Viral Motifs Research Articles

T-cell reactivity to GAD65 peptide sequences shared with coxsackie virus protein in recent-onset IDDM, post-onset IDDM patients and control subjects

GAD65 is one of the major autoantigens associated with insulin-dependent diabetes mellitus (IDDM). The two peptides p17 and p18 of GAD65 that share sequence similarity with coxsackie virus (amino acid sequence identity: PEVKEK) appeared to be the major determinants of GAD65 recognized preferably by T cells from new-onset IDDM patients and their first degree relatives. In contrast, in our study unrelated control subjects frequently recognized the two GAD peptides (55%, 16/29), similar to first degree relatives (41%, 12/29) and IDDM patients post-onset (68%, 15/22). However, recent-onset IDDM patients, responded less frequently (25%, 4/16) compared with IDDM patients post-onset (p < 0.03) or unrelated control subjects (borderline significant) confirming previous observations in humans and NOD mice that T-cell reactivity to GADp17/p18 at diabetes onset is decreased. Moreover, this study demonstrated a positive correlation of T-cell proliferation to GAD p17 (amino acid 247-266) and p18 (amino acid 260-279) with simultaneous responses to both peptides in 13% of all subjects tested (n = 97) (p < 0.001). T-cell proliferation to GAD p17 was higher than to p18 in recent-onset diabetic patients, first degree relatives and unrelated control subjects (p < 0.02, p < 0.004, p < 0.002, respectively). However, in post-onset IDDM patients, the two peptides were recognized equally well. Our results show that T-cell reactivity to GAD65, peptides homologous with coxsackie protein is very frequently observed, but not primarily associated with IDDM. The temporary decline of T-cell proliferation is not associated with the beta-cell destruction process, but with clinical manifestation. The positive correlation of reactivity to the two peptides in the viral motif implicates that PEVKEK is an immunogenic epitope.

A Novel Gene,G7e,Resembling a Viral Envelope Gene, Is Located at the Recombinational Hot Spot in the Class III Region of the Mouse MHC

DNA sequence analysis of a segment of 15 kb, situated betweenG7bandG7aand present in the mouse but absent in human, revealed about 11 kb of DNA harboring a large number of repetitive sequences and 4 kb harboring a novel gene,G7e.This gene is transcribed in lymphoid tissues, having a 3-kb mRNA. The cDNA sequence ofG7eshows stretches of nucleotide homology with murine leukemia virus (MuLV) envelope genes, and the predicted protein encompasses viral envelope motifs. The finding of a gene resembling MuLV envelope genes, flanked by a long terminal repeat andgag- andpol-like sequences, leads to the assumption thatG7bandG7ain the mouse were separated through the insertion of a provirus, an event that might have taken place even before speciation of rat and mouse. The 15-kb interval forms a part of a 50-kb region, betweenHsp70.3andG7,where recombination preferentially takes place. Several disease susceptibility genes have been mapped to this same interval. The position ofG7ein or in the vicinity of a recombinational hot spot might not be coincidental. The presence of adjacent putative recombination regulatory sequences is suggestive for the location of the crossover sites of recombination in this interval.

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin ( α Vβ 3/β 5) and fibronectin ( α 5 β 1) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.

Transcription factors interacting with herpes simplex virus alpha gene promoters in sensory neurons.

Interference with VP16-mediated activation of herpes virus immediate-early (or alpha) genes is thought to be the major cause of establishing viral latency in sensory neurons. This could be brought about by lack of a key activating transcription factor(s) or active repression. In this study we find that sensory neurons express all important components for VP16-mediated alpha gene induction, such as the POU transcription factor Oct-1, host cell factor (HCF) and GABP alpha/beta. However, Oct-1 and GABP alpha/beta are only present at low levels and the VP16-induced complex (VIC) appears different. We do not find protein expression of the transcription factor Oct-2, implicated by others as an alpha gene repressor. The POU factor N-Oct3 (Brn 2 or POU3F2) is also present in sensory neurons and binds viral TAATGARAT motifs with higher affinity than Oct-1, indicating that it may be a candidate repressor for competitive binding to TAATGARAT motifs. When transfected into HeLa cells, where Oct-1 and GABP alpha/beta are highly abundant, N-Oct3 represses model promoters with multimerized TAATGARAT motifs, but fails to repress complete alpha gene promoters. Taken together our findings suggest that modulation of alpha gene promoters could contribute to viral latency when low concentrations of the activating transcription factors Oct-1 and GABP alpha/beta prevail. Our data, however, refute the notion that competing Oct factors are able to block alpha gene transcription to achieve viral latency.

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.

Amino-aciduria and copper metabolism in hepatolenticular degeneration.

<h3>Abstract</h3> First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by germline-encoded pattern recognition receptors, followed by activation of the type I IFN system and establishment of an intracellular antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. Activin A mediates multiple innate and adaptive immune functions – including antiviral effects. However, how such effects are mediated and how activin might be triggered by viral infection have not been defined. Here we addressed this in vivo and in vitro, in humans and mice. Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant activin A and IFNα <i>in vitro</i>. Activin A mRNA, encoded by <i>INHBA</i>, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors <i>in vitro</i>, across multiple cell lines and in human peripheral blood mononuclear cells. <i>In vivo</i>, infection of mice with influenza A also upregulated <i>Inhba</i> mRNA in the lung; this local upregulation of <i>Inhba</i> is retained in MAVS knockout mice, indicating a role for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis. Together, these data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions. This model has implications for the development of targeted antiviral therapies, in addition to revealing novel facets of activin biology.