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D T Brown

Publications and source records attributed to D T Brown.

At least 55 records · Page 3Linked to original sources
PubMed1997

Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract.

We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.

Animals↗
PubMed1996

Mutations in the endo domain of Sindbis virus glycoprotein E2 block phosphorylation, reorientation of the endo domain, and nucleocapsid binding.

Exposure of the carboxyl terminus (endo domain) of Sindbis virus membrane glycoprotein E2 to the cell cytoplasm is critical for the interaction of the nucleocapsid with viral envelope proteins in modified cell membranes. We have shown that the endo domain of PE2/E2 is initially translocated into membranes of the endoplasmic reticulum and subsequently drawn back into the cell cytoplasm during virus assembly. We suggested that phosphorylation of PE2/E2 might be responsible for the reorganization of the PE2/E2 carboxyl terminus. To test this hypothesis, two potential phosphorylation sites in the endo domain of E2, Thr398 and Tyr400, were changed by site-directed mutagenesis. Virus structural proteins are produced at normal levels in BHK-21 cells transfected with RNA containing the double mutation, nucleocapsids are formed, and the envelope proteins are exported from the endoplasmic reticulum; however, no virus is produced. The double mutation prevents phosphorylation of PE2/E2, and electron microscopy of cells transfected with the double mutant RNA reveals no attachment of nucleocapsids to cell membranes. The double mutation blocks exposure of the carboxyl terminus of E2 to the cytoplasm. Revertants of the double mutant to virus production all restored tyrosine at position 400 and restored the ability of the E2 protein to be phosphorylated. Although the threonine at position 398 is conserved among the alphaviruses, no revertant restored threonine at this position.

Amino Acid Sequence↗
PubMed1996

Identification of a protein binding site on the surface of the alphavirus nucleocapsid and its implication in virus assembly.

BACKGROUND: Many enveloped viruses exit cells by budding from the plasma membrane. The driving force for budding is the interaction of an inner protein nucleocapsid core with transmembrane glycoprotein spikes. The molecular details of this process are ill defined. Alphaviruses, such as Sindbis virus (SINV) and Semliki Forest virus (SFV), represent some of the simplest enveloped viruses and have been well characterized by structural, genetic and biochemical techniques. Although a high-resolution structure of an alphavirus has not yet been attained, cryo-electron microscopy (cryo-EM) has been used to show the multilayer organization at 25 A resolution. In addition, atomic resolution studies are available of the C-terminal domain of the nucleocapsid protein and this has been modeled into the cryo-EM density. RESULTS: A recombinant form of Sindbis virus core protein (SCP) was crystallized and found to diffract much better than protein extracted from the virus (2.0 A versus 3.0 A resolution). The new structure showed that amino acids 108 to 111 bind to a specific hydrophobic pocket in neighboring molecules. Re-examination of the structures derived from virus-extracted protein also showed this 'N-terminal arm' binding to the same hydrophobic pocked in adjacent molecules. It is proposed that the binding of these capsid residues into the hydrophobic pocket of SCP mimics the binding of E2 (one of two glycoproteins that penetrate the lipid bilayer of the viral envelope) C-terminal residues in the pocket. Mutational studies of capsid residues 108 and 110 confirm their role in capsid assembly. CONCLUSIONS: Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

Alphavirus↗
PubMed1996

Assembly of the Sindbis virus spike protein complex.

The Sindbis virus glycoproteins E1 and E2 are organized into 80 trimers of heterodimers within the virus envelope. Using pulse-chase protocols and chemical crosslinkers, we have found that E1 and E2 precursor, PE2, rapidly assemble into heterodimers and then into trimers of heterodimers after translocation into the endoplasmic reticulum. E1 folds into its mature conformation within the endoplasmic reticulum via at least three intermediates differing in the configurations of their disulfide bonds. PE2 can pair with the second of these E1 folding intermediates. The remaining E1 folding steps, therefore, occur after E1-PE2 multimers begin to form. Quaternary interactions between E1 and PE2 may help guide the folding of E1. While no PE2 folding intermediates have yet been detected, we have found that PE2 transiently enters into large, noncovalent complexes or aggregates with other PE2 molecules and/or with unknown host factors prior to pairing with E1.

Animals↗
PubMed1996

Differential effect of H1 variant overexpression on cell cycle progression and gene expression.

To identify functional differences among non-allelic variants of the mammalian H1 linker histones a system for the overexpression of individual H1 variants in vivo was developed. Mouse 3T3 cells were transformed with an expression vector containing the coding regions for the H1c or H10 variant under the control of an inducible promoter. Stable, single colony transformants, in which the normal stoichiometry of H1 variants was perturbed, displayed normal viability, unaltered morphology and no long-term growth arrest. However, upon release from synchronization at different points in the cell cycle transformants significantly overproducing H10 exhibited transient inhibition of both G1 and S phase progression. Overexpression of H1c to comparable levels had no effect on cell cycle progression. Analysis of transcript levels for several cell cycle-regulated and housekeeping genes indicated that overexpression of H10 resulted in significantly reduced expression of all genes tested. Surprisingly, overexpression of H1c to comparable levels resulted in either a negligible effect or, in some cases, a dramatic increase in transcript levels. These results support the suggestion that functional differences exist among H1 variants.

3T3 Cells↗
PubMed1996

Events in the endoplasmic reticulum abrogate the temperature sensitivity of Sindbis virus mutant ts23.

Temperature-sensitive mutations in proteins produced at or heated to a nonpermissive temperature render the proteins defective in some aspect of their maturation into functional entities. The characterization of temperature-sensitive mutations in model proteins, such as virus membrane proteins, has allowed the elucidation of critical events in the maturation of virus membranes as well as in the intracellular folding, processing, and transport of membrane proteins in general. We have used a transport-defective, temperature-sensitive mutant of Sindbis virus, ts23, which has two amino acid changes in the envelope protein E1, to further examine requirements placed upon the glycoproteins for their export to the plasma membrane. Pulse-chase experiments in which we utilized the transport inhibitors monensin and brefeldin A allowed us to synthesize and assemble the glycoproteins of ts23 into export-competent heterodimers at the permissive temperature while concurrently blocking their export to the cell surface. After removal of the inhibitors and a shift to the nonpermissive temperature, we assayed for protein transport, cell-cell fusion, and infectious-particle production. Taken together, the data show that the irreversible loss of the temperature-sensitive phenotype of ts23 can be correlated with the folding of E1 and the formation of export-competent PE2-E1 heterodimers in the endoplasmic reticulum. Furthermore, we have found that E1 pairs with PE2 to form the heterodimer prior to the completion of E1 folding.

Animals↗
PubMed1996

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.

Disulfides↗
PubMed1996

Wear rates of various artificial tooth materials: a literature review.

Current artificial tooth materials include acrylic resin, improved resin, filled polyurethane dimethacrylate, and porcelain. The improved materials have been popularized by their ability to resist abrasion. A wide variety of abrasives, measuring instruments, and methods of wear testing make it difficult to assess study results that compare these materials. Clinical trials are needed to help establish a standard laboratory test.

Dental Materials↗
PubMed1995

Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue.

The filamentous fungus Fusarium lateritium is cyanide tolerant, due partly to the induction of the enzyme cyanide hydratase in the presence of cyanide. This enzyme catalyses the hydration of cyanide to formamide. The expression in Escherichia coli of a cDNA clone encoding cyanide hydratase is described. The cDNA cloned was expressed as a transcriptional fusion in the expression vector pKK233-2 and a high level of activity of cyanide hydratase was detected in E. coli. Site-directed mutagenesis of the cys-163 residue inactivated the enzyme.

Amino Acid Sequence↗
PubMed1995

Replication and tissue tropism of the alphavirus Sindbis in the mosquito Aedes albopictus.

We have examined the replication and tissue distribution of the alphavirus Sindbis in the mosquito Aedes albopictus. Parenteral inoculation of virus resulted in an acute infection accompanied by rapid virus replication and a persistent infection, during which total virus production was reduced. Acute and persistent phase virus RNA synthesis, virus production, and organ-specific distribution of infection were determined over an 18-day incubation period. Organs were classified as refractory (ovarioles, Malpighian tubules), cleared (head ganglia), transient (salivary glands, anterior midgut, posterior midgut, and thoracic muscle), or persistent (fat bodies-hemolymph, hindgut, and tracheole-associated cells) according to the onset, peak, and duration of Sindbis virus antigens within that particular organ. Virus was identified in respiratory tissue by immunological and ultrastructural methods. This represents a novel tropism for an alphavirus. These findings demonstrate that the cells of mature insect organs respond differently to virus infection. Correlations among virus replication, virus RNA synthesis, and organ-specific clearing of a pantropic infection were observed. We suggest that the underlying temporal and spatial kinetics that characterize this virus-invertebrate interaction may reflect a mechanism for the modulation of the arbovirus titer seen in the mosquito host.

Aedes↗
PubMed1995

A comparison of three methods for preparing reversible hydrocolloid duplicating material.

This study compared the effects of repeated microwave oven and double-boiler liquefactions and prolonged autoduplicator storage on four physical properties of one reversible hydrocolloid duplicating material. No significant differences were observed between the linear dimensional change and detail reproduction of the three liquefaction techniques. Tear strength differences most clearly distinguished the techniques and effects of aging. Fifteen double-boiler remeltings produced tear strength values lower than those recorded for material stored in an autoduplicator for 2 weeks. Thirty microwave melting cycles still produced tear strength values equivalent to that of the autoduplicator material. After 30 melting cycles the compressive strengths of the microwave and double-boiler materials were inferior to that of the stored autoduplicator material.

Alginates↗
PubMed1995

Involvement of the molecular chaperone BiP in maturation of Sindbis virus envelope glycoproteins.

Sindbis virus codes for two membrane glycoproteins, E1 and PE2, which assemble into heterodimers within the endoplasmic reticulum. We have examined the role of the molecular chaperone BiP (grp78) in the maturation of these two proteins. E1, which folds into its mature conformation via at least three intermediates differing in the configurations of their disulfide bonds, was found to interact strongly and transiently with BiP after synthesis. ATP depletion mediated by carbonyl cyanide m-chlorophenylhydrazone treatment results in the stabilization of complexes between BiP and E1. The depletion of intracellular ATP levels also greatly inhibits conversions between the E1 folding intermediates and results in the slow incorporation of E1 into disulfide-stabilized aggregates. These results suggest that the ATP-regulated binding and release of BiP have a role in modulating disulfide bond formation during E1 folding. In comparison with E1, very little PE2 is normally recovered in association with BiP. However, under conditions in which E1 folding is aberrant, increased amounts of PE2 become directly associated with BiP. The formation of these BiP-PE2 interactions occurs after E1 begins to misfold or fails to fold efficiently. We propose that nascent PE2 is stable prior to pairing with E1 for only a limited period of time, after which unpaired PE2 becomes recognized by BiP. This implies that the productive association of PE2 and E1 must occur within a restricted time frame and only after E1 has accomplished certain folding steps mediated by BiP binding and release. Kinetic studies which show that the pairing of E1 with PE2 is delayed after translocation support this conclusion.

Adenosine Triphosphate↗
PubMed1995

Aura alphavirus subgenomic RNA is packaged into virions of two sizes.

The alphavirus genome is 11.8 kb in size. During infection, a 4.2-kb subgenomic RNA is also produced. Most alphaviruses package only the genomic RNA into virions, which are enveloped particles with icosahedral symmetry, having a triangulation number (T) = 4. Aura virus, however, packages both the genomic RNA and the subgenomic RNA into virions. The genomic RNA is primarily packaged into a virion that has a diameter of 72 nm and which appears to be identical to the virions produced by other alphaviruses. The subgenomic RNA is packaged into two major, regular particles with diameters of 72 and 62 nm. The 72-nm-diameter particle appears to be identical in construction to virions containing genomic RNA. The 62-nm-diameter particle probably has T = 3. The large and small Aura virions can be partially separated in sucrose gradients. In addition to these two major classes of particles, there are other particles produced that appear to arise from abortive assembly. From these results and from previous studies of alphavirus assembly, we suggest that during assembly of alphavirus nucleocapsids in the infected cell there is a specific initiation event followed by recruitment of additional capsid subunits into the complex, that the triangulation number of the complex is not predetermined but depends upon the size of the RNA and interactions that occur during assembly, and that budding of assembled nucleocapsids results in the acquisition of an envelope containing glycoproteins arranged in a manner determined by the nucleocapsid.

Alphavirus↗
PubMed1994

Isolation and characterization of two replication-dependent mouse H1 histone genes.

Mice contain at least seven nonallelic forms of the H1 histones, including the somatic variants H1a-e and less closely related variants H1 degrees and H1t. The mouse H1 degrees and H1c (H1var.1) genes were isolated and characterized previously. We have now isolated, sequenced and studied the expression properties of two additional mouse H1 genes, termed H1var.2 and H1var.3. Extensive amino acid and nucleotide sequence comparisons were made between the two genes and other mammalian H1 histone genes. A high degree of nucleotide sequence identity was seen between the H1var.2, rat H1d and human H1b genes, even well beyond the coding region, indicating that these genes are likely homologues. Unlike the previously characterized mouse H1var.1 gene which produces both nonpolyadenylated and polyadenylated mRNAs, the H1var.2 and H1var.3 genes produce only typical, replication dependent, nonpolyadenylated mRNAs.

Amino Acid Sequence↗
PubMed1994

A 55-kDa protein induced in Aedes albopictus (mosquito) cells by antiviral protein.

We have previously shown that the antiviral protein (AVP) produced by Sindbis virus-infected Aedes albopictus (mosquito) cells (Riedel and Brown, J. Virol. 29, 51-60, 1979) blocks Sindbis viral RNA synthesis and stimulates its own production when applied to uninfected A. albopictus cells (Luo and Brown, Virology 194, 44-49, 1993). From a virus-sensitive (wild-type) mosquito cell line (U4.4) we produced a virus-resistant cell line (L4.4) by exposing U4.4 cells to the AVP. L4.4 cells constitutively produce AVP and fail to replicate viral RNA after infection with virus or transfection with purified viral RNA. In this study we have compared cellular proteins produced as the sensitive cells are temporally converted to the resistant phenotype following exposure to the AVP. A 55-kDa membrane protein associated with lysosomes is found in L4.4 and not in U4.4 cells. This protein is induced during the first 48 hr following treatment of U4.4 cells with the AVP. The correlation of the appearance of the 55-kDa protein with the onset of virus resistance implies that this AVP-induced protein may be responsible for the inhibition of viral RNA replication. Although virus RNA synthesis is blocked in the L4.4 cell line, we have found that the synthesis of nonstructural proteins takes place.

Aedes↗
PubMed1994

Mutations in an exposed domain of Sindbis virus capsid protein result in the production of noninfectious virions and morphological variants.

A critical event in the process of Sindbis virus assembly is the interaction of the virus nucleocapsid with the target membrane which has been modified by virus envelope glycoproteins. A specific association between the endodomain of the pE2/E2 glycoprotein and an unidentified domain in the capsid protein is responsible for this association. We have examined the nature of this association by the production of a mutant which has two amino acid substitutions in a domain of the capsid protein known to be exposed on the surface of the assembled nucleocapsid (Ser180/Gly183). These substitutions result in a mutant which is defective in production of infectious virus, especially at low (28 degrees) and high (39.5 degrees) temperatures. Although noninfectious, the mutant virus is as efficient at mediating cell-virus-cell fusion from without as is an equal amount of wild-type virus particles, suggesting that the envelope glycoproteins are functional and that the loss of infectivity may be due to an event after membrane fusion, possibly nucleocapsid uncoating. At low temperature but not high temperature, the mutant produces normal size virus particles (T = 4) and larger particles with a predicted triangulation number of 7. These data suggest that, at low temperature, the mutant capsid protein may assume two different conformations capable of assembling either T = 4 or T = 7 nucleocapsids.

Amino Acid Sequence↗
PubMed1994

The configuration of Sindbis virus envelope proteins is stabilized by the nucleocapsid protein.

We have previously provided evidence that a strong and precise interaction of the Sindbis virus nucleocapsid with the cytoplasmic tail of E2 is critical for the function of the mature virion and that changes in this association may affect infectivity (21). In this study, we examined the effects of temperature-sensitive defects in capsid protein on virus infectivity and glycoprotein function. The two Sindbis virus mutants chosen were ts2 and Ser180/Gly183, which contain amino acid substitutions in their capsid proteins. Heating these mutants to 60 degrees C results in a loss of infectivity significantly greater than the loss observed in wild type viruses. Viral glycoprotein-mediated fusion from without using heat-treated virions occurs normally with wild type viruses but is lost in the mutants. These results suggest that defects in the capsid protein can induce a loss of infectivity through secondary conformational changes in the virus envelope glycoproteins and that the function of the virus envelope is dependent upon specific capsid-E2 interactions.

Animals↗