Biomedical subjects

S S Broyles

Publications and source records attributed to S S Broyles.

At least 19 recordsLinked to original sources

PubMed1999

Transcription factor YY1 is a vaccinia virus late promoter activator.

Vaccinia virus has a DNA genome, yet replicates in the cytoplasmic compartment of the cell. We previously described the identification of a cellular protein having high affinity for vaccinia virus late promoter DNA. Sequence substitutions in the vaccinia I1L promoter were used to define a 5-nucleotide block at the transcription initiation site as essential for interaction with the protein. Within this sequence is the recognition motif for the nuclear transcription factor YY1. This factor regulates a multitude of cellular promoters, as an activator of transcription, as a repressor, or as an initiator element-binding protein. Antibodies directed against YY1 were used to show that YY1 copurified with the vaccinia late promoter-binding protein and was present in late promoter-protein complexes in gel supershift assays. Bacterially expressed YY1 also bound specifically to late promoter DNA. A dinucleotide replacement within the YY1 recognition motif directly adjacent to the transcription start site severely reduced the affinity of YY1 for the I1L promoter in vitro and impaired I1L promoter-dependent transcription in vivo. The intracellular localization of YY1 was shown by immunofluorescence microscopy to shift from primarily nuclear to the cytoplasm after vaccinia infection. These results indicate that YY1 has a positive role in the regulation of vaccinia virus late gene transcription and suggest that poxviruses have adapted cellular initiator elements as a means of regulating viral gene expression. This is the first identifiable cellular protein implicated in poxvirus transcription.

Base Sequence↗

PubMed1998

A cellular protein binds vaccinia virus late promoters and activates transcription in vitro.

Available evidence indicates that the transcription of the late class of vaccinia virus genes requires the participation of several virus-encoded proteins in addition to the viral RNA polymerase. In this report we describe the identification of a protein present in extracts of uninfected HeLa cells that binds avidly to viral late promoter DNA. The protein bound specifically to several different vaccinia virus late promoters but not an early nor an intermediate promoter. DNase I footprinting localized the protein's binding site to nucleotides surrounding the transcriptional start site of the I1L promoter. Optimal promoter binding required sequences in the highly conserved TAAAT motif at the transcriptional start site as well as sequences immediately upstream; however, one variation on the motif's sequence did not affect promoter binding by the protein. Partially purified late promoter binding protein (LPBP) was capable of stimulating the transcription activity of extracts depleted of LPBP on a late promoter-driven template, establishing LPBP as a transcription activator in vitro. These results suggest that a cellular protein is responsible for targeting vaccinia virus late promoters for initiation of transcription.

Binding Sites↗

PubMed1995

The DNA binding domain of the vaccinia virus early transcription factor small subunit is an extended helicase-like motif.

The vaccinia virus early transcription factor (VETF) is an ATP-dependent activator of the early class of viral genes. VETF is a heterodimeric protein that binds an initiator-like element surrounding the start site of transcription. Previous studies indicated that the small subunit of VETF contacts the promoter DNA. We have taken a mutational approach to determine sequences in the VETF small subunit that are important for DNA binding. Two types of sequences were targeted for mutation: ones resembling motifs that are conserved in the nucleic acid helicase family and positively charged residues in predicted alpha-helices. Mutations affecting transcription activation were clustered in two regions. One mutation that impaired DNA binding is located near the N-terminus within the putative ATP-binding pocket that comprises helicase domain I. DNA binding was also severely reduced by mutations in a sequence resembling helicase domain VI and two putative alpha-helices that flank this domain in the C-terminal third of the polypeptide. These results indicate that the DNA binding domain in the small subunit of VETF is not isolated within a separable domain as is the case with most transcription factors, but rather, spans most of the length of the 637 residue polypeptide. A model for VETF structure is suggested in which the active site for ATP hydrolysis is integrated within an extended DNA-binding domain such that the structure and function of each domain influences that of the other.

Binding Sites↗

PubMed1994

Critical phosphorylation sites for acetyl-CoA carboxylase activity.

Acetyl-CoA carboxylase (ACC) is rapidly regulated by reversible phosphorylation; phosphorylation inactivates ACC, whereas dephosphorylation activates the enzyme. Among protein kinases only cAMP-dependent protein kinase and 5'-AMP-dependent protein kinase can inactivate ACC; cAMP-dependent protein kinase phosphorylates Ser-77 and -1200; 5'-AMP-dependent protein kinase phosphorylates Ser-79, -1200, and -1215. In this report, the construction and expression of ACC cDNA containing the entire coding region (7.2 kilobase pairs) is described. In order to identify the critical phosphorylation site(s) for each protein kinase, we introduced site-specific mutations at Ser-77, -79, -1200, and -1215 of ACC cDNA and a series of mutated ACCs containing various combinations of these four mutated sites was expressed. By examination of the various mutant ACCs, we provided evidence that the effect of cAMP-dependent protein kinase is entirely mediated by the phosphorylation of Ser-1200 and that Ser-79 is important for 5'-AMP-dependent protein kinase action in vitro.

Acetyl-CoA Carboxylase↗

PubMed1994

Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus.

The major protein kinase activity from vaccinia virus core particles was purified to near homogeneity. The protein kinase is a 50-kDa polypeptide that is shown here to phosphorylate primarily seryl residues in alpha-casein, a casein kinase I-specific peptide substrate, and itself through autophosphorylation. The sequence of four peptides derived from the protein kinase demonstrated that it is encoded by the vaccinia virus F10L gene. Expression of the F10L gene product in bacteria as a fusion with glutathione S-transferase confirmed that the vaccinia F10L gene encodes the protein kinase. We have termed this enzyme vaccinia protein kinase 2 (VPK2) to distinguish it from the protein kinase encoded by the vaccinia B1R gene. Targeted disruption of the VPK2 gene with a positive selectable marker demonstrated that all viruses with a disrupted gene also possessed a wild-type gene, suggesting that VPK2 is essential for viability. The discovery of a second essential protein kinase encoded by vaccinia virus, in addition to a protein phosphatase, underscores the importance of protein phosphorylation in poxvirus biogenesis.

Amino Acid Sequence↗

PubMed1994

Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components.

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.

Adenosine Triphosphatases↗

PubMed1993

The DNA-dependent ATPase activity of vaccinia virus early gene transcription factor is essential for its transcription activation function.

Vaccinia virus early transcription factor (VETF) activates the transcription of early gene templates by the viral RNA polymerase. VETF is a heterodimeric protein that binds to transcription promoters and has an associated DNA-dependent ATPase activity. The small subunit of VETF has sequences resembling two motifs commonly found in ATPases: an A-type ATP binding motif and a DEAH box. To investigate the functional role of the ATPase activity, we have analyzed the effect of mutations in each of the putative ATPase motifs. Recombinant VETF was expressed in HeLa cells using a vaccinia virus/T7 RNA polymerase system. Simultaneous expression of both subunits of VETF was required to obtain soluble protein with promoter binding, DNA-dependent ATPase, and transcription activation functions. The mutants with altered ATPase motifs retained promoter binding activity but had no detectable ATPase activity and no ability to activate transcription. The DEAH box mutant was shown to dominantly repress transcription activation by wildtype VETF. These results indicate that the DNA-dependent ATPase activity of VETF is essential for its transcription activation function.

Adenosine Triphosphatases↗

PubMed1993

Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor.

Transcription of vaccinia virus early genes in vitro requires the virally encoded RNA polymerase and early transcription factor, VETF. VETF is a promoter-binding protein with DNA-dependent ATPase activity. We have investigated the functional role of VETF in transcription activation by analyzing the interaction between the RNA polymerase and promoter DNA. Using a gel shift assay, a novel protein-DNA complex was detected that required both RNA polymerase and VETF. The complex was suggested to be a transcription initiation complex by its ability to incorporate 32P-labeled nucleotides in combinations compatible with synthesis of a short RNA chain. Competition binding studies indicated that the RNA polymerase associated specifically with a viral early promoter. These experiments demonstrate that VETF activates transcription by directly recruiting the RNA polymerase to the promoter. Sedimentation analysis showed that VETF and RNA polymerase did not form a stable complex unless promoter DNA was present, indicating that protein-protein contacts are not the sole basis for initiation complex assembly. DNase I cleavage and methylation interference analyses revealed a hyperreactive site in the center of the promoter. Radiolabeling of RNA in the RNA polymerase-promoter complex did not occur when AMP-PNP (adenyl-5'-yl imidodiphosphate) was substituted for ATP, suggesting that ATP hydrolysis is required for the initiation of transcription. A model is proposed to account for these findings.

Adenosine Triphosphatases↗

PubMed1993

Vaccinia virus encodes a functional dUTPase.

The sequence of the vaccinia virus open reading frame F2L predicts a polypeptide with significant similarity to cellular dUTPases. To determine whether the F2L gene product has this activity, it was expressed in bacteria as a fusion with glutathione S-transferase. Affinity purified fusion protein was shown to hydrolyze dUTP yielding dUMP as the product. While the dUTPase was not completely dependent on the addition of divalent cations, its activity was stimulated markedly by Zn2+, Mg2+, and Mn2+. The nucleotide substrate specificity of the enzyme was limited to dUTP. These results demonstrate that vaccinia virus encodes a functional dUTPase whose role in viral infection is suggested to be the augmentation of DNA nucleotide precursors and the minimization of cytoplasmic dUTP concentrations.

Chromatography, Thin Layer↗

PubMed1993

The small subunit of the vaccinia virus early transcription factor contacts the transcription promoter DNA.

The vaccinia virus early transcription factor (VETF), in addition to the viral RNA polymerase, is required for efficient transcription of early genes in vitro. VETF is a heterodimeric protein that binds specifically to early gene promoters. In order to localize the VETF DNA binding domain, we have used photoreactive oligonucleotide probes with the sequence of the vaccinia virus growth factor promoter. The probes consisted of double-stranded oligonucleotides incorporating radiolabeled dAMP and 5-bromo-dUMP into sequences of the promoter known to contact VETF. Irradiation of a DNA probe having these nucleotides located upstream of the transcription start site in the presence of VETF resulted in the transfer of label to a polypeptide that comigrated with the small subunit of VETF. The label transfer reaction was shown to occur with the recombinant VETF small subunit in the absence of the large subunit. These results indicate that the small subunit comprises at least part of the VETF DNA binding domain and contacts the promoter in the region upstream of the transcription start site.

Base Sequence↗

PubMed1992

Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues.

Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.

Amidophosphoribosyltransferase↗

PubMed1992

The vaccinia virus B1R gene product is a serine/threonine protein kinase.

The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.

Adenosine Triphosphate↗

PubMed1991

Promoter DNA contacts made by the vaccinia virus early transcription factor.

Vaccinia virus RNA polymerase requires the heterodimeric protein, vaccinia early transcription factor (VETF), for transcription of early gene templates in vitro. We have analyzed the vaccinia growth factor promoter sequences interacting with VETF at the nucleotide level and provide evidence that the factor contacts the DNA at two separate sites. DNase I protection analysis showed that VETF was found to nucleotides -12 to -29 relative to the transcription initiation site, and also to nucleotides +8 to +10 downstream of the initiation site. The importance of both binding sites for stable complex formation was supported by methylation interference analysis. Using synthetic oligonucleotides encoding different parts of the vaccinia growth factor promoter, it was shown that nucleotides down-stream of the transcription initiation site are required for stable complex formation. Competition binding experiments demonstrated that only the upstream binding site contributes significantly to binding specificity. Binding to two separated DNA sequences results in a bend in the promoter DNA as demonstrated by electrophoretic mobility shift analysis of permuted DNA fragments. These findings suggest that VETF activates transcription by sequence specific binding and structural alteration of the promoter DNA helix.

Base Sequence↗

PubMed1991

A role for ATP hydrolysis in vaccinia virus early gene transcription. Dissociation of the early transcription factor-promoter complex.

Vaccinia virus RNA polymerase requires the vaccinia early transcription factor, VETF, for the in vitro initiation of transcription at early gene promoters in a reaction requiring ATP hydrolysis. VETF binds specifically to early gene promoters and has an associated DNA-dependent ATPase activity. The effect of ATP on the interaction of VETF with the promoter for the vaccinia growth factor gene promoter has been examined. ATP had no marked effect on the steady-state level of promoter binding but dramatically affected the kinetics of dissociation of VETF from the promoter. The half-life of the VETF-promoter complex was greatly reduced in the presence of ATP. The destabilization of the complex was specific for ATP and dATP, consistent with the substrate specificity of the VETF-associated ATPase. ADP or the non-hydrolyzable ATP analog adenylyl-imidodiphosphate did not destabilize the complex suggesting that ATP hydrolysis is obligatory for dissociation. These findings provide a link between the promoter binding and ATPase activities associated with VETF and suggest that the ATP-dependent dissociation of the VETF-promoter complex is an important event in the transcription of vaccinia virus early genes.

Adenosine Triphosphate↗

PubMed1991

A Tyr/Ser protein phosphatase encoded by vaccinia virus.

Protein tyrosine phosphorylation is associated with alterations in receptor activity, cellular proliferation and modulation of the cell cycle. Inappropriate tyrosine phosphorylation can lead to unrestrained cell growth and oncogenesis. Enzymes important in tyrosine dephosphorylation have also been described. Protein tyrosine phosphatases (PTPases) consist of two families. There is a receptor-like family of PTPases with an extracellular domain, transmembrane-spanning region and typically two repeated phosphatase domains. Proteins of the non-receptor-like family have a single catalytic phosphatase domain, show a substrate specificity for Tyr phosphate and will not hydrolyse Ser or Thr phosphate. Here we report that the vaccinia virus genome contains an open reading frame which shares amino-acid sequence identity with the PTPases. The purified protein encoded by the vaccinia virus H1 open reading frame expressed in bacteria hydrolyses substrates containing phosphotyrosine and phosphoserine. Mutagenesis of an essential Cys in the vaccinia phosphatase abolishes catalytic activity directed towards both substrates, suggesting that hydrolysis proceeds by a common mechanism. Understanding the function of the H1-encoded protein will help to define the role of the phosphatase in viral replication and pathogenesis.

Amino Acid Sequence↗

PubMed1990

Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase.

The polypeptide encoded by the vaccinia virus open reading frame D7R was synthesized in bacteria. Immunization of rabbits with the polypeptide resulted in antibodies that specifically recognized a virion polypeptide of 20,000 daltons. The immunoreactivity with the 20,000-dalton polypeptide was found to coincide with the virion-associated DNA-dependent RNA polymerase through DEAE-cellulose chromatography and glycerol gradient sedimentation. These results argue that the product of the vaccinia open reading frame D7R is a subunit of the viral RNA polymerase.

DNA-Directed RNA Polymerases↗

PubMed1990

Vaccinia virus gene encoding a 30-kilodalton subunit of the viral DNA-dependent RNA polymerase.

Antibody was raised against purified vaccinia virus RNA polymerase and used to screen a recombinant vaccinia virus-lambda gt11 library. The DNA from several immunopositive clones was shown by Southern hybridization to originate from the vaccinia virus HindIII E fragment. The nucleotide sequence of the RNA polymerase subunit gene predicts a polypeptide 287 amino acids in length and 30,000 daltons in mass. An early transcript with a 5' terminus just upstream of the putative initiation codon was identified by S1 nuclease protection and primer extension analyses, demonstrating that this RNA polymerase subunit is expressed as an early viral gene product. The RNA polymerase subunit was synthesized by a bacterial expression vector to demonstrate that it corresponds to the previously described 37,000-dalton RNA polymerase subunit.

Amino Acid Sequence↗

PubMed1990

Vaccinia virus gene encoding a component of the viral early transcription factor.

The gene product of the vaccinia virus open reading frame D6R was synthesized in bacteria and used to raise antiserum against the protein. Using the antiserum as a probe, we demonstrated that the D6R protein is a component of the virion particle, localized to the virus core structure. The D6R protein, purified from virions, has been shown to copurify with the vaccinia virus early transcription factor (VETF). The apparent molecular weight of the D6R polypeptide is identical to that of the smaller of the two VETF-associated polypeptides. Antibodies directed against D6R block both the early promoter-binding and DNA-dependent ATPase activities of VETF, supporting the identity of D6R as a VETF-associated polypeptide. An ATP-binding site was inferred near the amino terminus of the derived D6R amino acid sequence. Thus, the D6R polypeptide could be the source of the ATPase activity associated with VETF. The D6R gene was shown previously to belong to the late class of vaccinia virus genes. Synthesis of the VETF at late times after infection suggests a cascade model for vaccinia virus gene regulation in which class-specific transcription factors are synthesized at the previous phase of the infectious cycle.

Adenosine Triphosphatases↗