Pepscan mapping and fusion-related properties of the major phosphatidylserine-binding domain of the glycoprotein of viral hemorrhagic septicemia virus, a salmonid rhabdovirus

In order to define factors which are important for the development of hepatitis C virus (HCV) infection and disease in transplant patients, we examined the role of class II MHC antigen restriction in viral antigen presentation to support a hypothesis of the association of this disease with an autoimmune pathogenesis. A greater degree of histocompatibility match between these donors and their HCV-negative recipients was associated with a greater predisposition to recipient HCV liver disease (ALT elevation) posttransplant. The HCV carrier state could be identified with significant amplification of autologous mixed lymphocyte reactivity (AMLR) in both long-term hemodialysis and long-term renal transplant patients, but the AMLR was absent in end-stage liver disease patients with HCV-associated cirrhosis and was insignificantly elevated in these patients with persistent infection in the first 2 years after a new liver was transplanted. There was also a moderate reduction in autologous reactivity as well as serum HCV titers among renal transplant patients who displayed biochemical evidence of chronic liver disease as opposed to those who did not. This appeared later in the course of the disease. HCV RNA could be detected in peripheral blood mononuclear cells (PBMC) of only a portion of HCV-infected renal transplant patients and these showed significantly higher autologous reactivity. In contrast, despite the fact that observations were earlier after de novo liver transplantation, HCV RNA (i.e., earlier in the course of a new or recurrent disease process) was found in PBMC of all liver transplant recipients tested. The AMLR of noninfected laboratory volunteers could be amplified by preincubating their stimulating cells (APCs) with enriched HCV possibly in immune complex (pHCV-IC). This amplification appeared only with specific combinations of HCV strains with HLA DR serotypes. In addition, HCV-primed T cells could be generated to the virus which displayed accelerated activation kinetics. Liver infiltrating lymphocytes extracted from HCV-positive end-stage diseased livers had significantly higher proliferative and cytotoxic reactivity to autologous (HCV-infected) hepatocytes than the extracted lymphocytes responding to autologous hepatocytes from HCV-negative livers. These findings offer evidence of dynamic autoimmune mechanisms in the spectrum of progression of HCV disease and may help to predict the effect of intervention at various intervals in this progression in organ transplant recipients.     

Studies on the disulfide bonds of glycoprotein hormones. Complete reduction and reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone

Reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone (LH) after their complete reduction both in the presence and absence of denaturing agent yields a product which is indistinguishable from the native subunit in its electrophoretic pattern on polyacrylamide gels and in its ability to recombine with the beta subunits of both luteinizing hormone and thyrotropin. The circular dichroism spectrum of the reoxidized alpha subunit is essentially identical to that of native alpha subunit except that its maximum at 233 nm is smaller than observed with native LHalpha. The intact hormone preparations obtained by recombination of reoxidized alpha subunit with native LH-beta exhibit electrophoretic patterns in polyacrylamide gels, elution profiles on gel filtration, binding activities to a membrane fraction from rat testes, and circular dichroism spectra identical to those of native LH and recombinants of native LH-alpha with the beta subunit. Recombinants of native or reoxidized LH-alpha with the beta subunit of thyrotropin are also indistinguishable in their electrophoretic patterns on polyacrylamide gels and in their in vivo activities of stimulating 32P uptake in thyroids of day-old chicks. While this study does not preclude that the alpha subunit may be biosynthesized as part of a larger precursor protein, the data demonstrate that sufficient information is present in the linear sequence of the alpha subunit to allow folding and formation of disulfide bonds to yield a functional alpha subunit.     

Disulfide bonds in recombinant human platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulfide linkages

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.     

Structure analysis of the rice yellow stunt rhabdovirus glycoprotein gene and its mRNA

The nucleotide sequence of the glycoprotein (G) gene of rice yellow stunt rhabdovirus (RYSV) was determined. The G gene is 2158 nucleotides long and contains an open reading frame of 2007 nucleotides encoding a protein with a calculated molecular mass of 75,358 Da. Furthermore, the 5'- and 3'-terminal sequences of the G protein mRNA were defined by the RACE method. Non-viral nucleotides appear to be present at the 5' end of G mRNA. The G protein contains an N-terminal signal peptide of 32 amino acids, C-terminal transmembrane and cytoplasmic domains, ten potential glycosylation sites and four stretches of a-d hydrophobic heptad-repeats.     

Sindbis virus membrane fusion is mediated by reduction of glycoprotein disulfide bridges at the cell surface

We have examined the role of thiol-disulfide exchange reactions during the penetration of cells by Sindbis virus. The protein-protein association that form the rigid icosahedral lattice of the Sindbis virus envelope have been shown to be stabilized by disulfide bridges, and reduction of these critical disulfide bridges during cell penetration may be the mechanism by which the rigid protein lattice is disrupted prior to fusion (R. Anthony and D. T. Brown, J. Virol. 65:1187-1194, 1991; R. Anthony, A. Paredes, and D. T. Brown, Virology 190:330-336, 1992). Reduction of disulfide bridges occurs at near neutral pHs via thiol-disulfide exchange reactions, and these reactions can be blocked by covalent modification of the thiol involved. In this study, the effects of the reducing agent 2-mercaptoethanol on Sindbis virus-mediated cell-cell fusion from without and the effects of the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) on Sindbis virus penetration were determined. The presence of exogenous reducing agent was found to induce fusion from without under conditions unfavorable to both typical Sindbis virus-mediated fusion from without and cysteine-mediated thiol-disulfide exchange reactions. In addition, the thiol-alkylating reagent was found to inhibit Sindbis virus entry when present during infection. These results are consistent with a model for Sindbis virus entry in which reduction of critical disulfide bridges at the cell surface disrupts the rigid protein-protein associations of the envelope, allowing membrane fusion and release of the viral genome into the cell.     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

Characterization of simian-human immunodeficiency virus envelope glycoprotein epitopes recognized by neutralizing antibodies from infected monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Viral hemorrhagic septicemia virus, Ichthyophonus hoferi, and other causes of morbidity in Pacific herring Clupea pallasi spawning in Prince William Sound, Alaska, USA

A recently proposed Jovanovic-Freundlich isotherm model for single component adsorption without lateral interactions on heterogeneous surfaces is extended to account for lateral interactions and for competitive adsorption. The model is tested using previously reported single component and competitive adsorption data of 2-phenylethanol and 3-phenylpropanol on ODS-silica with methanol-water as the mobile phase. A comparison is made regarding the ability of the Jovanovic-Freundlich and Langmuir-Freundlich models to predict competitive equilibria using the single component identified parameters. Fair predictions of the competitive data were obtained when using heterogeneous-surface models which do not take into account the possible interactions of phenylalcohols in the adsorbed phase via hydrogen bonding. Markedly improved predictions were obtained with models which account simultaneously for the two main sources of adsorbed phase nonideal behavior, i.e. adsorbate-adsorbate interactions and heterogeneity of the adsorbent surface.     

Determination of the regulatory disulfide bonds of NADP-dependent malate dehydrogenase from Pisum sativum by site-directed mutagenesis

The light-mediated reversible activation of NADP-dependent malate dehydrogenase (NADP-MDH) from Pisum sativum can be simulated in vitro by reducing the inactive oxidized enzyme with dithiothreitol. Since the gross structure and the dimeric state of the enzyme are unaffected by the state of oxidation, the redox modulation cannot be attributed to inter-subunit disulfide bridges. In order to identify intra-chain cystine cross bridges that might be candidates responsible for the activation reaction, site-directed mutagenesis experiments were performed, substituting alanine for up to four exposed cysteine residues. Mutants were expressed in freshly transformed EcoB cells and purified to homogeneity. As indicated by the activation behavior (by dithiothreitol-mediated thioldisulfide exchange), disulfides C23-C28 in the N-terminal and C364-C376 in the C-terminal part of the polypeptide chain are involved in the light-induced modulation of the activity of the wild type enzyme. A mutant of the enzyme lacking the N-terminal 45 residues confirms this result. Electrophoretic mobility and FPLC prove the wild type enzyme and its mutants to be dimeric; differences refer to the packing of the N- and C-terminal portions of the enzyme in its oxidized and reduced state. The kinetics of the redox modulation differ, depending on the solvent conditions and the mode of activation. After elimination of the N-terminal disulfide bond, sigmoidal activation profiles are no longer observed, suggesting a slow conformational rearrangement in the N-terminal portion of the wild type enzyme to be rate-limiting in the course of reductive activation. For the wild type, this finding can be mimicked in the presence of non-denaturing concentrations of guanidinium-chloride.     

Roles of individual disulfide bonds in the stability and folding of an omega-conotoxin

Although it contains only 25 amino acid residues, omega-conotoxin MVIIA folds into a well-defined three-dimensional structure that is stabilized by 3 disulfide bonds. To assess the contributions of the disulfides to folding and stability, three analogues, each with one pair of disulfide-bonded Cys residues replaced with Ala, were prepared and characterized. The analogues also contained a C-terminal Gly residue that is believed to be present when the peptide folds in vivo and has been shown previously to stabilize the native structure. Circular dichroism spectra and biological assays of the analogues indicated that removing any one of the disulfides greatly destabilized the native conformation. The two disulfides in each analogue were also reduced much more rapidly than in the native form with three disulfides. When the analogues were fully reduced and allowed to form disulfides in the presence of oxidized and reduced glutathione, the native disulfides were not formed in preference to non-native disulfides, further indicating that the forms with two-native disulfides are not significantly stabilized by noncovalent interactions. However, the measured equilibrium constants for disulfide formation indicate that forming any two of the three native disulfides leads to an effective concentration of approximately 25-50 M for the two remaining thiols. The two-disulfide analogues thus appear to represent a stage of folding in which the polypeptide is constrained to a distribution of relatively compact conformations that greatly favor formation of the third disulfide and the final folded structure.     

Characterization of Cu,Zn superoxide dismutase from the bathophile fish, Lampanyctus crocodilus

PURPOSE: To determine if CT scan provides morphologic criteria allowing differentiation between primary renal cell cancer of the clear type (PRCCCT) and renal metastasis (RM). MATERIALS AND METHODS: Twenty cases of PRCCCT and 20 cases of RM from various origins (excluding lymphomatous origin) were retrospectively analyzed by two independent readers. CT scans were evaluated with respect to tumor size, shape, location and number of tumors, encapsulation, contour sharpness, presence of calcification, and extrarenal involvement by tumor. RESULTS: A tumor size > 3 cm was more frequently seen in PRCCCT than in RM (100% vs 70% respectively, P < .05). Rounded shape was more frequent in PRCCCT than in RM (90% vs 50% respectively, P < .01). Encapsulation was more frequent in PRCCCT than in RM (70% vs 10%, P < .01, respectively). Calcification was depicted only in PRCCCT (35% vs 0%, P < .01). Enlarged perirenal lymph nodes were less frequent in PRCCCT than in RM (20% vs 55% respectively, P < 0.05). CONCLUSION: Results of our study show that a constellation of morphologic features suggest the diagnosis of PRCCCT rather than that of RM. Our study suggests that calcification is highly specific for the diagnosis of PRCCCT. However, percutaneous biopsy remains indicated to ascertain the diagnosis.     

Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.     

Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.     

Stabilization of ribonuclease HI from Thermus thermophilus HB8 by the spontaneous formation of an intramolecular disulfide bond

To identify factors that contribute to the thermal stability of ribonuclease HI (RNase HI) from Thermus thermophilus HB8, protein variants with a series of carboxyl-terminal truncations and Cys --> Ala mutations were constructed, and their thermal denaturations were analyzed by CD. The results indicate that Cys41 and Cys149 contribute to the protein stability, probably through the formation of a disulfide bond. Peptide mapping analysis for the mutant protein with only two cysteine residues, at positions 41 and 149, indicated that this disulfide bond is partially formed in a protein purified from Escherichia coli in the absence of a reducing reagent but is fully formed in a thermally denatured protein. These results suggest that the thermal stability of T. thermophilus RNase HI, determined in the absence of a reducing reagent, reflects that of an oxidized form of the protein. Comparison of the thermal stabilities and the enzymatic activities of the wild-type and truncated proteins, determined in the presence and absence of a reducing reagent, indicates that the formation of this disulfide bond increases the thermal stability of the protein by 6-7 degreesC in Tm and approximately 3 kcal/mol in DeltaG without seriously affecting the enzymatic activity. Since T. thermophilus RNase HI is present in a reducing environment in cells, this disulfide bond probably is not formed in vivo but is spontaneously formed in vitro in the absence of a reducing reagent.     

Characterization of Orungo virus, an orbivirus from Uganda and Nigeria

Orungo virus was studied in cell culture and mice by light and electron microscopy. The virus developed in the cytoplasm of infected cells in mouse brain and cell culture in association with a specific viral granular matrix and accompanying filaments. Virus particle size was 63 nm with a core diameter of 34 nm. Most particles were released from infected cells by lysis, but some budded through membranes and assumed a "pseudoenvelope". In its morphology and mode of morphogenesis, Orungo virus was indistinguishable from other described orbiviruses.     

Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain

Phloridzin-insensitive D-glucose uptake into enterocytes isolated sequentially from along the crypt-villus axis showed the majority of transport activity to reside in cells from the upper third of the villus. In contrast, total postnuclear glucose transporter 2 (GLUT2) protein content of the cells was high even close to the crypt and was almost constant for the upper 80% of the villi. A 4 h lumenal perfusion in vivo with 100 mM D-glucose prior to harvesting the enterocytes produced a 2- to 3-fold increase in phloridzin-insensitive D-glucose uptake which extended down 70% of the villus. Vascular infusion in vivo with either 800 pM gastric inhibitory polypeptide (GIP) or glucagon-like peptide-2 (GLP-2) prior to harvesting enterocytes produced the same response as lumenal glucose, while glucagon like peptide-1 (GLP-1) had no effect. Inclusion of 30 microM brefeldin A (BFA), an inhibitor of protein trafficking, in the lumenal perfusate produced a small, but not significant, increase in the control uptake profile along the villus in isolated enterocytes. However, BFA significantly reduced the upregulation induced by lumenal glucose and vascular GIP and blocked the stimulation produced by vascular GLP-2. Biotinylation of surface proteins and isolation with protein A indicated that there was no change in the membrane abundance of GLUT2 after GLP-2 treatment. These results are discussed in relation to the role of gastrointestinal peptide hormones in controlling intestinal hexose transport and the possibility of protein trafficking being involved in mediating the upregulation of GLUT2 activity in the enterocyte basolateral membrane.     

Characterization of the mRNA encoding a proline-rich 37-kilodalton glycoprotein from the excretory-secretory products of Trichostrongylus colubriformis

A glycoprotein, with apparent molecular weight in SDS-polyacrylamide gels of 37 kDa, has been isolated from the excretory-secretory (ES) products of the adult stage of Trichostrongylus colubriformis, a parasitic nematode. This protein is the major ES product recognized in immunoblots by lymph from a naturally infected sheep. A synthetic oligonucleotide, based on peptide sequence data from a digest of the purified protein was used to successfully screen a cDNA library. A cDNA clone was isolated which encoded a presumptive protein precursor of 220 amino acids that contained a 63 amino acid region of which more than 35% of the residues were proline, three peptide sequences determined from the natural component, and three potential N-glycosylation sites, consistent with the protein being isolated from the lectin-bound fraction of the adult ES products. The presumptive, processed, amino terminus encoded by the cDNA clone was preceded by a signal-like, hydrophobic-rich region of 16 amino acids.