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H D Klenk

Publications and source records attributed to H D Klenk.

At least 127 records · Page 7Linked to original sources
PubMed1992

Marburg virus, a filovirus: messenger RNAs, gene order, and regulatory elements of the replication cycle.

The genome of Marburg virus (MBG), a filovirus, is 19.1 kb in length and thus the largest one found with negative-strand RNA viruses. The gene order - 3' untranslated region-NP-VP35-VP40-GP-VP30-VP24-L-5' untranslated region-resembles that of other non-segmented negative-strand (NNS) RNA viruses. Six species of polyadenylated subgenomic RNAs, isolated from MBG-infected cells, are complementary to the negative-strand RNA genome. They can be translated in vitro into the known structural proteins NP, GP (non-glycosylated form), VP40, VP35, VP30 and VP24. At the gene boundaries conserved transcriptional start (3'-NNCUNCNUNUAAUU-5') and stop signals (3'-UAAUUCUUUUU-5') are located containing the highly conserved pentamer 3'-UAAUU-5'. Comparison with other NNS RNA viruses shows conservation primarily in the termination signals, whereas the start signals are more variable. The intergenic regions vary in length and nucleotide composition. All genes have relatively long 3' and 5' end non-coding regions. The putative 3' and 5' leader RNA sequences of the MBG genome resemble those of other NNS RNA viruses in length, conservation at the 3' and 5' ends, and in being complementary at their extremities. The data support the concept of a common taxonomic order Mononegavirales comprising the Filoviridae, Paramyxoviridae, and Rhabdoviridae families.

Base Sequence↗
PubMed1992

Carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size.

Comparison of the haemagglutinins (HA) of the pathogenic avian influenza viruses A/FPV/Dutch/27 (H7N7) and A/FPV/Rostock/34 (H7N1) revealed 94.7% nucleotide and 93.8% amino acid sequence homologies. Six of the seven N-glycosidic oligosaccharides of the Rostock HA are at the same positions as the six carbohydrates of the Dutch strain. The additional oligosaccharide side chain of the Rostock strain, which is of the complex type, is attached to asparagine149 in antigenic epitope B. The accessibility of this antigenic epitope has been analysed by using rabbit antisera raised against synthetic peptides comprising amino acids 143-162. The carbohydrates of the HA of the Rostock strain have been modified (i) to truncated cores by expression in insect cells using a baculovirus vector, (ii) to oligomannosidic side chains by growth in the presence of the trimming inhibitor methyldeoxynojirimycin and (iii) to a single N-acetylglucosamine residue by removal of the oligomannosidic sugar with endo-beta-N-acetylglucosaminidase H. Neither the authentic nor the modified oligosaccharides allowed antibody binding, as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Reactivity was observed, however, after complete removal of the carbohydrate from HA of the Rostock strain by digestion with peptide-N-glycosidase F. HA of the Dutch strain was reactive without prior peptide-N-glycosidase F treatment. These results demonstrate that a single N-acetyl-glucosamine at asparagine149 is sufficient to prevent recognition of the peptide epitope.

Amino Acid Sequence↗
PubMed1992

Carbohydrate structure of Marburg virus glycoprotein.

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.

Amidohydrolases↗
PubMed1992

Sequence analysis of the Marburg virus nucleoprotein gene: comparison to Ebola virus and other non-segmented negative-strand RNA viruses.

The first 3000 nucleotides from the 3' end of the Marburg virus (MBG) genome were determined from cDNA clones produced from genomic RNA and mRNA. Identified in the sequence was a short putative leader sequence at the extreme 3' end, followed by the complete nucleoprotein (NP) gene. The 5' end of the NP mRNA was determined as was the polyadenylation site for the NP gene. The transcriptional start (3' UUCUUCUUAUAAUU..) and termination (3' ..UAAUUCUUUUU) signals of the MBG NP gene are very similar to those seen with Ebola virus (EBO). In comparison to other non-segmented negative-strand RNA viruses, filovirus transcriptional signals are most similar to members of the Paramyxovirus and Morbillivirus genera. In vitro translation of a run-off transcript containing the entire MBG NP coding region produced an authentic NP. Sequence comparisons of the 3' end of the MBG and EBO genomes revealed weak nucleotide sequence similarity, but the predicted sequence of the first 400 amino acids of these viruses showed a high degree. This homology is encoded in divergent nucleotide sequences through different codon usages and substitutions of similar amino acids. A small region in the middle of the MBG and EBO NP sequences was found to contain a significant amino acid homology with NPs of paramyxoviruses and to a lesser extent with rhabdoviruses. Specific sites of conserved sequence are contained in hydrophobic domains and may have a common function. Alignments of the entire NP amino acid sequences of these viruses also suggest that filoviruses are more closely related to paramyxoviruses than to rhabdoviruses.

Amino Acid Sequence↗
PubMed1992

Secretion of fowl plague virus haemagglutinin from insect cells requires elimination of both hydrophobic domains.

In the present study we have investigated the role of the hydrophobic domains of the fowl plague virus (FPV) haemagglutinin (HA) on its intracellular transport and maturation in insect cells. To this end processing of full-length HA (A+) has been compared to that of two truncated forms lacking either the cytoplasmic domain and the transmembrane domain (A-) or lacking the entire HA2 subunit, i.e. the transmembrane domain and the fusion peptide (HA2-). All glycosylation sites present on A- and HA2- were glycosylated, indicating that both truncated forms were completely translocated in the endoplasmic reticulum. Unlike A+, A- and HA2- did not form trimers as indicated by cross-linking, gradient centrifugation and studies employing conformation-specific antibodies. Whereas HA2- was efficiently secreted, A- was retained in the cells in an apparently membrane-bound form. The data show that the carboxy-terminal transmembrane region is essential for the formation and stability of the trimers of the FPV HA. These observations also indicate that, under certain conditions, the fusion peptide of the FPV HA can serve as a membrane anchor.

Animals↗
PubMed1992

Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice.

Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.

Amino Acid Sequence↗
PubMed1992

Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.

Amino Acid Sequence↗
PubMed1991

Intraepidemic variants of influenza virus H3 hemagglutinin differing in the number of carbohydrate side chains.

During the epidemic outbreak in the region of Greifswald in the winter 1974/75, we found influenza virus variants which showed differences in the electrophoretic mobility of HA. Among the 25 isolates 13 were of slower and 12 of higher mobility. HA1 of 6 isolates was studied by determining the number of the carbohydrate side chains and by direct sequencing of vRNA. Evidence is presented that variants showing a slower electrophoretic mobility of HA1 had consistently acquired a seventh carbohydrate side chain at Asn 126 in epitope A. All the isolates differed from the reference strain A/Port Chalmers/1/73 by the loss of the oligosaccharide at Asn 81. The field strain A/Dresden/3/71 possessed only 5 oligosaccharides in HA1. These results suggest that changes in glycosylation are an important mechanism in the structural variation underlying antigenic drift of HA.

Antigenic Variation↗
PubMed1991

Retarded processing of influenza virus hemagglutinin in insect cells.

When expressed in Spodoptera frugiperda cells by a baculovirus vector, the hemagglutinin of fowl plague virus has been found to contain palmitic acid in covalent hydroxylamine-sensitive linkage, indicating that these cells have the capacity to acylate foreign proteins at cysteine residues. Centrifugation on sucrose density gradients and immune precipitation with conformation-specific antibodies were used to compare trimerization of the hemagglutinin in insect cells and in fowl plague virus-infected MDCK cells. Trimerization of the hemagglutinin was incomplete in insect cells, and the kinetics of this reaction were about three times slower than in vertebrate cells. Similarly, post-translational proteolytic cleavage occurred in insect cells with a half-time of 90 min, and a substantial fraction of the hemagglutinin persisted in uncleaved form. In contrast, hemagglutinin was almost completely cleaved in MDCK cells, and the half-time of cleavage was only 30 min. The data indicate that in insect cells trimerization and, as a result, the subsequent processing steps of the hemagglutinin, are retarded and less efficient. The possible roles of aberrant glycosylation, acidic milieu, and lack of other influenza virus proteins in hemagglutinin trimerization are discussed.

Acylation↗
PubMed1991

Isolated HE-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity.

Bovine coronavirus (BCV) and hemagglutinating encephalomyelitis virus (HEV) from swine were found to grow to high titers in MDCK I cells, a subline of Madin Darby canine kidney cells. Virus grown in these cells was used to isolate and purify the HE-protein. This protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of BCV. Here we show that HEV contains this enzyme, too. The glycoproteins were solubilized by treatment of virions with octylglucoside. Following centrifugation through a sucrose gradient the surface proteins S and HE (hemagglutinin-esterase) were obtained in purified form. After removal of the detergent by dialysis, HE formed rosettes as shown by electron microscopy. The purified HE protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. HE protein released from the viral membrane failed to agglutinate red blood cells. However, it was found to recognize glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of BCV, HEV, and influenza C virus. The purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses.

Acetylesterase↗
PubMed1991

Glycosylation and oligomerization of the spike protein of Marburg virus.

The oligosaccharide side chains of the glycoprotein of Marburg virus (MW 170,000) have been analyzed by determining their sensitivity to enzymatic degradation and their reactivity with lectins. It was found that they consist of N- and O-glycans. Studies employing chemical cross-linking showed that the glycoprotein is present as a homotrimer in the viral envelope.

Cross-Linking Reagents↗
PubMed1991

Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus.

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.

Amino Acid Sequence↗
PubMed1991

The M2 protein of influenza A virus is acylated.

The M2 protein of influenza A virus, a 97 amino acid integral membrane protein expressed on the surface of infected cells, is covalently modified with long chain fatty acids. The fatty acid bond is sensitive to treatment with neutral hydroxylamine and mercaptoethanol, which indicates a labile thioester type linkage. Thin-layer chromatographic fatty acid analysis of [3H]myristic and [3H]palmitic acid-labelled M2 protein shows that palmitic acid is the predominant fatty acid linked to this polypeptide. Palmitoylation of M2 occurs post-translationally and causes an upward shift in the SDS-PAGE mobility of the protein.

Acylation↗
PubMed1991

Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin.

The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.

Acylation↗
PubMed1991

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.

Amino Acid Sequence↗
PubMed1991

Modification of the cleavage activation of the influenza virus hemagglutinin by site-specific mutagenesis.

Factors determining cleavability of influenza virus hemagglutinin which is activated by ubiquitous cellular endoproteases were analysed by carrying out site-directed mutagenesis on the cloned hemaglutinin genes of strains A/FPV/Rostock/34 (subtype H7) and A/Port Chalmers/1/73 (subtype H3). Substitutions at the cleavage site of the H7 hemagglutinin indicate that the tetrapeptide Arg-X-Lys/Arg-Arg is the minimal consensus sequence recognized by the ubiquitous proteases. The H3 hemagglutinin also became susceptible to these enzymes, when additional arginines were inserted at the cleavage site. Three arginines were sufficient, when the carbohydrate was removed, whereas four additional arginines are needed when this carbohydrate was present, indicating that the accessibility of the cleavage motif is important for the protease. The appropriate localization of the basic cleavage motif within the amino acid sequence and the spatial structure of the hemagglutinin precursor is an additional prerequisite for cleavage.

Amino Acid Sequence↗