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Biomedical subjects

H D Klenk

Publications and source records attributed to H D Klenk.

At least 181 records · Page 10Linked to original sources
PubMed1985

The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase.

The nature of the receptor-destroying enzyme (RDE) of influenza C virus has been elucidated by analyzing its effect on the haemagglutination inhibitors rat alpha 1-macroglobulin (RMG) and bovine submandibulary mucin (BSM), respectively. The inhibitory activity of both compounds is abolished by incubation with influenza C virus. After inactivation, RMG and BSM were found to contain reduced amounts of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) and increased amounts of N-acetylneuraminic acid (Neu5Ac). H.p.l.c. analysis revealed that purified Neu5,9Ac2 is converted to Neu5Ac by incubation with influenza C virus. These results demonstrate that RDE of influenza C virus is neuraminate-O-acetylesterase [N-acyl-9(4)-O-acetylneuraminate O-acetylhydrolase (EC 3.1.1.53)]. The data also indicate that haemagglutination-inhibition (HI) by RMG and BSM and most likely virus attachment to cell surfaces involves binding of influenza C virus to Neu5,9Ac2.

Acetylesterase↗
PubMed1985

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.

Glycopeptides↗
PubMed1985

Neuraminic acid is involved in the binding of influenza C virus to erythrocytes.

Neuraminidases of both viral and bacterial origin have been reported to be unable to destroy the cellular receptor for influenza C virus on chicken erythrocytes, in contrast to the receptors for influenza A and B virus. However, under appropriate conditions neuraminidases from both Vibrio cholerae and Clostridium perfringens were able (i) to make chicken red blood cells resistant against agglutination by influenza C virus and (ii) to reduce the hemagglutination-inhibiting activity of rat serum. Both effects were abolished in the presence of the neuraminidase inhibitor 2,3-dehydro-2-deoxyneuraminic acid (DDN). These results indicate that contrary to previous assumptions sialic acid may very well be an essential component of the receptor for influenza C virus.

Animals↗
PubMed1985

Rat alpha 1 macroglobulin inhibits hemagglutination by influenza C virus.

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.

Animals↗
PubMed1985

Haemagglutinin transport mutants.

Two mutants (ts1 and ts651) with a temperature sensitive defect in the intracellular transport of the haemagglutinin from the rough endoplasmic reticulum to the plasma membrane have been analysed. Nucleotide sequencing of the haemagglutinin revealed with each mutant two point mutations that are located in the stem region of the molecule.

Base Sequence↗
PubMed1985

Increased infectivity of extracellular adenovirus type 12.

Human adenovirus type 12 was propagated on human embryonic kidney cells, and the specific infectivities of intra- and extracellular virus particles were compared between 48 and 104 h after infection. Released virions exhibited a specific infectivity of up to 10 times higher than that of intracellular particles. The increased infectivity was apparently not due to enhanced rates of adsorption or penetration of extracellular virus. There may be a delay in the onset of viral DNA replication in intracellular virus-infected cells. Differences in the composition of intra- and extracellular virions were not recognized. Differences might also be sought in late expression or assembly of progeny virions or both. The data indicated that the virions released from the infected cells differed from those retained in the nucleus with respect to their specific infectivities. Active mechanisms of virus release have not yet been investigated.

Adenoviruses, Human↗
PubMed1984

Studies on the adaptation of influenza viruses to MDCK cells.

The amino acid sequences and biological properties of the haemagglutinin of three variants of the influenza virus X-31 (H3N2) selected for their capacity to grow in MDCK cells are reported. In two variants, amino acid substitutions at HA1 residues 8 and 144 correlated with the loss of a site for glycosylation and specific changes in antigenicity, respectively. In all three variants substitution of an arginine residue for histidine at HA1 position 17 was correlated with increased pH optima of haemolysis. The importance of this substitution for cleavage of the haemagglutinin precursor required to produce infectious virus is discussed in relation to the three-dimensional structure of X-31 haemagglutinin.

Adaptation, Physiological↗
PubMed1984

Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187.

At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.

Animals↗
PubMed1984

A mutant of fowl plague virus (influenza A) with an altered glycosylation pattern in its hemagglutinin.

A temperature-sensitive mutant (ts 1/1) with a defect in the hemagglutinin (HA) gene, which was obtained by undiluted passage of fowl plague virus (FPV) at 33 degrees, is described. At 33 degrees proteolytic cleavage of the abnormal HA yielded an altered HA2 (XHA2) which migrated ahead of the NS1 protein and lacked the complex oligosaccharide side chain. At the nonpermissive temperature of 40 degrees, the migration of the HA of ts 1/1 from the rough endoplasmic reticulum (RER) via the Golgi apparatus to the cell surface was rate limiting for virus maturation. The HA was only slowly cleaved and migrated during polyacrylamide gel electrophoresis ahead of the HA of wild type FPV. Some revertants of ts 1/1 exhibited the same protein pattern as the mutant, others resembled wild type FPV, while one revertant gave rise to a mixture of HA2 and XHA2 at 40 degrees. These results suggest that (1) the loss of the complex oligosaccharide side chain is not responsible for the ts phenotype, (2) the mutation is presumably not at the site where the oligosaccharide side chain is linked to the protein backbone, and (3) ts 1/1 presumably carries a mutation located in RNA segment 4, which by pseudoreversion (suppressor mutation) in the same gene leads to different ts+ phenotypes.

Animals↗
PubMed1984

Biological functions of monospecific antibodies to envelope glycoproteins of Newcastle disease virus.

Monospecific antisera to HN and F glycoproteins of Newcastle disease virus were prepared, and their effects on the biological activities of the virus were investigated. Anti-HN serum inhibited hemagglutinating and neuraminidase activity, as well as hemolysis. Anti-F serum had no effect on hemagglutination or neuraminidase but inhibited hemolysis and virus-induced cell fusion. Anti-HN serum was highly neutralizing, while neutralization by anti-F serum was very inefficient in conventional plaque reduction tests, although both sera were estimated to contain comparable amounts of antibody reacting with the virus as indicated by complement fixation and immuno-diffusion tests. The neutralizing activity of anti-F serum was greatly enhanced by the addition of anti-IgG serum or fresh guinea pig serum, whereas that of anti-HN serum was little enhanced. Anti-HN serum incorporated in the agar overlay suppressed the development of plaques to some degree, while anti-F serum had little effect. The combination of anti-HN and anti-F sera resulted in a marked decrease in the number and size of plaques, demonstrating the synergistic effect of the two species of antibody in the containment of the spread of viral infection.

Animals↗
PubMed1984

Carbohydrates of influenza virus. V. Oligosaccharides attached to individual glycosylation sites of the hemagglutinin of fowl plague virus.

The carbohydrate side chains of the hemagglutinin of fowl plague virus (A/FPV/Rostock/34 (H7N1] have been localized by a procedure involving fragmentation of the polypeptide with cyanogen bromide and various proteases. The positions of the fragments were determined by radioactive labeling of the sugars and of specific amino acids. Side chains of the complex type I are attached to asparagine residues 12, 28, 123, 149, and 478. A mannose-rich (type II) side chain is linked to asparagine 406. Asparagine 231 is not glycosylated. The side chains attached to asparagine residues 12, 123, 149, and 478 contain sulfate. Glycopeptides derived by Pronase digestion from the individual attachment sites have been analyzed by their affinity to concanavalin A and Lens culinaris agglutinin. The results indicate that each glycosylation site has a typical set of heterogeneous oligosaccharides. Comparison of the glycosylation patterns of the hemagglutinins of FPV and other influenza A viruses reveals that the glycosylation sites at asparagine residues 12, 28, and 478, which are located at the base of the spike, are highly conserved. Mannose-rich side chains appear to be located preferentially at interfaces between the three monomers of a spike or between the globular and fibrous domains of a monomer.

Acetylglucosaminidase↗
PubMed1984

The carboxyterminus of the hemagglutinin-neuraminidase of Newcastle disease virus is exposed at the surface of the viral envelope.

The amino-terminal and the carboxy-terminal amino acids of the hemagglutinin-neuraminidase glycoprotein of the Ulster strain of Newcastle disease virus have been analyzed before and after proteolytic activation of the precursor HNo (Mr approximately 82K). The amino termini of HNo and of the large cleavage fragment HN (approximately 74K) obtained by in vivo and in vitro proteolysis could not be sequenced by Edman degradation. This indicates that in both instances the amino termini are blocked. The carboxy termini of HNo and HN are different as demonstrated by end-point digestion with carboxypeptidase A. Furthermore, a small cleavage fragment (approximately 9K) of HNo that was removed from the virion after trypsin treatment could be purified by HPLC. In contrast to HN, this fragment displays a free amino terminus susceptible to Edman degradation. These data indicate that conversion of HNo involves removal of a 9K glycopeptide from the carboxy-terminal end. Thus, it has to be concluded that, unlike most other viral glycoproteins, the hemagglutinin-neuraminidase is inserted in the envelope with its carboxy terminus exposed at the surface of the virus particle.

Amino Acid Sequence↗
PubMed1984

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1.

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

Carbohydrate Sequence↗
PubMed1984

[Significance of hemagglutinins for the pathogenicity of avian influenza viruses].

In addition to acute viral diseases, persistent infections have attained considerable interest in recent years. Such persistent infections are characterized by extended time periods in which the infecting virus remains within the organism before the eventual appearance of manifest symptoms. These infections may be evoked by a variety of virus species resulting in a diversity of pathogenic reactions and clinical manifestations. The mechanisms of viral persistence, where known, also appear to be quite diverse. As far as space permits, some examples of persistent infections will be presented and the mechanisms of the pathogenesis of the resulting diseases will be discussed.

Amino Acid Sequence↗