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H D Klenk

Publications and source records attributed to H D Klenk.

At least 19 recordsLinked to original sources
PubMed1998

Release of viral glycoproteins during Ebola virus infection.

Maturation and release of the Ebola virus glycoprotein GP were studied in cells infected with either Ebola or recombinant vaccinia viruses. Significant amounts of GP were found in the culture medium in nonvirion forms. The major form represented the large subunit GP1 that was shed after release of its disulfide linkage to the smaller transmembrane subunit GP2. The minor form were intact GP1,2 complexes incorporated into virosomes. Vector-expressed GP formed spikes morphologically indistinguishable from spikes on virus particles, indicating that spike assembly is independent of other viral proteins. Analysis of a truncation mutant revealed an early and almost complete release of GP1,2 molecules, showing that membrane anchoring is mediated by the carboxy-terminal hydrophobic domain of GP2. We have also compared wild-type virus which requires transcriptional editing for synthesis of full-length GP with a variant that does not depend on editing. Both viruses released comparable amounts of GP1, but the variant expressed only minute amounts of the small, soluble GP which is the expression product of nonedited mRNA species of the GP gene. The abundant shedding of soluble GP1 may play an important role in the immunopathology of Ebola hemorrhagic fever in experimentally and naturally infected hosts.

Cell Line↗
PubMed1998

Processing of the Ebola virus glycoprotein by the proprotein convertase furin.

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.

Animals↗
PubMed1998

How to overcome resistance of influenza A viruses against adamantane derivatives.

We tested two approaches to overcoming resistance of influenza A viruses against adamantane derivatives. First, adamantane derivatives that interfere with the ion channel function of the variant M2 protein of amantadine-resistant viruses may prevent drug resistance, if they are used in mixture with amantadine. Second, amantadine acts on the M2 protein (at low concentrations) and indirectly on the hemagglutinin (at concentrations at least 100 times higher). Identifying and using a drug that reacted with both targets at the same concentration might reduce development of resistance, since, in this case, two mutations, one in each target protein would be necessary at once. Such a double mutation is assumed to be a rare event. We evaluated forty adamantane derivatives and two related compounds to determine whether they interfered with plaque formation by influenza A strains, including A/Singapore/1/57 (H2N2). Variants resistant to drugs that interfered at low concentrations (approximately 1 microg/ml; e.g. amantadine) were cross-resistant with each other, but were sensitive to those agents effective at high concentrations (8 microg/ml; e.g. memantine). The former group of compounds act on the ion channel; the corresponding escape mutants tested had amino acid replacements at positions 27, 30 or 31 of the M2 protein. Hemagglutinin was the indirect target of the latter group of compounds. Variants resistant to these agents lacked amino acid replacements within the ion channel of the M2 protein and the mutants tested had amino acid replacements in the hemagglutinin. Although we failed to identify compounds that interacted with the ion channel of amantadine-resistant variants and inhibited their replication, we were able to construct at least two compounds that interfered with both the ion channel and the hemagglutinin at about the same concentration. After passage in the presence of these compounds, we either failed to obtain any drug-resistant mutants or those obtained had amino acid replacements in the ion channel of the M2 protein and the hemagglutinin.

Adamantane↗
PubMed1998

Elongation of the cytoplasmic tail interferes with the fusion activity of influenza virus hemagglutinin.

The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.

Amino Acids↗
PubMed1997

Histones as a target for influenza virus matrix protein M1.

Matrix protein M1 purified from influenza A and B viruses has been analyzed for its ability to specifically interact with cellular proteins by immune coprecipitation and by an in vitro binding assay on nitrocellulose on PVDF membranes. When M1 was mixed with lysates of uninfected cells there was selective binding of histones H2A, H2B, H3, and H4. Week binding of H1 was also observed. The binding specificity of M1 was confirmed by using purified histones. The M1-histone complexes were dependent on pH and ionic strength, indicating electrostatic interactions. Chemical cleavage of M1 by formic acid into an N-terminal 9-kDa fragment and a C-terminal 18-kDa fragment did not abolish interaction with histones. However, after treatment with 1 M sodium chloride cleaved M1 no longer bound to histones, whereas uncleaved M1 showed an increased binding activity after salt treatment. These findings suggest that both N- and C-terminal domains of M1 are involved in histone binding and that conformation of M is an important factor in this interaction. The data support the notion that there is specific interaction of M1 with nucleosomes during the nuclear phase of influenza virus replication.

Animals↗
PubMed1997

Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin-Darby canine kidney cells (type I).

gp40 has been recently identified as a major apical cell-surface sialoglycoprotein of type-I Madin-Darby canine kidney cells, a cell line widely used for the study of polarized transport. The determination of two internal amino acid sequences of the purified glycoprotein by Edman degradation enabled us to isolated the cDNA encoding the 18.6 kDa protein backbone of gp40. Sequence analysis revealed that gp40 is a type-I membrane protein which has several characteristics in common with glycophorin A and other mucin-type glycoproteins. At least 14 serine/threonine residues were found to be used for O-glycosylation. No potential sites for N-glycosylation were detected. gp40 turned out to represent the canine homologue of a cell-surface antigen expressed by various epithelial and non-epithelial cells in rat and mouse. Potential O-glycosylation sites, transmembrane and cytoplasmic domains were found to be highly conserved in the three species. gp40 was detected in canine lung, intestine, kidney, brain and heart but not in liver and spleen. The subline II of Madin-Darby canine kidney cells was found not to express gp40. Stable expression of gp40 in transfected type-II cells revealed that gp40 is predominantly delivered to the apical plasma membrane. N-Glycans and a glycosylphosphatidylinositol anchor, both proposed apical targeting signals, are absent from gp40, indicating that other determinants are responsible for its polarized transport.

Amino Acid Sequence↗
PubMed1997

Emergence of subtype Zaire Ebola virus in Gabon.

Gabon has recently been struck three times by Ebola hemorrhagic fever. The first isolate originating from the 1994 outbreak has been subjected to molecular characterization of its GP and VP24 genes. Sequence analysis demonstrates that the agent, Gabon-94 virus, belongs to subtype Zaire of Ebola virus. The isolate is closely related to the Kikwit-95 isolate, and both viruses seem to have evolved from a progenitor virus different from that of the Zaire-76 isolates. The relatively close relationship of all subtype Zaire viruses isolated at different geographical locations and up to 20 years apart suggests an extreme conservation in the yet unknown natural reservoir of Ebola viruses. The level of genetic variability in the human host might be different as indicated by the comparison of isolates from a single outbreak (Mayinga-76 and Eckron-76), but needs further investigation on clinical material of patients by PCR since both isolates have different levels of passages in tissue culture.

Animals↗
PubMed1997

Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides.

Intracellular transport, glycosylation, tetramerization and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a half-time of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPV-infected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs described to date and contains only three potential N-glycosylation sites, which are all located in the globular head domain. Elimination of each of the three N-glycosylation sites revealed that the two oligosaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose-rich form. The glycosylation mutants showed also that oligosaccharides at positions 124 and 213 of FPV NA modulate enzymatic activity. Transport of NA is not influenced by single elimination of any of the three oligosaccharide attachment sites. Mutational analysis of the three Cys residues not involved in intrachain disulfide pairing revealed that Cys-49 in the stalk of the NA molecule is responsible for the formation of disulfide-linked dimers. Analysis of cysteine mutants of FPV NA also demonstrated that disulfide-linked dimers are not absolutely necessary for the formation of enzymatically active tetramers but may stabilize the quaternary structure of NA.

Animals↗
PubMed1997

Inactivation of inhibitors by the receptor-destroying enzyme of influenza C virus.

The importance of the receptor-destroying enzyme of influenza C virus for inactivation of inhibitors was analysed. Using three different inhibitors (rat serum, bovine submandibulary mucin and bovine brain gangliosides) inhibition of virus infection was observed only at an inhibitor concentration that was about 100-fold higher than the maximum concentration of inhibitor that could be inactivated by the receptor-destroying enzyme of a given amount of virus. From our data and other observations we conclude that the receptor-destroying enzyme is not required to inactivate inhibitors.

Acetylesterase↗
PubMed1997

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.

Animals↗
PubMed1997

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.

Animals↗
PubMed1997

Oligosaccharides in the stem region maintain the influenza virus hemagglutinin in the metastable form required for fusion activity.

The influenza A virus hemagglutinin (HA) has three conserved oligosaccharides located in the stem region at asparagine residues 12, 28, and 478. The biological role of these oligosaccharides has been investigated by mutational analysis of HA of fowl plague virus that was expressed from a simian virus 40 vector in the presence of ammonium chloride for protection from acid denaturation in the trans-Golgi network. Resistance to endoglycosidase H and cleavage of HA into the subunits HA1 and HA2 have been analyzed as markers for intracellular transport. Cell surface exposure has been determined by hemadsorption following neuraminidase treatment, by immunofluorescence staining, and by fluorescence-activated cell sorter analysis. When all three stem oligosaccharides were removed, transport was almost completely blocked. When two of the three stem oligosaccharides, particularly those at asparagine residues 12 and 28, were missing, HA was transported to the surface but showed extremely low fusion activity. With mutants lacking one stem oligosaccharide, fusion was reduced to a lesser extent. Removal of stem oligosaccharides resulted also in an increase in the pH optimum required for fusion. On the other hand, no reduction in fusion activity was observed when oligosaccharides in the head region of the HA spike were removed. These results indicate that the conserved oligosaccharides in the stem stabilize HA in the form susceptible to the conformational change necessary for fusion.

Glycosylation↗
PubMed1996

Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

Animals↗
PubMed1996

Termini of all mRNA species of Marburg virus: sequence and secondary structure.

The 3' and 5' ends of Marburg virus (MBG)-specific mRNA species have been determined using reverse transcription-PCR, rapid amplification of cDNA ends, or the reverse ligation-mediated PCR procedure after removal of cap structures with tobacco acid pyrophosphatase. The polyadenylation sites of all MBG-specific mRNAs were strictly conserved and corresponded to the predicted transcriptional stop signals of genomic RNA. Determination of the 5' ends of the mRNA species showed that mRNA synthesis started precisely at the first nucleotide of a highly conserved transcriptional start site. The 5' ends of the mRNA species can build a stable secondary structure with the conserved nucleotides always located in the stem region of a hairpin. Nucleotide substitutions in the conserved 5' regions are accompanied by compensatory mutations of the complementary nucleotide thus leading to a conservation of the secondary structures. Compensatory mutations were also found when 5' ends of mRNA of MBG strain Musoke were compared with MBG strain Popp or the closely related Ebola virus, indicating that the secondary structures will be conserved even if the sequence is altered.

Base Sequence↗
PubMed1996

Emerging and reemerging of filoviruses.

Filoviruses are causative agents of a hemorrhagic fever in man with mortalities ranging from 22 to 88%. They are enveloped, nonsegmented negative-stranded RNA viruses and are separated into two types, Marburg and Ebola, which can be serologically, biochemically and genetically distinguished. In general, there is little genetic variability among viruses belonging to the Marburg type. The Ebola type, however, is subdivided into at least three distinct subtypes. Marburg virus was first isolated during an outbreak in Europe in 1967. Ebola virus emerged in 1976 as the causative agent of two simultaneous outbreaks in southern Sudan and northern Zaire. The reemergence of Ebola, subtype Zaire, in Kikwit 1995 caused a worldwide sensation, since it struck after a sensibilization on the danger of Ebola virus disease. Person-to-person transmission by intimate contact is the main route of infection, but transmission by droplets and small aerosols among infected individuals is discussed. The natural reservoir for filoviruses remains a mystery. Filoviruses are prime examples for emerging pathogens. Factors that may be involved in emergence are international commerce and travel, limited experience in diagnosis and case management, import of nonhuman primates, and the potential of filoviruses for rapid evolution.

Animals↗
PubMed1996

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1,2-N-acetylglucosaminyltransferase I.

Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Animals↗