Three structural polypeptides coded for by minite virus of mice, a parvovirus

Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step.     

Comparative analysis of the virion structural proteins of 3 nuclear polyhedrosis viruses

The composition of structural proteins of virions of nuclear polyhedrosis viruses (NPV) of Barathra brassicae L., Lymantria dispor L. and Aporia crataegi L. was studied by polyacrylamide gel-SDS electrophoresis (PAGE-SDS). The object of the study included fractions of virion bundles in the envelope. All the baculoviruses under study were shown to have in virion structural proteins 23-24 polypeptides with molecular weights ranging from 12500 to 95000 daltons. Alongside with polypeptides of similar molecular weight, each baculovirus was shown to have a specific set of structural proteins allowing them to be readily identified. Specific distribution of major polypeptides of the virions was found. All the protein fractions were readily reproducible in repeated analyses with the exception of two minor polypeptides of NPV of Barathra brassicae L. The study showed that the composition of structural proteins of virions may be an important criterion for identification of baculoviruses under identical electrophoresis conditions.     

Anti-lipid A monoclonal antibody centoxin (HA-1A) binds to a wide variety of hydrophobic ligands

This note describes the binding specificities of four lipid A monoclonal antibodies (MAbs) including Centoxin (HA-1A); these MAbs display similar binding properties. MAbs reacted with lipid A and heat-killed smooth bacteria, whereas no reactivity was observed with smooth lipopolysaccharide (LPS). Immunoblotting of bacterial extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs bound to many polypeptide bands including the molecular weight markers. Denaturation of bovine serum albumin (BSA) by boiling or dithiothreitol treatment unmasked antibody epitopes. In addition, binding both to a hydrophobic aliphatic C12 chain covalently coupled to BSA and to single-stranded DNA was observed. The polyreactivity of these clones is most likely mediated by a preferential reactivity with hydrophobic molecular patches.     

Serum albumin. A CAP survey

The results of a 1974 survey of albumin measurements as performed by more than 1,300 laboratories are presented. The most widely used methods are the dye-binding technics: bromcresol green (BCG) and 2-(4'-hydroxyazobenzene) benzoic acid (HABA). These are followed by electrophoresis and salt fractionation. All methods yielded comparable albumin concentrations except electrophoresis, which manifested a consistent low bias. This close agreement is attributed, in part, to the normal-range concentration of albumin in the test specimen. Type of standardization, i.e., commercial serum, bovine serum albumin, human serum albumin, or pooled serum, did not appear to be a factor in the estimation of albumin in the normal serum submitted for analysis. Surprisingly, interlaboratory variation, from method means, was the lowest for salt fractionation and electrophoretic technics.     

The virion host shutoff protein of herpes simplex virus inhibits reporter gene expression in the absence of other viral gene products

The virion host shutoff (vhs) function of herpes simplex virus induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. Previous studies have shown that disruption of the UL41 gene abrogates vhs activity, but have not determined whether the UL41 polypeptide is the direct inducer of mRNA degradation or whether it is the only virion component required for this activity. In this paper we report that transfection of cells with UL41 inhibits expression of a cotransfected CAT reporter gene and that the inhibition is not dependent upon other viral genes. Inhibition of CAT expression was due to UL41-dependent reduction of CAT mRNA levels. UL41 alleles encoding polypeptides that lacked vhs activity during virus infections exhibited a similar lack of activity in transfected cells. The results indicate that the UL41 polypeptide is the direct inducer of host mRNA degradation following virus infection and that it is the only virion component directly required for this activity. A 382-amino-acid nonsense polypeptide missing the last 107 residues of UL41 lacked inhibitory activity, but was packaged into virions, while a 343-amino-acid nonsense polypeptide lacked both inhibitory activity and the ability to be packaged.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Comparative study of the oncornavirus A and D proteins of human J-96 cells by the methods of isoelectric focusing and polyacrylamide gel electrophoresis

Three peaks of 14C-radioactivity with buoyant densities of 1.23--1.24, 1.26 and 1.29 g/ml were detected in a cytoplasmic extract of J-96 cells upon equilibrium centrifugation in sucrose gradient. Electron microscopy of the 1.23--1.24 g/ml buoyant density fraction revealed particles 60--80 nm in diameter showing morphology characteristic of oncornavirus A. Isoelectric focusing in polyacrylamide gel showed polypeptides of extracellular D virus and oncornavirus A to differ in isofocusing points (pI). Proteins of extracellular D virus were localized in zones with pH 3.7, 4.0, 4.4, 4.7, 5.6, 6.5, 8.1, 9.45, and 10.0; polypeptide of intracytoplasmic oncornavirus A had the following isofocusing points: 4.0, 4.9, 6.7, 7.3, 9.0, 9.45 and over 10.0. Electrophoresis of polypeptides of D virus and intracellular oncornavirus A revealed differences in the molecular weights of the components. No proteins with molecular weights of 10,000, 12,000, 15,000, and 27,000 dalton characteristic of the extracellular D virus were found in oncornavirus A virions. The analysis of protein patterns obtained in parallel experiments of isoelectric focusing and polyacrylamide gel electrophoresis suggests that oncornaviruses A and D of J-96 cells differ in the characteristics (pI and molecular weight) of the structural polypeptide components.     

Comparison of methods for separating proteins using bisalbuminemia as a model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and indentification procedures for evaluating serum albumin variants were compared. The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholines prevented the complete isolation of the chicken albumin. The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken anti-turkey sera.     

Proteolytic activation of tick-borne encephalitis virus by furin

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.     

Partitioning of proteins in dextran/hydrophobically modified dextran aqueous two-phase systems

Partitioning of proteins was studied in aqueous two-phase systems composed of the polymers dextran and hydrophobically modified dextran. The modified dextrans were benzoyl dextran with a degree of substitution of 0.17 and valeryl dextran with a degree of substitution of 0.20. Phase diagrams for the systems of dextran/benzoyl dextran and dextran/valeryl dextran were determined at room temperature. The proteins studied were beta-galactosidase, bovine serum albumin, beta-lactoglobulin, lysozyme, myoglobin and cytochrome C. The partition coefficients of a series of salts were determined in dextran/benzoyl dextran two-phase systems. The addition of salts had strong effect on the partitioning of proteins. This effect was related to protein net charge and the position of the ions in the Hofmeister series. Cross partitioning of bovine serum albumin was studied in a dextran/benzoyl dextran aqueous two-phase system.     

Evaluation of the radiologic findings on hysterosalpingography by selective hydrotubation with flexible hysterofiberscope

In order to assess the influence of the protein charge on its partitioning in poly(ethyleneglycol)/salt aqueous two-phase systems, three bovine serum albumin derivatives with isoelectric points of 5.50, 6.20 and 6.85 were obtained by chemical modification of the protein with a soluble carbodiimide and glycine O-methyl ester and separation of the derivative mixture by liquid isoelectric focusing. The modification reaction was mild enough to preserve the tertiary structure of the proteins, as judged by circular dichroism and fourth derivative UV spectra. The surface hydrophobicity of the bovine serum albumin derivatives was identical, as measured by hydrophobic interaction chromatography. Partitioning of the derivatives in poly(ethyleneglycol)/phosphate and poly(ethyleneglycol)/citrate aqueous two-phase systems between pH 5.2 and 6.5 indicates that partitioning is not dependent on the protein charge in the poly(ethyleneglycol)/salt systems studied.     

Relative affinities of bilirubin for serum albumins from different species

Relative equilibrium constants ("affinity ratios") of complexes of bilirubin with a molar excess of charcoal-treated serum albumins from different species (human, bovine, rabbit and chicken) in aqueous solution, were estimated by circular dichroism measurements in the visible region at 26-27 degrees C, pH 7.4, and in the presence of 0.1 M NaCl. By variation of the mol ratios of the components of pairs of different bilirubin-serum albumin complexes showing circular dichroic bands of opposite sign, the apparent association constants of complexes of bilirubin with either human or chicken albumin were found to be greater by factors between 6 and 17 than those of bovine or rabbit albumins. The usefulness in the determination of affinity ratios is illustrated by the evaluation of single equilibrium constants of systems of high-ligand affinity from those of relatively lower affinity, the latter of which are more readily amendable to direct experimental measurement.     

Human immunodeficiency virus type 1 replication is modulated by host cyclophilin A expression levels

Human immunodeficiency virus type 1 (HIV-1) Gag and the cellular protein cyclophilin A form an essential complex in the virion core: virions produced by proviruses encoding Gag mutants with decreased cyclophilin A affinity exhibit attenuated infectivity, as do virions produced in the presence of the competitive inhibitor cyclosporine. The A224E Gag mutant has no effect on cyclophilin A affinity but renders HIV-1 replication cyclosporine resistant in Jurkat T cells. In contrast, A224E mutant virus is dead in H9 T cells, although replication is rescued by cyclosporine or by expression in cis of a Gag mutant that decreases cyclophilin A-affinity. The observation that disruption of the Gag-cyclophilin A interaction rescues A224E mutant replication in H9 cells prompted experiments which revealed that, relative to Jurkat cells, H9 cells express greater quantities of cyclophilin A. The resulting larger quantity of cyclophilin A shown to be packaged into virions produced by H9 cells is presumably disruptive to the A224E mutant virion core. Further evidence that increased cyclophilin A expression in H9 cells is of functional relevance was provided by the finding that Gag mutants with decreased cyclophilin A affinity are dead in Jurkat cells but capable of replication in H9 cells. Similarly, cyclosporine concentrations which inhibit wild-type HIV-1 replication in Jurkat cells stimulate HIV-1 replication in H9 cells. These results suggest that HIV-1 virion infectivity imposes narrow constraints upon cyclophilin A stoichiometry in virions and that infectivity is finely tuned by host cyclophilin A expression levels.     

Zinc(II) and copper(II) binding to serum albumin. A comparative study of dog, bovine, and human albumin

Metal binding strategies employing low molecular weight chelators and equilibrium dialysis were used to investigate several unresolved aspects of zinc and copper binding to serum albumin. Direct measurement of histidine binding to bovine serum albumin when the histidine is presented either as a metal-chelate or alone provides no evidence for an albumin-metal-histidine ternary complex. Using previously determined intrinsic constants for Zn(II) and Cu(II), we have measured zinc binding to bovine serum albumin in the presence of saturating amounts of copper. The results of these experiments unambiguously show that zinc and copper bind at separate noninteracting sites on this protein. The intrinsic constants for zinc and copper binding to dog serum albumin have been determined. Contrary to previous reports, we find that dog serum albumin has a specific high affinity site for copper, log10K 10.17 for Cu(II) compared to 6.85 for Zn(II) at the separate site.     

Study of herpes simplex virus maturation during a synchronous wave of assembly

Production of an infectious herpes simplex virus (HSV) particle requires sequential progression of maturing virions through a series of complex assembly events. Capsids must be constructed in the nucleus, packaged with the viral genome, and transported to the nuclear periphery. They then bud into the nuclear membrane to acquire an envelope, traffic through the cytoplasm, and are released from the cell. Most of these phenomena are very poorly defined, and no suitable model system has previously been available to facilitate molecular analyses of genomic DNA packaging, capsid envelopment, and intracellular virion trafficking. We report the development of such an assay system for HSV type 1 (HSV-1). Using a reversible temperature-sensitive mutation in capsid assembly, we have developed conditions in which an accumulated population of immature capsids can be rapidly, efficiently, and synchronously chased to maturity. By assaying synchronized scaffold cleavage, DNA packaging, and acquisition of infectivity, we have demonstrated the kinetics with which these events occur. Kinetic and morphological features of intranuclear and extranuclear virion trafficking have similarly been examined by indirect immunofluorescence microscopy and electron microscopy. This system should prove a generally useful tool for the molecular dissection of many late events in HSV-1 biogenesis.     

Inhibition of HIV type 1 infectivity by coexpression of a wild-type and a defective glycoprotein 120

An amino acid substitution (D --> K) in the C3 region of HIV-1 gp120 has previously been shown to inhibit binding of virions to CD4+ cells. We have introduced the same mutation into the HIV-1 isolate LAV-I(BRU), in which the mutation is denoted D373K. Here we show that the D373K envelope protein is processed and incorporated into virus particles, but that D373K virions have no detectable infectivity (below 0.1% relative to wild type). When D373K and the wild-type envelope gene were cotransfected in 293 cells at a 4:1 ratio, the resultant infectivity of the HIV-1 supernatant was reduced more than 100-fold. When the same ratio of plasmids was tested in COS-1 cells the inhibition of HIV-1 was an order of magnitude less than observed in 293 cells. COS-1 and 293 cells differed in that only 293 cells displayed saturation of virus production with respect to the envelope protein. Our data fit a simple model: when virion formation is saturated with envelope protein, expression and incorporation of a defective envelope protein imply a corresponding dilution of wild-type protein on the surface of virions. The cooperative function of wild-type envelope proteins is subsequently compromised, and a trans-dominant inhibition of virus infectivity is observed.     

A new structural transition of serum albumin dependent on the state of Cys34. Detection by 1H-NMR spectroscopy

1. Reactions of fatty-acid-free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(I) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(I) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 1H-NMR spectroscopy. 2. In reactions of albumin with auranofin, tetraacetylthioglucose and dithiothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disulphides with cysteine were produced. It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione. The above reactions lead to deblocking by thiol-disulphide interchange reactions. No release of glutathione from albumin was detected. 3. Changes in the His H epsilon 1 regions of the 1H-NMR spectra show that albumin exists in two structural forms dependent on whether the side-chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation. We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed. 4. The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(I) as a heavy atom derivative in X-ray crystallographic studies of albumins.     

Comparative analysis of sociality in lion tamarins (Leontopithecus rosalia) and marmosets (Callithrix kuhli): Responses to separation from long-term pairmates.

The purpose of the present study was to examine responses to a short-term separation from the partner in 2 species of pair-bonding primates: lion tamarins (Leontopithecus rosalia) and marmosets (Callithrix kuhli). Observations were conducted on well-established pairs during a preseparation baseline, a 1-hr separation, and a postseparation reunion. Lion tamarins responded to separation with increased levels of distress, relative to marmosets. Affiliative behavior increased in marmosets upon reunion, whereas affiliative behavior decreased upon reunion in lion tamarins. In marmosets, there were more trials in which sexual interactions were recorded during the postseparation reunion, whereas in lion tamarins, no increases were observed. The results indicate that the mechanisms responsible for the maintenance of social and sexual relationships may differ in these 2 species. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant

The envelope of immature vaccinia virions consists of a lipoprotein bilayer upon which a precise curvature is imposed by acquisition of an external scaffold of spicules. Self-assembly of this tegument was examined employing our ts 6757 mutant, which induces accumulation of immature envelopes at the restrictive temperature. With ts 6757 the envelope bilayers were also assembled into an alternative membrane configuration in the form of flexible cylinders or tubes of uniform width, lacking the spicule coat. Such tubes became extensions of or were continuous with the spherical virion envelopes. The approximately 65 kDa spicule protein, L65, product of gene D13L on the HindIII map, generally designated as a late protein, was expressed as an early function in presence of hydroxyurea, an inhibitor which entirely blocked vaccinia DNA synthesis without stopping assembly of immature envelopes. Labeling of thin sections by immunogold for electron microscopy demonstrated that L65 is present at the surface of immature virions, consistent with the position of spicules on envelopes. Transiency of the spicule scaffold was documented by (a) absence of L65 from intracellular mature virions (IMV) and (b) rapid turnover of L65 during ts 6757 virus replication at the permissive temperature but conservation of this protein at restrictive temperature, as demonstrated in pulse-chase experiments. Time-related decrease in MW of L65 to a smaller polypeptide is interpreted as evidence suggesting that the spicules attached to the envelope are assembled from a higher MW precursor.     

Amino acid sequences of hemoglobin beta chains of five species of pinnipeds: Neophoca cinerea, Otaria byronia, Eumetopias jubatus, Pusa hispida, and Pagophilus groenlandica

Pinnipeds (Otariidae, Odobenidae, and Phocidae) in the order Carnivora have one or two types (Hb I and Hb II) of hemoglobin components. These hemoglobins consist of identical beta chains and different alpha chains. We determined the complete amino acid sequences of the hemoglobin beta chain of three species of Otariidae (Australian sea lion, South American sea lion, and northern sea lion) and two species of Phocidae (ringed seal and harp seal) from intact beta chain and chemical cleavage fragments. The sequences are similar to beta chains of the already known sequences of pinnipeds. These sequences were compared with those of other carnivores (Mustelidae, Ursidae, Canidae, and Felidae) and adult human hemoglobin beta chain. Using Artiodactyla (pig) as an outgroup, we find that the tree constructed by means of phylogenetic analysis shows that Odobenidae is closest to Otariidae, and that Otariidae and Odobenidae are closer to Mustelidae than to Phocidae.