Optical fiber immunosensor for the detection of IgG antibody to Rift Valley fever virus in humans.

This paper describes the development and evaluation of an optical fiber immunosensor (OFIS) for the detection of IgG antibody to Rift Valley fever virus (RVFV) in humans. The OFIS was based on a sandwich enzyme-linked immunosorbent assay (S-ELISA) format, whereby gamma-irradiated RVFV and control antigens were immobilized on the optical fiber surface coated with a mouse anti-RVFV antibody. Data sets derived from field-collected sera in Africa (n=242) were dichotomized according to the results of a virus neutralization test. Compared to standard colorimetric S-ELISA, the OFIS technique was more sensitive in detecting smaller quantity of specific IgG to RVFV in human sera. At cut-off value selected at a 95% accuracy level by the two-graph receiver operating characteristic analysis, the OFIS diagnostic sensitivity was 97.22% and diagnostic specificity 98.86%. Our results demonstrate that the OFIS technology reported here is highly accurate, simple to perform and has the potential to be used in a portable format.

Vaccinia virus interactions with the cell membrane studied by new chromatic vesicle and cell sensor assays.

The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.

Development of a highly sensitive, field operable biosensor for serological studies of Ebola virus in central Africa.

We describe herein a newly developed optical immunosensor for detection of antibodies directed against antigens of the Ebola virus strains Zaire and Sudan. We employed a photo immobilization methodology based on a photoactivatable electrogenerated poly(pyrrole-benzophenone) film deposited upon an indium tin oxide (ITO) modified conductive surface fiber-optic. It was then linked to a biological receptor, Ebola virus antigen in this case, on the fiber tip through a light driven reaction. The photochemically modified optical fibers were tested as an immunosensor for detection of antibodies against Ebola virus, in animal and human sera, by use of a coupled chemiluminescent reaction. The immunosensor was tested for sensitivity, specificity, and compared to standard chemiluminescent ELISA under the same conditions. The analyte, anti-Ebola IgG, was detected at a low titer of 1:960,000 and 1:1,000,000 for subtypes Zaire and Sudan, respectively. While the same serum tested by ELISA was one order (24 times) less sensitive.

Long-acting follicle-stimulating hormone analogs containing N-linked glycosylation exhibited increased bioactivity compared with o-linked analogs in female rats.

The effects of altering the number and type of additional carbohydrate moieties on the pharmacokinetic and pharmacodynamic properties of FSH were examined in this report. A series of single-chain follitropins, containing variable numbers of additional N- (or O-) linked carbohydrates, were designed and expressed in Chinese hamster ovary cells. Proper folding, efficient receptor binding, and signal transduction were confirmed by in vitro assays. Pharmacokinetic and pharmacodynamic parameters were evaluated in immature female Sprague Dawley rats. Increasing the number of glycosylation sites with either N- (or O-) linked moieties extended the elimination half-life as much as 2-fold compared with recombinant human FSH (rhFSH). However, there was a maximum elimination half-life such that further glycosylation provided no additional lengthening of the half-life. Conversely, biopotency, as assessed by inhibin A levels 74 h post injection, and follicle production were significantly higher for the N-linked analogs. Rats stimulated with the longest acting analogs (either N- or O-linked) showed significantly higher ovarian weights than rats receiving a single injection of rhFSH. The analog containing four additional N-linked sites (rhFSH-N4) had the greatest number of large, preovulatory follicles. Although the half-life of rhFSH-N4 displayed no further enhancement beyond the other longest acting analogs, this analog exhibited significantly increased biopotency in rats. This work provides the basis for the generation of a series of reagents potentially useful for therapeutic applications.

Development and characterization of a long-acting recombinant hFSH agonist.

BACKGROUND: Fusion of the carboxyterminal peptide (CTP) of hCG to FSH results in a follitropin agonist with an extended half-life, presumably due to the four O-oligosaccharides on the CTP. Alternatively, an rhFSH analogue containing additional N-linked carbohydrate is described in this report. METHODS: A DNA sequence containing two N-oligosaccharide signal sequences was ligated into a vector containing hFSHbeta- and alpha-subunit encoding cDNA, and expressed in CHO-K1 cells. In-vitro bioactivity of the single-chain hormone was assessed in CHO cells expressing the hFSH receptor. Pharmacokinetic values were derived from serial serum assays of the analogue in immature female rats following a single i.v. injection. In-vivo bioactivity was assessed by measuring ovarian weight gain 3 days post-injection. RESULTS: rhFSH-N2 and native rhFSH induced comparable levels of cAMP in vitro. t(1/2) for native rhFSH, rhFSH-CTP and rhFSH-N2 were 3.7, 7.1 and 7.3 h respectively. Rats receiving rhFSH-N2 had a mean +/- SD ovarian weight 3 days post-i.v. injection (22 +/- 3.6 mg) significantly greater than rats receiving rhFSH and saline (16.7 +/- 1.5 and 15.3 +/- 0.47 mg respectively, P < 0.05). CONCLUSIONS: rhFSH-N2 has prolonged half-life and increased bioactivity compared with native rhFSH. This rhFSH agonist, and other analogues containing additional N-oligosaccharides may have important clinical applications.

Development of a fluorescence based high throughput assay for antagonists of the human chorionic gonadotropin receptor extracellular domain: analysis of peptide inhibitors.

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

High-level bacterial expression of a natively folded, soluble extracellular domain fusion protein of the human luteinizing hormone/chorionic gonadotropin receptor in the cytoplasm of Escherichia coli.

We have expressed the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and glutathione reductase genes. The chimeric protein isolated following induction of expression is purified in a soluble form and binds hCG with an affinity approximating that of native receptor. This truncated form of the receptor displays the same specificity as intact hLH/CG receptor and does not bind human follicle stimulating hormone. This cytoplasmically produced protein is expressed at levels that exceed 10 mg/L. Expression of properly folded extracellular domain of the hLH/CG receptor in the cytoplasm of E. coli allows the facile and economic purification of large quantities of material. This will facilitate the determination of the structure of the hormone-binding domain of the glycoprotein receptor as well as the production of epitope-specific antibodies.

Expression and characterization of recombinant beta-subunit hCG homodimer.

We have linked two human chorionic gonadotropin (hCG) beta-subunit cDNAs in tandem such that the expressed fusion protein consists of two mature beta-subunits joined through the carboxy terminal peptide of the first beta-subunit. A single glycine residue is inserted between the two subunits in the fusion protein. Chinese hamster ovary (CHO) cells transformed with a clone that contains the fused cDNAs express and secrete a protein that is consistent with it being a beta-hCG homodimer protein. These beta-homodimer molecules can recombine with two free alpha-subunits indicating that both beta-subunits within the homodimer are likely folded in their native conformation. Our data also suggest that the two beta-subunits fold upon each other as a globular protein and do not appear to exist as a simple fusion of two linear beta-subunits. Furthermore, the two beta-monomer subunits in the fusion protein form a stable homodimer that can bind and activate the hLH/CG receptor specifically. Recombination of the fusion protein with alpha-subunits appears to favor an arrangement where two alpha-subunits combine with a single molecule of the fusion protein. The recombined molecule consists of four subunits and is comparable to two tethered hCG moieties, which constitutes a hCG dimer. This hormone dimer can bind and activate the hLH/CG receptor with an activity approximating that of native hCG.

Structural and molecular studies of human chorionic gonadotropin and its receptor.

Human chorionic gonadotropin (hCG) is a placental hormone that stimulates secretion of the pregnancy-sustaining steroid progesterone. It and other glycoprotein hormones are disulfide-rich heterodimers that share a common alpha chain and distinctive beta chains specific to their particular G protein-linked receptors. We determined the structure of partially deglycosylated hCG at 2.6 A resolution from multiwavelength anomalous diffraction (MAD) measurements of a selenomethionyl hCG crystal. We have also begun three- and four-dimensional structural studies on the biologically active hormone and have determined the structure of the carbohydrate attached to the alpha-subunit. Despite little sequence similarity limited to 10% identity, the alpha and beta subunits of hCG maintain strikingly similar tertiary folds, with cystine-knot motifs at cores of extended hairpin loops. Structural and sequence comparisons indicate an evolutionary homology between the glycoprotein hormone chains and other cystine-knot proteins, notably PDGF, TGF-beta, and NGF. This structural similarity has led us to speculate that early hCG secretion has a broader role than solely the stimulation of the corpus luteum; indeed, levels of hCG, which rise rapidly in the circulation after implantation, are greater than the levels necessary for corpus luteum function. One such role of hCG or its subunits could be as a growth factor that facilitates endometrial receptivity. Our studies of hCG have also identified structural variants, notably in the carbohydrate moiety, that are distinctive for patients with a variety of disorders of pregnancy, including hydatidiform mole and choriocarcinoma. We have also focused our efforts on using information gleaned from the structure of hCG for the design of drug-like molecules that might serve as either agonists or antagonists of hCG. To facilitate these experiments, we have designed a rapid screen for the identification of molecules that might bind the hCG receptor by identifying compounds that disrupt binding of hCG to its receptor. This screen employs a filamentous phage that displays the extracellular domain of the hCG receptor on its surface. Thus far, we have identified a few compounds that disrupt binding of hCG with its receptor at a concentration of approximately 1 micromolar. These "lead" molecules are currently being modified in an attempt to identify a molecule that can disrupt binding of hCG at nanomolar concentrations.

Mutations in the alpha1 subunit of an L-type voltage-activated Ca2+ channel cause myotonia in Caenorhabditis elegans.

The control of excitable cell action potentials is central to animal behavior. We show that the egl-19 gene plays a pivotal role in regulating muscle excitation and contraction in the nematode Caenorhabditis elegans and encodes the alphal subunit of a homologue of vertebrate L-type voltage-activated Ca2+ channels. Semi-dominant, gain-of-function mutations in egl-19 cause myotonia: mutant muscle action potentials are prolonged and the relaxation delayed. Partial loss-of-function mutations cause slow muscle depolarization and feeble contraction. The most severe loss-of-function mutants lack muscle contraction and die as embryos. We localized two myotonic mutations in the sixth membrane-spanning domain of the first repeat (IS6) region, which has been shown to be responsible for voltage-dependent inactivation. A third myotonic mutation implicates IIIS4, a region involved in sensing plasma-membrane voltage change, in the inactivation process.

Filamentous phage displaying the extracellular domain of the hLH/CG receptor bind hCG specifically.

We have expressed the extracellular binding domain of the human LH/CG receptor as a fusion with the cpIII filamentous phage coat protein. These fusion phage are able to specifically bind ELISA plates coated with hCG. Preincubation of the phage with hCG before exposure to the coated plates inhibited binding of phage, whereas human FSH had no effect. Furthermore, addition of anti-hLH/CG receptor antisera inhibited binding of phage to the plates. We have also tested the effect of monoclonal antibodies to hCG on phage binding. Monoclonal antibodies that can bind hCG when it is bound to native receptor do not inhibit phage binding. These include B105 and A109 Alternatively, B107 and B109 have an inhibitory effect on phage binding. They cannot bind hCG when it is bound to receptor and are likely directed against epitopes at or near the receptor binding interface. Therefore, these fusion phage exhibit the same binding specificity as the wild-type receptor and likely display the extracellular domain of the hLH/CG receptor on their surface in the native conformation. Phage display is a potentially useful tool for studying the hLH/CG receptor structure and in screening for synthetic compounds that compete with hormone for receptor binding.

Three-dimensional structures of gonadotropins.

Most secreted proteins are modified post-translationally with the addition of carbohydrate. It has been difficult to use crystallography to solve the structures of these proteins due to the inherent heterogeneity of the carbohydrate. The structure of the chemically deglycosylated form (hydrogen fluoride treated) of human chorionic gonadotropin (hCG) has been solved through crystallographic techniques. Unfortunately this form of hCG is not biologically active, and exhibits immunochemical differences from native hormone. In addition, subunit interactions appear altered after chemical deglycosylation as indicated by the increased thermal stability of the HF-treated hormone. The Asn 52 glycan on the alpha-subunit of hCG has been identified as being required for biological activity, it is, therefore, of physiological importance to determine the structure of the hormone with its carbohydrate intact. Also, it has not been possible to obtain crystals of the individual glycosylated subunits of hCG. Therefore an alternative method to solve the structure of the biologically active form of the hormone in solution as well as its separated subunits is necessary. Structural information utilizing NMR techniques can be obtained from native hCG subunits in solution if they can be uniformly labeled with 13C and 15N isotopes. We have developed a universal nonradioactive isotope, labeling medium enriched in 13C and 15N which can be used to express uniformly labeled hCG from Chinese hamster ovary cells suitable for solving the structure of the individual subunits and ultimately that of the native, biologically active hormone. The isotopically labeled recombinant hCG and its purified subunits are essentially identical to urinary hCG on comparison by biochemical, immunochemical, biological activity and the ability of the isolated subunits to recombine to form a biologically active dimer. Mass spectrometric analysis and preliminary structural NMR data indicate that the labeling is uniform and there is greater than 90% incorporation, sufficient for complete structural determination studies. This labeled growth medium represents a technological advance which will enable the rapid solution of the structures of the other glycoprotein hormones, as well as other glycoproteins which have proven unsuitable for crystallographic study.

The Caenorhabditis elegans gene lin-17, which is required for certain asymmetric cell divisions, encodes a putative seven-transmembrane protein similar to the Drosophila frizzled protein.

Mutations in the gene lin-17 result in the disruption of a variety of asymmetric cell divisions in Caenorhabditis elegans. We have found that lin-17 encodes a protein with seven putative transmembrane domains. The LIN-17 protein is most similar to the Drosophila Frizzled protein and its vertebrate homologs. Studies using a lin-17-green fluorescent protein translational fusion indicate that lin-17 is expressed in mother cells before asymmetric cell divisions and in both daughter cells after the divisions. Our results suggest that lin-17 encodes a receptor that regulates the polarities of cells undergoing asymmetric cell divisions and raise the possibility that the LIN-17 protein acts as a receptor for the Wnt protein LIN-44, which also controls asymmetric cell divisions.

The palindromic LTR-LTR junction of Moloney murine leukemia virus is not an efficient substrate for proviral integration.

We generated viral constructs to test the hypothesis that the major substrate on retroviral DNA that is utilized for proviral DNA integration is the palindromic sequence, termed the LTR-LTR junction, normally present in circular molecules formed by joining the two termini of linear proviral DNA. Recombinant viral genomes were built which carried a selectable marker and an extra copy of the LTR-LTR junction from a cloned circular provirus. The junction sequence in each case was positioned such that its use during integration would lead to an easily detected, aberrantly integrated proviral DNA. Analysis of DNA from cells infected with the virus constructs showed that the introduced junction sequence is used at least 1,000-fold less efficiently than the natural sequences at the ends of the genome. This suggests that a linear or more exotic DNA intermediate is most likely the true precursor for the integration reaction.

Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains.

During B cell development V kappa gene rearrangement seems to occur only in mu-positive pre-B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre-B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre-B cells.

Mutants of murine leukemia viruses and retroviral replication.

The analysis of retroviral mutants has played a critical role in the development of our understanding of the complex viral life cycle. The most fundamental result of that analysis has been the definition of the replication functions encoded by the viruses. From a biochemical examination of a particular step in the life cycle it is difficult to determine, for example, whether that step is catalyzed by a viral or a host enzyme; but the isolation of a viral mutant defective in that step can firmly establish that a viral function is involved. In this way many facts about the viruses have been established. We know that reverse transcriptase is encoded by the virus; that RNAase H and DNA polymerase activities reside on the same gene product; that processing of many precursor proteins is mediated by a viral proteinase; and that establishment of the integrated provirus requires a viral protein. The list of functions mediated by viral enzymes has largely been defined by the mutants isolated and studied in various laboratories. The second significant result of the studies of viral mutants has been the assignation of the replication functions to particular viral genes, and then more specifically to particular domains of these genes. Mutants and viral variants have been essential in the determination, for example, that the gag protein is the critical gene product for the assembly of a virion particle; that the env protein is the determinant of species specificity of infection; or that the LTR is a major determinant of tissue tropism and leukemogenicity. The subdivisions of functions within a given gene have similarly hinged on mutants. Genetic mapping was needed to establish that P30 is the most important region for assembly; that the proteinase and integrase functions reside, respectively, in the 5' and 3' portions of the pol gene; and that the glycosylated gag protein is dispensable for replication. A third important area of knowledge has depended heavily on viral mutants: the determination of host functions and proteins that interact with viral proteins. Variant viruses with altered or restricted host ranges serve to define differences between pairs of different host cells, and the mapping of the viral mutations serves to define the viral protein important in that interaction with the host. These studies are only in their infancy, but it is clear that substantial efforts will be made to further analyze these host functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Insertion mutagenesis of embryonal carcinoma cells by retroviruses.

Mutagenesis was studied in cultured F9 embryonal carcinoma cells infected with a variant of Moloney murine leukemia virus. Proviral insertion induced the inactivation of the hypoxanthine phosphoribosyltransferase locus, and the virus was used to isolate the mutated genes rapidly. Mutagenesis by these methods may be useful for the genetic dissection of the various mammalian cell phenotypes.

Construction and recovery of viable retroviral genomes carrying a bacterial suppressor transfer RNA gene.

The integration of retroviral genomes into cellular DNA can induce mutations by altering the expression of nearby cellular genes and can serve to identify the gene affected. The construction of a retrovirus that stably carries a suppressor transfer RNA gene from Escherichia coli has allowed facile recovery of the viral genome in vectors marked with amber mutations. This virus can be used for rapid isolation of cellular sequences at the site of proviral insertion.

Reverse transcription of retroviral genomes: mutations in the terminal repeat sequences.

The process of reverse transcription of retroviral genomes begins with the synthesis of a short DNA molecule near the 5' end of the RNA template. This molecule, termed minus-strand strong-stop DNA, is then translocated to the 3' end of the viral RNA by means of a repeated sequence, the R region, present at both ends of the template. The translocation should result in the transfer of genetic information from the 5' R region to the 3' R region. We have generated a series of mutants of Moloney murine leukemia virus with alterations in the R regions by in vitro mutagenesis of a cloned DNA copy of the viral genome. The altered DNAs were introduced into mouse cells by transfection, and the translocation of the mutations during viral replication was assessed. Some mutations were not transferred from the 5' R region to the 3' R region; these results were not in accord with current models for reverse transcription. The results can be explained if DNA molecules shorter than strong-stop DNA, formed by premature termination of synthesis, are sometimes translocated. A number of mutants with large deletions in the R region were tested and were able to replicate with normal strong-stop DNA translocation. Thus, short stretches of homology can be used by the virus to carry out strong-stop translocations.

A temperature-sensitive mutation constructed by "linker insertion" mutagenesis.

The in vitro mutagenesis of cloned DNAs allows the formation of virtually any specific mutation, but no method has been found which might routinely lead to the important phenotype of temperature sensitivity. We have studied three linker insertion mutations in the envelope gene of Moloney murine leukemia virus (M-MuLV), and found that one was exquisitely temperature-sensitive for plaque formation. We suggest that the construction of short insertion mutations may be a fruitful approach for the generation of temperature-sensitive phenotypes in cloned genes.