Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection.

The effects of monospecific antibodies to the viral glycoprotein with hemagglutinating and neuraminidase activity (HN) and the viral glycoprotein with membrane-fusing activity (F) of the paramyxovirus simian virus 5 (SV5) on the spread of infection in two cell types have been investigated. In CV-1 cells, infection can spread by either released progeny virus adsorbing to and infecting other cells, or by fusion of an infected cell with an adjacent cell as a result of the cell-fusing activity of the F glycoprotein. In these cells, antibodies specific for the HN glycoprotein prevented the dissemination of infection by released infectious virus, but spread by cell fusion was not inhibited. Antibodies to the F glycoprotein completely prevented the spread of infection in these cells. In Madin-Darby bovine kidney cells, which are relatively resistant to SV5-induced fusion, antibodies to either the HN or F glycoproteins were capable of preventing the dissemination of infection. These results indicate that effective immunological prevention of the spread of paramyxovirus infection requires the presence of antibodies that inactivate the F glycoprotein. This requirement for anti-F antibodies has obvious implications for the design of effective paramyxovirus vaccines and provides an explanation for previous failures of formalin-inactivated paramyxovirus vaccines as well as additional insight into the possible immunopathological mechanisms involved in the atypical and severe infections that have occurred in individuals who received inactivated paramyxovirus vaccines and were subsequently infected by the virus.

Use of DNA end joining activity of a Xenopus laevis egg extract for construction of deletions and expression vectors for HIV-1 Tat and Rev proteins.

We have developed a cloning strategy which combines conventional T4 DNA ligation with the highly efficient nonhomologous DNA end joining (EJ) activity of an extract from Xenopus laevis eggs. The nonhomologous EJ activity allowed the rapid construction of deletion mutants by the intramolecular rejoining of nonhomologous DNA ends generated for the purpose of deleting restriction fragments from the vector. The combined use of T4 DNA ligase for intermolecular ligation and X. laevis egg extracts for intramolecular nonhomologous EJ proved to be a powerful tool, as demonstrated here for the construction of expression vectors for HIV-1 Tat and Rev.

Stepwise deletion of the HIV type 1 glycoprotein 41 N terminus leads to an increasing export of microvesicles containing uncleaved Env glycoprotein.

Deletion of two or more amino acid residues from the N terminus of HIV-1 gp41 leads to an increasing loss of cleavability of the envelope (Env) precursor on introduction of an env-expressing vector into HeLa-T4+ cells. In protein analysis, this is paralleled by the appearance of a second form of uncleaved Env precursor that is terminally sialylated. Cell-derived microvesicles that preferentially incorporate this form of Env precursor were found in the culture medium. The same applies to a mutant with a nonfunctional cleavage site, indicating that a pathway by which uncleaved Env glycoprotein leaves the cell exists. The amount of exported glycoprotein is augmented as compared with wild-type Env. Transfection with a wild-type Env-expressing vector leads to the presence of extracellular microvesicles that contain only the transmembrane domain of HIV-1 Env. Microvesicles derived from wild-type Env and mutant Env contain sialylated glycoproteins that are resistant to exo- and endoglycosidase treatment unless the particles have been previously lysed by detergent. This raises the possibility that the C-terminal domains of the glycoproteins are exposed on the surface of the exported microvesicles.

Natural variation in the amino acid sequence around the HIV type 1 glycoprotein 160 cleavage site and its effect on cleavability, subunit association, and membrane fusion.

To assess the natural variation of the structure of the cleavage site as well as the N-terminal region of gp41 for the cytopathogenicity of HIV-1, syncytium-inducing (SI) and non-syncytium-inducing (NSI) virus isolates were obtained from HIV-1-infected patients. In addition, the coreceptor usage of the isolates was determined by infection of primary macrophages and PM-1 cells. DNA sequences encoding the C-terminal 41 amino acid residues of gp120 and the 64 amino acid N-terminal residues of gp41 were amplified by the polymerase chain reaction and inserted into the Env expression vector pNLA1. When transfected into HeLa-T4(+) cells, all the recombinant plasmids, including those with inserts from NSI isolates, led to the formation of processed glycoprotein and to syncytium formation. One construct displayed significant lowered fusion capacity and had an amino acid exchange in the first position of the gp41 N terminus (gp41, 512A-->S) leading to a decreased association of the SU and TM subunits. Four constructs derived from two isolates of the same patient showed an unusual gp41 N terminus (gp41, 514G-->P) and a slightly diminished fusion capacity due to a decreased cleavability. This indicates that the major determinants for the SI and NSI phenotypes are not located around the gp160 cleavage site and that the N terminus of gp41 plays a minor role in the processing and fusion capacity of wild-type HIV-1 isolates.

Oxygen-regulated expression of TGF-beta 3, a growth factor involved in trophoblast differentiation.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

Effects of the antirheumatic remedy hox alpha--a new stinging nettle leaf extract--on matrix metalloproteinases in human chondrocytes in vitro.

Inflammatory joint diseases are characterized by enhanced extracellular matrix degradation which is predominantly mediated by cytokine-stimulated upregulation of matrix metalloproteinase (MMP) expression. Besides tumour necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta) produced by articular chondrocytes and synovial macrophages, is the most important cytokine stimulating MMP expression under inflammatory conditions. Blockade of these two cytokines and their downstream effectors are suitable molecular targets of antirheumatic therapy. Hox alpha is a novel stinging nettle (Urtica dioica/Urtica urens) leaf extract used for treatment of rheumatic diseases. The aim of the present study was to clarify the effects of Hox alpha and the monosubstance 13-HOTrE (13-Hydroxyoctadecatrienic acid) on the expression of matrix metalloproteinase-1, -3 and -9 proteins (MMP-1, -3, -9). Human chondrocytes were cultured on collagen type-II-coated petri dishes, exposed to IL-1beta and treated with or without Hox alpha and 13-HOTrE. A close analysis by immunofluorescence microscopy and western blot analysis showed that Hox alpha and 13-HOTrE significantly suppressed IL-1beta-induced expression of matrix metalloproteinase-1, -3 and -9 proteins on the chondrocytes in vitro. The potential of Hox alpha and 13-HOTrE to suppress the expression of matrix metalloproteinases may explain the clinical efficacy of stinging nettle leaf extracts in treatment of rheumatoid arthritis. These results suggest that the monosubstance 13-HOTrE is one of the more active antiinflammatory substances in Hox alpha and that Hox alpha may be a promising remedy for therapy of inflammatory joint diseases.

Isolation and purification of the envelope proteins of Newcastle disease virus.

A procedure has been developed for the isolation of Newcastle disease virus (NDV) envelope proteins. The two surface glycoproteins and the non-glycosylated membrane protein were solubilized with 2% Triton X-100 and 1 m KCl. Removal of the KCl by dialysis yielded by precipitation a pure preparation of the non-glycosylated membrane protein, which is insoluble in solutions of low ionic strength. The soluble fraction consisting of the two glycoproteins possessed full neuraminidase and hemagglutinating activities. The two glycoproteins could be separated by rate zonal sedimentation in a sucrose gradient containing 1% Triton X-100 and 1 m KCl. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 1, was 9.3s, and that of the smaller, virus protein 2, was 6.1s. Both hemagglutinating and neuraminidase activities were associated with virus protein 1; virus protein 2 had neither activity. The results suggest that both activities reside on a single NDV glycoprotein. Similar results were obtained previously with another paramyxovirus, simian virus 5. These findings suggest that the association of hemagglutinating and neuraminidase activities with one glycoprotein is a general property of the paramyxovirus group.

Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression.

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.

Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.

The conformations of the helical nucleocapsids of the paramyxoviruses Sendai virus and simian virus 5, and of a rhabdovirus, vesicular stomatitis virus, have been found to vary extensively with changes in salt concentration. In 10 mM sodium phosphate buffer at pH 7.2, the nucleocapsids are loosely coiled or almost completely extended; with increasing concentrations of NaCl they become more tightly coiled and less flexible. Under isotonic conditions (150 mM) the Sendai virus nucleocapsid is moderately tightly coiled but still curved and apparently flexible, whereas at 400 mM or higher it is very tightly coiled, with the appearance of a rigid rod. These salt-dependent changes in conformation were also found with nucleocapsids composed of proteolytically cleaved protein subunits. Because of the effect of salt concentration, and the fact that it may change during the preparation of negatively stained samples of electron microscopy, it was necessary to fix that nucleocapsids before negative staining to preserve their original conformation. The striking changes in nucleocapsid conformation in response to the ionic milieu indicate the plasticity of its helical structure and suggest that changes in the microenvironment of the nucleocapsid could influence its conformation during viral RNA transcription and replication or during virus assembly by budding, processes in which changes in the coiling of the nucleocapsid or its flexibility could be important.

Histomorphological and lectin-histochemical confirmation of the antidegenerative effect of diclofenac in experimental osteoarthrosis.

The integrity of cartilage matrix depends on the homeostasis of synthetic and degradative processes. Any disturbance of the rate of synthesis and catabolism may alter the amount of matrix components (e.g. proteoglycans). Based upon a biochemically induced osteoarthrosis (OA) in the knee joints of rats we investigated the histomorphological alterations under therapy with diclofenac sodium by histological-histochemical grading. Lectin-binding techniques using labelled wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), and peanut agglutinin (PNA) were applied to analyze the cellular as well as the extracellular glycoconjugates in situ. Lectin-binding patterns quantitatively describe the topographical localization of structural components of the cartilage matrix which carry certain sugar residues. The therapy of the experimental OA with diclofenac sodium (2.0 mg/kg s.c.) led to a marked reduction of cartilage degenerations. Our results indicate antidegenerative properties of this compound. These findings are consistent with the fluorescent analytical data which show a stimulating effect on the anabolic activity of chondrocytes in the osteoarthritic joints under the treatment with diclofenac sodium. Fluorescein isothiocyanate labelled lectins are useful histochemical tools to determine alterations in the integrity of cartilage by their specific binding patterns, because zonal differentiation in physiological function and morphological structure of cartilage tissue as well as cellular, pericellular, and interterritorial local events are characterized.(ABSTRACT TRUNCATED AT 250 WORDS)

Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site.

Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F1 and F2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P.W. Choppin [1976). Virology, 265-277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60-70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.

Genetically modified mouse models in studies on cutaneous wound healing.

Genetically modified mice have become an invaluable tool in modern biomedical and basic research. This review provides an overview of knockout and transgenic mice studied with regard to their cutaneous wound healing properties. In addition, several gene transfer studies are briefly introduced, which have further highlighted our knowledge on individual gene function in wound healing.

Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer.

The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.

Fusion of Sendai virus with liposomes: dependence on the viral fusion protein (F) and the lipid composition of liposomes.

The characteristics of fusion of the membrane of Sendai virus with that of liposomes has been investigated using two different methods to monitor the fusion reaction. The first method, which permits quantitation of lipid fused with virus, depends on separation by centrifugation of unfused liposomes from those fused with virus. The second involves the digestion after fusion of internal viral proteins by trypsin contained in liposomes; this assay is completely independent of exchange of lipid between liposomal and viral membranes in the absence of fusion. A fusion-inactive mutant virus, pa-cl, with an uncleaved F protein served as the appropriate control in these experiments. It was found that fusion of the virus with liposomes that contained no protein required cleavage of the F protein; such cleavage was previously shown to be required for fusion of the virus with cell membranes. This indicates the relevance of this model system for studies of fusion. Kinetic studies indicated that at neutral pH fusion was 88% complete in 10 min at 37 degrees. Investigation of the effects of liposomal lipid composition indicated that the presence of cholesterol in the liposomal membrane was required for fusion; a 0.3-0.4-mole fraction of cholesterol was optimal. The presence of neuraminic acid in the membrane was not essential for fusion. The results obtained are compatible with previous evidence suggesting a hydrophobic interaction between the cleaved F protein and the target membrane during fusion.

Enhancement of membrane-fusing activity of sendai virus by exposure of the virus to basic pH is correlated with a conformational change in the fusion protein.

The effect of pH on the membrane-fusion activity of Sendai virus was examined (pH 5.0-9.5) by using, as assays of activity, hemolysis of chicken erythrocytes and the fusion of baby hamster kidney (BHK-21) cells. Exposure of virus to basic pH increased fusion activity; the optimum pH was found to be approximately equal to 9.0. All assays were carried out at pH 7.0, and the virus retained enhanced fusion activity after it was exposed to basic pH and returned to neutral pH. The enhanced fusion activity was correlated with an irreversible conformational change in the fusion protein (F protein) of the virus, as demonstrated by a change in the circular dichroism spectrum of the protein.