Biomedical subjects

H D Klenk

Publications and source records attributed to H D Klenk.

At least 109 records · Page 6Linked to original sources

PubMed1994

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.

Amino Acid Sequence↗

PubMed1994

Baculovirus infection does not alter N-glycosylation in Spodoptera frugiperda cells.

Spodoptera frugiperda cells are the standard host system for baculovirus-vector-mediated expression of recombinant glycoproteins. In an attempt to explore their ability to produce complex N-glycans containing terminal neuraminic acid, we tested both mock- and AcMNPV-infected SF9 cells. To elucidate the structures of the carbohydrate chains of cellular glycoproteins, radiolabeled oligosaccharides were liberated by treatment with endo-H and glycopeptidase F. When the endo-H resistant material was subjected to sequential degradation with exoglycosidases, only truncated carbohydrates with the structures Man3GlcNAc2 and Man3[Fuc]GlcNAc2 were found. There was no evidence for the presence of neuraminic acid and complex N-glycans. The results indicate that SF9 cells have only a limited capacity to process N-glycans. Infection with AcMNPV has no significant effect on glycosylation in these cells.

Amidohydrolases↗

PubMed1993

Methyl alpha-glycoside of N-thioacetyl-D-neuraminic acid: a potential inhibitor of influenza A virus. A 1H NMR study.

The binding of influenza A virus hemagglutinin to its cell surface receptor, alpha-linked 5-N-acetylneuraminic acid (sialic acid), was studied in solution. The effect of structural modifications introduced into the N-acetyl group of the sialic acid on the binding was monitored by determining the dissociation constants by proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl alpha-glycoside of N-thioacetylneuraminic acid showed high, whereas the corresponding N-methylcarbamoylneuraminic acid exhibited relatively low binding affinity towards the hemagglutinin.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Role of the M2 protein in influenza virus membrane fusion: effects of amantadine and monensin on fusion kinetics.

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 microM AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 microM monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25 degrees, but were very prominent at 45 degrees. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.

Amantadine↗

PubMed1993

Molecular biology and evolution of filoviruses.

The family Filoviridae contains extremely pathogenic human viruses causing a fulminating, febrile hemorrhagic disease. Filoviruses are enveloped, filamentous particles with a nonsegmented negative-strand RNA genome showing the gene arrangement 3'-NP-VP35-VP40-GP-VP30-VP24-L-5'. Genes are flanked by highly conserved transcriptional signals and are generally separated by variable intergenic regions. They are transcribed into monocistronic polyadenylated messenger RNAs which contain relatively long 5' and 3' untranslated regions. Seven structural proteins are encoded by the genome of which four form the helical nucleocapsid (NP-VP35-VP30-L), two are membrane-associated (VP40-VP24), and one is a transmembrane glycoprotein (GP). Comparison of filovirus genomes with those of other nonsegmented negative-strand RNA viruses suggest comparable mechanisms of transcription and replication and a common evolutionary lineage for all these viruses. Sequence analyses of single genes, however, showed that filoviruses are more closely related to paramyxoviruses, particularly human respiratory syncytial virus. These data support the concept of the taxonomic order Mononegavirales for all nonsegmented negative-strand RNA viruses and the classification of Marburg virus, Ebola virus, and Reston virus in the family Filoviridae, separate from the families Paramyxoviridae and Rhabdoviridae.

Animals↗

PubMed1993

Antibody response to the M2 protein of influenza A virus expressed in insect cells.

A recombinant baculovirus expressing the M2 protein from influenza A/Ann Arbor/6/60 (H2N2) virus (AA60 virus) was constructed. The expressed M2 protein was recognized by a monoclonal antibody specific for the M2 protein and comigrated with the M2 protein from cells infected with AA60 virus on SDS-polyacrylamide gels. Immunofluorescence studies indicated that the expressed M2 protein was present on the surface of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus. Immunoassays using the expressed M2 protein were able to detect antibodies to the M2 protein in serum samples from humans and ferrets infected with influenza A viruses.

Animals↗

PubMed1993

Production of the M2 protein of influenza A virus in insect cells is enhanced in the presence of amantadine.

Recombinant baculoviruses that express the M2 protein from the genes of either the amantadine-sensitive, influenza A/Ann Arbor/6/60 virus or a laboratory-derived, amantadine-resistant mutant of this virus were constructed. Addition of amantadine or rimantadine at 2 micrograms/ml to cultures of Sf9 cells infected with the recombinant baculoviruses increased the yield of the M2 protein from the amantadine-sensitive virus approximately 10-fold, but did not increase the yield of the M2 protein from the amantadine-resistant virus. Flow cytometry demonstrated that the increased production of M2 in the presence of amantadine resulted in increased cell surface expression of the M2 protein. Pulse-chase experiments indicated that whereas the rate of synthesis of the M2 protein increased in the presence of amantadine, the M2 protein was stable in both the presence and absence of amantadine. Addition of amantadine to Sf9 cells as late as 72 h after infection with the recombinant virus increased the production of M2 protein. These data suggest that the M2 protein exerts some biological activity in Sf9 cells.

Amantadine↗

PubMed1993

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.

Animals↗

PubMed1993

Marburg virus gene 4 encodes the virion membrane protein, a type I transmembrane glycoprotein.

Gene 4 of Marburg virus, strain Musoke, was subjected to nucleotide sequence analysis. It is 2,844 nucleotides long and extends from genome position 5821 to position 8665 (EMBL Data Library, emnew: MVREPCYC [accession no. Z12132]). The gene is flanked by transcriptional signal sequences (start signal, 3'-UACUUCUUGUAAUU-5'; termination signal, 3'-UAAUUCUUUUU-5') which are conserved in all Marburg virus genes. The major open reading frame encodes a polypeptide of 681 amino acids (M(r), 74,797). After in vitro transcription and translation, as well as expression in Escherichia coli, this protein was identified by its immunoreactivity with specific antisera as the unglycosylated form of the viral membrane glycoprotein (GP). The GP is characterized by the following four different domains: (i) a hydrophobic signal peptide at the amino terminus (1 to 18), (ii) a predominantly hydrophilic external domain (19 to 643), (iii) a hydrophobic transmembrane anchor (644 to 673), and (iv) a small hydrophilic cytoplasmic tail at the carboxy terminus (674 to 681). Amino acid analysis indicated that the signal peptide is removed from the mature GP. The GP therefore has the structural features of a type I transmembrane glycoprotein. The external domain of the protein has 19 N-glycosylation sites and several clusters of hydroxyamino acids and proline residues that are likely to be the attachment sites for about 30 O-glycosidic carbohydrate chains. The region extending from positions 585 to 610 shows significant homology to a domain observed in the envelope proteins of several retroviruses and Ebola virus that has been suspected to be responsible for immunosuppressive properties of these viruses. A second open reading frame of gene 4 has the coding capacity for an unidentified polypeptide 112 amino acids long.

Amino Acid Sequence↗

PubMed1993

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.

Biological Transport↗

PubMed1993

Replication of Marburg virus in human endothelial cells. A possible mechanism for the development of viral hemorrhagic disease.

Marburg and Ebola virus, members of the family Filoviridae, cause a severe hemorrhagic disease in humans and primates. The disease is characterized as a pantropic virus infection often resulting in a fulminating shock associated with hemorrhage, and death. All known histological and pathophysiological parameters of the disease are not sufficient to explain the devastating symptoms. Previous studies suggested a nonspecific destruction of the endothelium as a possible mechanism. Concerning the important regulatory functions of the endothelium (blood pressure, anti-thrombogenicity, homeostasis), we examined Marburg virus replication in primary cultures of human endothelial cells and organ cultures of human umbilical cord veins. We show here that Marburg virus replicates in endothelial cells almost as well as in monkey kidney cells commonly used for virus propagation. Our data support the concept that the destruction of endothelial cells resulting from Marburg virus replication is a possible mechanism responsible for the hemorrhagic disease and the shock syndrome typical of this infection.

Base Sequence↗

PubMed1992

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.

Amino Acid Chloromethyl Ketones↗

PubMed1992

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.

Affinity Labels↗

PubMed1992

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion.

Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F1-R. In addition, T-9, a mutant with bipolar budding phenotype of F1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.

Animals↗

PubMed1992

Budding site of Sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice.

Wild-type Sendai virus fusion (F) glycoprotein requires trypsin or a trypsin-like protease for cleavage-activation in vitro and in vivo, respectively. The virus is pneumotropic in mice and buds at the apical domain of bronchial epithelial cells. On the other hand, the F protein of the protease-activation host range mutant, F1-R, is cleaved by ubiquitous proteases present in different cell lines and in various organs of mice. F1-R causes a systemic infection in mice and the mutant buds bipolarly at the apical and basolateral domains of infected epithelial cells. The enhanced cleavability of the F protein of F1-R has been shown to be a primary determinant for pantropism. Additionally, it has been postulated that bipolar budding of F1-R is required for the systemic spread of the virus and it has been attributed to mutations in the matrix (M) protein of F1-R (Tashiro et al., Virology 184, 227-234, 1991). In this study protease-activation mutants (KD series) were isolated from wild-type virus. They were revealed to bud at the apical domain, and the F protein was cleaved by ubiquitous proteases in mouse organs. The KD mutants were exclusively pneumotropic in mice following intranasal infection, whereas they caused a generalized infection when inoculated directly into the circulatory system. Comparative nucleotide sequence analysis of the F gene of the KD mutants revealed that the deduced amino acid substitutions responsible for enhanced cleavability of the F protein occurred removed from the cleavage site. Mutations were not at all found in the M gene of the KD mutants analyzed, in support of the role of the M protein of F1-R and of a revertant T-9 derived from the latter in bipolar budding. These results suggest that bipolar budding is necessary for the systemic spread of F1-R from the lungs and that apical budding by wild-type virus and the KD mutants leads to respiratory infections. Differential budding at the primary target of infection, in addition to the cleavage-activation of the F protein in mouse organs, is therefore also a determinant for tropism and pathogenicity of Sendai virus in mice.

Amino Acid Sequence↗

PubMed1992

The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses.

The nucleotide sequence of the L gene of Marburg virus, strain Musoke, has been determined. The L gene has a single long open reading frame encoding a polypeptide of 2330 amino acids (MW 267,175) that represents the viral RNA-dependent RNA polymerase. The putative transcription start signal (3'CUACCUAUAAUU 5') and the termination signal (3' UAAUUCUUUUU 5') of the gene could be identified. Computer-assisted comparison of the L protein with L proteins of other nonsegmented negative-stranded RNA viruses (Paramyxoviridae: Sendai virus, Newcastle disease virus, human parainfluenza 3 virus, measles virus, human respiratory syncytial virus; Rhabdoviridae: vesicular stomatitis virus, rabies virus) revealed significant homologies primarily in the N-terminal half of the proteins. We have identified three common conserved boxes (A, B, and C) among filo-, paramyxo-, and rhabdovirus L proteins, which are probably involved in the polymerase function. The L proteins can be divided into an N-terminal half, which seems to accommodate the common enzymatic sites, and a C-terminal half carrying virus specific peculiarities. The data presented here suggest a common evolutionary history for all nonsegmented negative-stranded RNA viruses and show that filoviruses are more closely related to paramyxo- than to rhabdoviruses.

Amino Acid Sequence↗

PubMed1992

A single point mutation of the influenza C virus glycoprotein (HEF) changes the viral receptor-binding activity.

From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

Amino Acid Sequence↗

PubMed1992

Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R.

The hemagglutinin of influenza virus A/FPV/Rostock/34 (H7) was altered at its multibasic cleavage site by site-directed mutagenesis and assayed for proteolytic activation after expression in CV-1 cells. The results indicated that the cellular protease responsible for activation recognizes the tetrapeptide motif R-X-K/R-R that must be presented in the correct sequence position. Studies on plaque variants of influenza virus A/fowl/Victoria/75 (H7N7) showed that alteration of the consensus sequence resulted in a loss of pathogenicity for chickens.

Amino Acid Sequence↗