Biomedical subjects

C M Horvath

Publications and source records attributed to C M Horvath.

28 records · Page 2Linked to original sources

PubMed1996

The antiviral state induced by alpha interferon and gamma interferon requires transcriptionally active Stat1 protein.

Both of the latent transcription factors activated by alpha interferon or gamma interferon contain the Stat1 protein. The role of Stat1 in virus interference mediated by interferons was directly examined by using cultured cells expressing Stat1 protein and its variants. In the absence of Stat1, no antiviral state is established. Full complementation of the antiviral state requires full-length Stat1 which is phosphorylated on both tyrosine 701 and serine 727. The closely related signal transducer and activator of transcription protein Stat3 cannot substitute for the antiviral properties of Stat1.

Antiviral Agents↗

PubMed1996

Interactions between STAT and non-STAT proteins in the interferon-stimulated gene factor 3 transcription complex.

The first STAT-containing transcription factor to be studied, the alpha-interferon-induced ISGF3, is composed of a Stat1:2 heterodimer and a weak DNA-binding protein, p48, that is a member of a growing family of proteins similar to the so-called interferon regulatory factor (IRF-1). The p48 and Stat1:2 heterodimer do not associate stably in the absence of DNA, but we show that amino acids approximately 150 to 250 of Stat1 and a COOH-terminal portion of p48 exhibit physical interaction, implying contact that stabilizes ISGF3. Moreover, amino acid exchanges within the Stat1 contact region diminish or abolish the functional activity of Stat1. This protein interaction domain may be important in other STAT proteins to recruit partners to multiprotein transcription factors.

DNA-Binding Proteins↗

PubMed1995

A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain.

Stat1 and Stat3 are two members of the ligand-activated transcription factor family that serve the dual functions of signal transducers and activators of transcription. Whereas the two proteins select very similar (not identical) optimum binding sites from random oligonucleotides, differences in their binding affinity were readily apparent with natural STAT-binding sites. To take advantage of these different affinities, chimeric Stat1:Stat3 molecules were used to locate the amino acids that could discriminate a general binding site from a specific binding site. The amino acids between residues approximately 400 and approximately 500 of these approximately 750-amino-acid-long proteins determine the DNA-binding site specificity. Mutations within this region result in Stat proteins that are activated normally by tyrosine phosphorylation and that dimerize but have greatly reduced DNA-binding affinities.

Amino Acid Sequence↗

PubMed1994

Transcription factor ISGF-3 formation requires phosphorylated Stat91 protein, but Stat113 protein is phosphorylated independently of Stat91 protein.

Transcription factor ISGF-3 is a multiprotein, interferon alpha-activated transcription complex consisting of a 48-kDa DNA-binding protein and two proteins termed Stats (for signal transducers and activators of transcription) that become phosphorylated on tyrosine in the cell cytoplasm, a 113-kDa and either a 91- or 84-kDa polypeptide, the latter two of which arise from differentially spliced mRNAs. Using cell lines lacking the Stat91 or Stat84 proteins, we show that mutations in several different sites in the 91-kDa protein block the interferon alpha-induced phosphorylation of the 91-kDa protein and subsequent ISGF-3 formation. Although correct tyrosine phosphorylation on residue 690 of the Stat113 protein occurs independent of the Stat91/84 protein, the Stat113 phosphoprotein by itself moves to the cell nucleus much less efficiently in the absence of phosphorylated Stat91/84 protein.

Amino Acid Sequence↗

PubMed1994

Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions.

Stat91 (a 91 kd protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands, including interferon alpha (IFN-alpha) and IFN-gamma. We report here that the inactive Stat91 in the cytoplasm of untreated cells is a monomer and that upon IFN-gamma-induced phosphorylation it forms a stable homodimer. Only the dimer is capable of binding to a specific DNA sequence directing transcription. Through dissociation and reassociation assays, we show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions. Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes.

Amino Acid Sequence↗

PubMed1993

Analysis of the trk NGF receptor tyrosine kinase using recombinant fusion proteins.

Nerve growth factor (NGF) represents a family of structurally related trophic factors, including brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5. These neurotrophin factors interact with two classes of receptors, the trk receptor tyrosine kinase family, and the low affinity p75 neurotrophin receptor. To study potential ligand-receptor interactions, recombinant trk fusion proteins have been constructed, and pan-trk polyclonal antisera directed against the cytoplasmic tyrosine kinase domain have been generated. The recombinant proteins were assessed for in vitro kinase activity and for the ability of K-252a to inhibit phosphorylation. Antibodies made against the fusion protein recognize all trk family members, and are effective in immunoprecipitation of affinity-crosslinked receptors. Comparative crosslinking indicates that NGF can recognize all trk receptor members, illustrating the large number of potential ligand-receptor interactions between neurotrophins and their receptors.

Animals↗

PubMed1992

Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed.

Amino Acid Sequence↗

PubMed1992

Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia.

The fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the paramyxovirus simian virus 5 (SV5) were expressed individually or coexpressed in CV-1 cells by using SV40-based vectors and recombinant vaccinia viruses. The extent of detectable fusion in a syncytium formation assay was found to be affected by the expression system used. In addition, when HN was coexpressed with F, it was found that the expression vector system influenced the contribution of HN in forming syncytia. The abilities of the SV5, human parainfluenza virus type 3, and Newcastle disease virus F glycoproteins to cause fusion, when expressed alone or coexpressed with HN, were directly compared by using the SV40-based vector system in CV-1 cells. The F proteins exhibited various degrees of fusion activity independent of HN expression, but the formation of syncytia could be enhanced to different extents by the coexpression of the homotypic HN protein.

Cell Line↗

PubMed1991

Diversity of coding strategies in influenza viruses.

Influenza viruses have exploited a variety of strategies to increase their genome coding capacities. These include unspliced, spliced, alternatively spliced and bicistronic mRNAs, translation from overlapping reading frames and a coupled stop-start translation of tandem cistrons.

Base Sequence↗

PubMed1990

Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.

Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.

Animals↗