of a BZLF1 promoter-driven reporter construct in an EBV-negative BL cell line can be inhibited by addition of cyclosporin A. Thus, analogous to activation of TNF-alpha gene in Akata cells, anti-Ig induction of the BZLF1 promoter is most likely mediated by calcineurin and probably involves translocation to the nucleus of a transcription factor sequestered in the cytoplasm. As such, immunosuppressants may be useful probes for dissecting B cell activation pathways involved in regulating EBV gene transcription.

Antibodies, Anti-Idiotypic↗
PubMed1995

Regulation of T-cell antigen receptor (TCR) alpha-chain expression by TCR beta-chain transcripts.

The TCR is an alpha beta heterodimer, a part of the multimeric structure through which physiological T-cell activation occurs. The expression of TCR alpha chain is greatly diminished in a beta-chain-deficient mutant Jurkat cell line (J.RT3-T3.5). The relationship between the expression of the TCR alpha and beta chains has been examined by stable transfection of a series of TCR beta-chain mutant constructs into this mutant cell line. The level of alpha-chain transcript was dramatically upregulated by the expression of the beta chain and specifically by a transcript of the beta-chain variable region alone, including a transcript in which the ATG start codon was mutated. The downregulation of the endogenous alpha-chain transcripts in mutants cells lacking complete beta-chain transcripts occurred primarily at the posttranscriptional level. This evidence for a regulatory function of the TCR beta-chain gene represents an unusual regulatory pathway in which the transcript of one gene is required for the optimal expression of another gene.

Amino Acid Sequence↗
PubMed1995

Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein.

Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class II binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class II binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity.

Amino Acid Sequence↗