Processing of influenza virus hemagglutinin in insect cells.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
H D Klenk
Publications and source records attributed to H D Klenk.
Isolation and characterization of the acetylesterase of hemagglutinating encephalomyelitis virus (HEV).
Explore the source record for details and available documents.
Acetylesterase↗
Differential reactivity of bovine coronavirus (BCV) and influenza C virus with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2)-containing receptors.
Explore the source record for details and available documents.
Coronaviridae↗
Nucleotide sequence analyses of the genes encoding the HN, M, NP, P, and L proteins of two host range mutants of Sendai virus.
Comparative nucleotide sequence analyses of the genome of Sendai virus (strain Z) and two host range mutants, ts-f1 and F1-R, previously described revealed that the ts defect of ts-f1 can be attributed to two nucleotide exchanges in the NP gene. These exchanges lead to a single amino acid substitution. A single base pair change was found in both the P and L genes of F1-R, but not of ts-f1. Both host range mutants have the two same exchanges in the M gene. These additional mutations are discussed concerning their significance in the pantropic properties of the host range mutants.
Animals↗
The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.
The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.
Animals↗
Inhibition of endoproteolytic cleavage of cytomegalovirus (HCMV) glycoprotein B by palmitoyl-peptidyl-chloromethyl ketone.
Endoproteolytic cleavage of glycoprotein B (gB) of human cytomegalovirus (HCMV) is inhibited by palmitoylated peptidyl-chloromethyl ketone (palFAKR-CEK) at concentrations above 30 microM. Inhibitor treatment of HCMV-infected human fibroblasts neither interfered with exposure of gB on the plasma membrane, detected by surface membrane immunostaining, nor reduced production of intracellular infectious viral progeny. Release of infectious virus, on the other hand, was impaired.
Amino Acid Chloromethyl Ketones↗
The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids.
We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.
Acylation↗
Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus.
The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.
Acetylesterase↗
A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein.
The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.
Amino Acid Sequence↗
The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.
This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.
Animals↗
Monoclonal antibodies to three structural proteins of Newcastle disease virus: biological characterization with particular reference to the conformational change of envelope glycoproteins associated with proteolytic cleavage.
Monoclonal antibodies (MAbs) to the haemagglutinin-neuraminidase (HN), fusion (F) and matrix (M) proteins of Newcastle disease virus were prepared and characterized. At least three non-overlapping or partially overlapping antigenic sites were delineated on the HN, three on the F and three on the M proteins by competitive binding assays. Antigenic sites on the HN and F proteins roughly represented functional domains defined by serological tests. Two antigenic sites on the F protein were involved in virus neutralizing and haemolysis-inhibiting activity. These antigenic determinants were readily affected by treatment with certain surfactants and acetone. Proteolytic cleavage of the HN and F proteins was associated with conformational change, revealed by altered reactivity with MAbs and by altered topological arrangements of some epitopes. None of the anti-M MAbs inhibited any biological activities of the virus.
Animals↗
Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells.
A protease activation mutant of Sendai virus, F1-R, causes a systemic infection in mice, whereas wild-type virus is exclusively pneumotropic (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). Budding of F1-R has been observed bidirectionally at the apical and basolateral surfaces of the bronchial epithelium of mice and of MDCK cells, whereas wild-type virus buds apically (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J. T. Seto, J. Virol. 64:3627-3634, 1990). In this study, wild-type virus was shown to be produced primarily from the apical site of polarized MDCK cells grown on permeable membrane filters. Surface immunofluorescence and immunoprecipitation analyses revealed that transmembrane glycoproteins HN and F were expressed predominantly at the apical domain of the plasma membrane. On the other hand, infectious progeny of F1-R was released from the apical and basolateral surfaces, and HN and F were expressed at both regions of the cells. Since F1-R has amino acid substitutions in F and M proteins but none in HN, the altered budding of the virus and transport of the envelope glycoproteins might be attributed to interactions by F and M proteins. These findings suggest that in addition to proteolytic activation of the F glycoprotein, the differential site of budding, at the primary target of infection, is a determinant for organ tropism of Sendai virus in mice.
Animals↗
Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium.
Wild-type Sendai virus is exclusively pneumotropic in mice, while a host range mutant, F1-R, is pantropic. The latter was attributed to structural changes in the fusion (F) glycoprotein, which was cleaved by ubiquitous proteases present in many organs (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). These studies were extended by investigating, by use of an organ block culture system of mice, whether differences exist in the susceptibility of the lung and the other organs to the viruses and in proteolytic activation of the F protein of the viruses. Block cultures of mouse organs were shown to synthesize the viral polypeptides and to support productive infections by the viruses. These findings ruled out the possibility that pneumotropism of wild-type virus results because only the respiratory organs are susceptible to the virus. Progeny virus of F1-R was produced in the activated form as shown by infectivity assays and proteolytic cleavage of the F protein in the infected organ cultures. On the other hand, much of wild-type virus produced in cultures of organs other than lung remained nonactivated. The findings indicate that the F protein of wild-type virus was poorly activated by ubiquitous proteases which efficiently activated the F protein of F1-R. Thus, the activating protease for wild-type F protein is present only in the respiratory organs. These results, taken together with a comparison of the predicted amino acid substitutions between the viruses, strongly suggest that the different efficiencies among mouse organs in the proteolytic activation of F protein must be the primary determinant for organ tropism of Sendai virus. Additionally, immunoelectron microscopic examination of the mouse bronchus indicated that the budding site of wild-type virus was restricted to the apical domain of the epithelium, whereas budding by F1-R occurred at the apical and basal domains. Bipolar budding was also observed in MDCK monolayers infected with F1-R. The differential budding site at the primary target of infection may be an additional determinant for organ tropism of Sendai virus in mice.
Animals↗
Use of a sialic acid analogue to analyze the importance of the receptor-destroying enzyme for the interaction of influenza C virus with cells.
Influenza C virus uses 9-O-acetyl-N-acetylaneuraminic acid (9-O-acetyl-Neu5Ac) as a receptor determinant for attachment to cells. The virus contains an acetylesterase which releases acetyl residues from position C-9 of sialic acid thereby inactivating the receptors. A synthetic sialic acid analogue, 9-N-acetyl-Neu5Ac, was attached to cell surface glycoconjugates by purified sialyltransferase and analyzed for its ability to substitute the 9-O-acetylated sialic acid. Erythrocytes which have been modified to contain either 9-O-acetyl-Neu5Ac or 9-N-acetyl-Neu5Ac were agglutinated by influenza C virus to the same titer. However, in contrast to the 9-O-acetyl group the 9-N-acetyl residue is resistant to cleavage by the viral acetylesterase. This characteristic property (recognition as a receptor determinant by influenza C virus, but resistance against the action of the receptor-destroying enzyme) makes this synthetic analogue a valuable tool to analyze the role of the receptor-destroying enzyme for an influenza C virus infection.
Acetylesterase↗
Inhibition of proteolytic activation of influenza virus hemagglutinin by specific peptidyl chloroalkyl ketones.
Lysates of cultured cells have been analyzed for arginine-specific endoproteases using peptidyl-p-nitroanilides as chromogenic substrates. The enzymes present in MDBK, MDCK, VERO, BHK, and chick embryo cells required lysine-arginine or arginine-arginine pairs as cleavage sites, whereas chorioallantoic membrane cells contained, in addition, an activity that could cleave at a single arginine. The effect of peptidyl chloroalkyl ketones on the activation of the fowl plague virus hemagglutinin by the proteases specific for paired basic residues has been investigated. When virions containing uncleaved hemagglutinin were incubated with lysates of uninfected cells, cleavage was completely inhibited by peptidyl chloroalkyl ketones containing paired basic residues at a concentration of 1 mM. In contrast a compound containing a single arginine had no inhibitory activity. When dibasic peptidyl chloroalkyl ketones were added to infected cell cultures, cleavage of hemagglutinin and multiple cycles of virus replication were inhibited at 10 mM. However, a 100- to 200-fold increase of the inhibitory activity in intact cells could be achieved by N-terminal acylation. These studies suggest a potential role of peptidyl chloroalkyl ketones as antiviral agents.
Amino Acid Chloromethyl Ketones↗
Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2).
Six variants that form plaques in chick embryo cells in the absence of trypsin have been isolated from the apathogenic avian influenza virus A/chick/Pennsylvania/1/83 (H5N2). Unlike the wild-type, the plaque variants contain a hemagglutinin that is cleaved in chick embryo cells and MDCK cells. The variants differ also from the wild-type in their pathogenicity for chickens. Nucleotide sequence and oligosaccharide analysis of the hemagglutinin have revealed that, unlike natural isolates with increased pathogenicity (Y. Kawaoka et al., 1984, Virology 139, 303-316; Y. Kawaoka and R. G. Webster, 1985, Virology 146, 130-137), the variants obtained in vitro have retained an oligosaccharide at asparagine 11 that is believed to interfere with the cleavage site of the wild-type. However, all variants showed mutations in the hemagglutinin resulting in an increased number of basic groups at the cleavage site. These observations demonstrate that masking of the cleavage site by an oligosaccharide is overcome by an enhancement of the basic charge at the cleavage site.
Amino Acid Sequence↗
Synthesis of biologically active influenza virus hemagglutinin in insect larvae.
The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.
Administration, Oral↗
Serine 71 of the glycoprotein HEF is located at the active site of the acetylesterase of influenza C virus.
The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293-1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with 3H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the 3H-DFP was subjected to sequence analysis. The amino acids determined from the NH2-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that 3H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.
Acetylesterase↗