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S J Talbot

Publications and source records attributed to S J Talbot.

At least 19 recordsLinked to original sources
PubMed2001

Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site.

We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

Base Sequence↗
PubMed1999

Transcriptional analysis of human herpesvirus-8 open reading frames 71, 72, 73, K14, and 74 in a primary effusion lymphoma cell line.

We examined the transcription and splicing of open reading frames (ORFs) 71 (K13)-74 of human herpesvirus-8 (HHV-8) in the primary effusion lymphoma cell line BCP-1 (latently infected with HHV-8), using a combination of NORTHERN blot analysis, RT-PCR, and rapid amplification of cDNA ends (PCR-RACE). The three genes encoded by ORFs 71, 72, and 73 [viral FLICE inhibitory protein (v-FLIP), v-cyclin, latent nuclear antigen (LNA)] are transcribed from a common transcription start site in BCP-1 cells uninduced (latent) or induced (lytic) with n-butyrate. The resulting transcript is spliced to yield a 5.32-kb message encoding LNA, v-cyclin, and v-FLIP and a 1.7-kb bicistronic message encoding v-cyclin and v-FLIP. The two genes encoded by ORFs K14 and 74 (v-Ox2 and v-GPCR) are transcribed as a 2.7-kb bicistronic transcript that is induced with n-butyrate. A small (149-bp) intron is spliced from the intragenic noncoding region immediately before the v-GPCR initiating codon. Examination of sequence elements in the promoter of the LNA/v-cyclin/v-FLIP operon revealed TAATGARAT and Octamer binding motifs characteristic of herpesvirus immediate-early genes. Sequence elements in the v-Ox2/v-GPCR promoter included AP1 and Zta-like (EBV Zebra transactivator) binding motifs consistent with the n-butyrate induction of this operon.

Base Sequence↗
PubMed1997

CD4-independent infection by HIV-2 (ROD/B): use of the 7-transmembrane receptors CXCR-4, CCR-3, and V28 for entry.

We have assayed a variety of 7tm chemokine receptors (CCR-2b, CCR-3, CCR-4, CCR-5, CXCR-1, CXCR-4) and two orphan 7tm receptors (V28 and EBI.1) for their ability to allow infection of CD4-negative feline kidney CCC cells by the HIV-2 strains ROD/A and ROD/B. We found that ROD/B was able to use CXCR-4 transiently expressed in CCC cells, and infection by ROD/A was enhanced 15-fold in the presence of sCD4. Feline CCC cells also became permissive to ROD/B and ROD/A entry when transiently transfected with the chemokine receptor CCR-3 or the orphan 7tm receptor V2B, when cultured in the presence of sCD4. Entry of ROD/A into CCC cells expressing CCR-3 could be blocked by 800 ng/ml eotaxin, the natural ligand for CCR-3.

Animals↗
PubMed1997

The cyclin encoded by Kaposi's sarcoma-associated herpesvirus stimulates cdk6 to phosphorylate the retinoblastoma protein and histone H1.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.

Animals↗
PubMed1997

CD4-independent infection by human immunodeficiency virus type 2 strain ROD/B: the role of the N-terminal domain of CXCR-4 in fusion and entry.

The human immunodeficiency virus type 2 (HIV-2) strain ROD/B can efficiently use the 7tm chemokine receptor CXCR-4 as a primary receptor to enter CD4-negative cells. We have stably expressed CXCR-4 on mink lung Mv-1-lu and feline kidney CCC cells (normally restrictive to HIV entry) and have shown efficient fusion, entry, and replication of ROD/B. Mutation of the two N-linked glycosylation sites on CXCR-4 (N11-->I, and N176-->Q) or pretreatment of CCC or Mv-1-lu cells expressing wild-type CXCR-4 with the glycosylation inhibitor tunicamycin increased fusion and entry by ROD/B. Deletion of portions of the N terminus of CXCR-4 resulted in a 3- to 10-fold decrease in cell-free infection by ROD/B and complete inhibition of cell-cell fusion by both ROD/B and another HIV-2 strain, CBL23. These data suggest that the N-terminal domain of CXCR-4 is involved in but is not essential for the efficient fusion of ROD/B with CD4-negative cells. Deletion of the C-terminal (intracellular) domain of CXCR-4 did not significantly affect entry by ROD/B, indicating that intracellular signalling through this domain does not play a significant role in entry by HIV-2.

Amino Acid Sequence↗
PubMed1995

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.

Base Composition↗
PubMed1995

Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B).

The human immunodeficiency virus type 2 (HIV-2) strain LAV-2/B is able to infect a variety of human cell lines via a CD4-independent pathway. We have used the glycosylation inhibitors tunicamycin, swainsonine, and deoxymannojirimycin to further characterize this putative alternative receptor for HIV-2 (LAV-2/B). These antibiotics resulted in an increase (5- to 30-fold) in the susceptibility of a variety of CD4- human cell lines to infection by LAV-2/B (RD, HeLa, HT29, Rsb, Heb7a, Hos, and Daudi). Several nonprimate cell lines (mink Mv-1-lu, rabbit SIRC, hamster a23, mouse NIH 3T3, cat CCC, and rat HSN) remained resistant to infection by LAV-2/B after treatment with glycosylation inhibitors, suggesting that they do not express the HIV-2 CD4-independent receptor. Two of these nonprimate cell lines are readily infected by HIV-2 when they express CD4 (Mv-1-lu and CCC). Treatment of human cells with neuraminidase had no effect on subsequent infection by LAV-2/B, suggesting that the increase in susceptibility to infection of deglycosylated cells is not due to a change in the electrostatic charge of the cell surface. Treatment of RD CD4- cells and HeLa CD4+ cells with a variety of proteases resulted in a 75 to 90% decrease in infection by LAV-2/B when compared with untreated cells. Taken together, all these data suggest that HIV-2 can utilize a membrane glycoprotein other than CD4 to attach and fuse with a variety of human cells.

Animals↗
PubMed1994

Gel retardation analysis of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli.

C5 protein binds specifically and with high affinity to M1 RNA to form the ribonuclease P holoenzyme of Escherichia coli. The interactions between the two subunits of the enzyme have been studied in vitro by a gel retardation assay. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant (Kd) for the specific interactions of the subunits in the holoenzyme complex is < or = 0.4 nM. C5 protein also has nonspecific affinity for M1 RNA and a variety of other RNA molecules with Kd values in the order of 10-40 nM. Scatchard analysis of binding data suggests the existence of two modes of interaction between C5 protein and M1 RNA--one high-affinity and one low-affinity mode. Regions of M1 RNA essential for formation of the specific complex with C5 protein have been defined by deletion analysis and footprinting methods. Our data show that regions of M1 RNA that interact with C5 protein are clustered into three main areas that are localized between nucleotides 41-99, 168-198, and 266-287.

Bacterial Proteins↗
PubMed1994

Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli.

A gel retardation assay has been used to examine the kinetic and equilibrium properties of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli. The interaction is relatively insensitive to the identity of the monovalent anions present and to pH in the range 7.0-9.0, but it has a more critical requirement for specific monovalent and divalent cations: NH4+, K+, Mg2+, Ca2+, and Mn2+ all promote efficient formation of the complex. A positive delta S (+6.4 cal mol-1 deg-1) and a negative delta H (-11.3 kcal mol-1) combine to give a delta G equal to -13.3 kcal mol-1 at 37 degrees C in 0.42 M salt. The binding reaction is sensitive to the concentration of monovalent and divalent cations, with the affinity increasing with increasing ionic strength (delta log Ka/delta log [NH4+] = +2.7 +/- 0.1). The dependence of Kd on the ionic strength and the positive delta S suggests that hydrophobic and stacking interactions contribute significantly to the formation of the RNase P holoenzyme.

Amino Acid Sequence↗
PubMed1993

Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid.

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

Amino Acid Sequence↗
PubMed1991

Hyperreactivity of adenines and conformational flexibility of a translational repression site.

We have used a diethylpyrocarbonate (DEPC) modification [(1976) Prog. Nucl. Acids Res. 16, 189-262] to probe the accessibility of adenines essential for coat protein binding in the MS2 translational operator [(1983) Biochemistry 22, 2601-2610, 2610-2615, 4723-4730; (1987) Biochemistry 26, 1563-1568]. The essential adenines are apparently hyperreactive with this reagent relative to other sites within the same molecule. Variation of ionic strength, pH and divalent cation concentrations reveal the existence of two distinct conformers of the RNA operator as judged by DEPC reactivity. We propose that the hyperreactivity observed is due to the participation of neighbouring bases in the DEPC modification reaction and can be used as a novel structural probe.

Adenine↗
PubMed1990

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex.

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.

Base Composition↗
PubMed1990

Histidine-stimulated acid secretion in the conscious rat is mediated by amino acid uptake system N.

The characteristics of sodium-dependent, histidine-stimulated gastric acid secretion were studied in the conscious gastric-fistulated rat. Intragastric L-histidine, but not glutamine, asparagine, glutamic acid or glycine, stimulated acid secretion. The histidine-stimulated acid secretion was inhibited by the presence of glutamine or asparagine, but not by glutamic acid or glycine. The substitution of lithium for sodium in the gastric perfusate was tolerated. Together with previous data, the results indicate that an N-like amino acid uptake system mediates histidine-stimulated acid secretion in the conscious rat.

Amino Acids↗
PubMed1989

Modeling loop structures in proteins and nucleic acids: an RNA stem-loop.

We have used a novel modeling technique, based on combining information from several preexisting structures, to generate a three-dimensional (3D) model for the RNA stem-loop responsible for translational repression of the MS2 RNA bacteriophage replicase. Specific features of the model have been tested experimentally by chemical and enzymatic structural probes; results from these experiments have been used to "improve" the model by fixing initial assumptions. The new model and chemical modification data are in part consistent, and further predictions are being tested. The modeling algorithm has a wide range of potential applications, particularly to loop regions in proteins and nucleic acids.

Algorithms↗