Heiner Niemann

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.

On Enteropathogenic Bovine Coronavirus

Cornonaviruses cause severe and economically important intestinal infections in newborn calves which become manifest as enteritis with diarrhea and additional clinical consequences. Studies concerning the pathogenesis of enteropathogenic bovine coronavirus (EBC-virus) enteritis have led to a better understanding of morphological alterations of the intestinal tract as well as of the individual cells, helping to explain the functional disorders observed with this disease (for references see Storz and Rott, 1980). Relatively little is known, however, about structural details of the virus and about the biochemical events of EBC-virus replication, principally a result of the difficulties encountered in growing the virus in tissue culture. After we succeeded in adapting EBC-virus strain L-9 isolated by Mebus et al. (1973) to grow in bovine fetal thyroid or brain cells in reasonable amounts it became possible to study some of the many unanswered questions. As a beginning, efforts were made to determine optimal growth conditions in order to provide an in vitro source for viral antigen. In addition, biochemical analysis of the virus was undertaken.

Primary structure of Klebsiella serotype K22 capsular polysaccharide: Another glycan containing 4-O-[(S-1-carboxyethyl]-d-glucuronic acid

The primary structure of the acidic capsular polysaccharide isolated from Klebsiella serotype K22 has been investigated using methylation analysis, hydrolysis, bacteriophage-borne enzyme degradation, and n.m.r. spectroscopy. The repeating unit comprises the chain disaccharide----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1---- substituted by 4-O-[(S)-1-carboxyethyl]-beta-D-GlcpA-(1----6)-alpha-D-Glcp-(1---- at O-4 of the galactose. The galactose carries an O-acetyl group on position 6.

PROCESSING OF THE HEMAGGLUTININ

SUMMARY Processing of the hemagglutinin involves transport from the rough endoplasmic reticulum to the plasma membrane, glycosylation, and proteolytic cleavage. The tight coupling of these events is demonstrated in experiments in which mutants of fowl plague virus (FPV) with a temperature sensitive defect in transport have been analyzed. Two groups of such mutants have been characterized. With the first group (ts1, ts227), the hemagglutinin is arrested in the rough endoplasmic reticulum. With the second group (ts482, 532, 651), the hemagglutinin migrates to the Golgi apparatus, but does not reach the plasma membrane. The distribution of the carbohydrate side chains on the FPV hemagglutinin has been elucidated. HA 1 has 4 type I side chains attached to asparagine residues 12, 28, 123, and 149. The potential attachment site at asparagine 231 is not glycosylated. HA2 has a type II chain at asparagine 406 and a type I chain at asparagine 478. Comparison with other hemagglutinins demonstrates that the side chains in positions 12, 28, and 478 are conserved. The cleavage site has been analyzed with H3, H7, and H10 hemagglutinins. The available evidence indicates that proteolytic activation involves the action of a trypsin-like protease followed by the action of an exopeptidase of the carboxypeptidase B type: After in vitro cleavage with trypsin, v^iich is paralleled by activation of infectivity, the N-terminus of HA 2 and presumably the C-terminus of HA. are identical to those obtained after in vivo cleavage. This indicates that the carboxypeptidase B activity is present in the virion. After cleavage with the non- activating enzymes thermolysin and chymo trypsin, the cleavage site is shifted by a few amino acids. These observations indicate that activation of infectivity requires a highly specific amino acid sequence at the cleavage site.

Detection of fms-oncogene-specific tyrosine kinase activity in human leukemia cells

SummaryThe c-fms protooncogene encodes the receptor for the colony-stimulating factor 1 of macrophages. Its transforming counterpart, the v-fms oncogene has previously been recognized as the transforming gene of the McDonough strain of feline sarcoma virus. We have isolated rabbit antisera against a 115-kDa recombinant polypeptide containing the 926 carboxy-terminal amino acids of the v-fms protein. All antibodies recognized the cytoplasmic domain of the v-fms protein, which is 95% homologous to the corresponding domain of human c-fms proteins. These sera were applied in a survey of various human cancer cell lines, such as peripheral blood mononuclear (HL60) and choriocarcinoma (BeWo) cells, as well as leukemic cells from 58 patients with acute myelocytic, chronic myelocytic or acute lymphocytic leukemias (AML, CML, ALL). Significantly enhanced levels of fms-specific tyrosine kinase activity were detected in 12-O-tetradecanoylphorbol-13-acetate-induced HL60 and in BeWo cells, and in 7 out of 24 samples from AML patients, whereas no activity could be detected in 9 ALL or in 25 CML cell preparations. The AML cells were classified according to the FAB criteria. The highest incidence of increased fms activity was found in cells assigned to the M4 class (four out of five cases). While no activity was found in material belonging to FAB classes M2 or M3, one of the two cases of the M5 class was kinase-positive. Interestingly, two out of seven cases of the M1 class cells exhibited enhanced levels of fms kinase. These data suggest that the determination of the fms kinase may be useful to subdivide the M1 class of the FAB classification into monocytic and non-monocytic precursor leukemia cells.

Inhibition of glycosylation processing alters the growth parameters of cells transformed by the oncogene of Simian Sarcoma virus

SummaryNormal rat kidney (NRK) cells transformed by the v-sis oncogene of Simian Sarcoma virus (SSV) were treated with the glucosidase I inhibitor castanospermine. The inhibitor did not change cell morphology, but specific growth parameters such as serum- and anchorage-independence were lost.

The Characterization of Influenza A Viruses by Carbohydrate Analysis

The hemagglutinin and the neuraminidase of influenza viruses are glycoproteins of great importance for the biology of the virus and the nature of the disease. They play an essential role in the initiation of infection. It is generally agreed that the hemagglutinin is responsible for adsorption of the virus to the cell surface. In addition, the hemagglutinin is involved in penetration by triggering fusion of the viral envelope with cellular membranes. There is evidence that the neuraminidase also takes part in the latter reaction (Huang et al. 1980a, b, 1981). Both glycoproteins are also the surface antigens of the virus and due to their structural variability, they appear to be directly responsible for the uncontrolled recurrence of influenza epidemics in man. It is therefore not surprising that the structure of the hemagglutinin, as the major surface antigen, has been analyzed in detail. The complete amino acid sequences of a whole series of influenza A virus hemagglutinins have been determined (for review see Ward 1981), and the tertiary structure of the molecule has also been elucidated (Wilson et al. 1981).

Glycoprotein E1 of Coronavirus A59: A New Type of Viral Glycoprotein

Glycosylation of viral glycoproteins as a co- and posttranslational event has been studied in a large number of viral systems (for review see Klenk and Rott, 1980). From these data a general picture can be drawn:

Transformation of chicken fibroblasts by the v-fms oncogene

The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membrane-associated tyrosine kinase (gp140v-fms) which is closely related, both structurally and functionally, to the c-fms-specified receptor for the macrophage colony stimulating factor (CSF-1). In mammalian fibroblasts, the natural producers of CSF-1, expression of v-fms leads to cell transformation. To study the interaction between CSF-1 and gp140v-fms molecules in a cell system that does not produce endogenous cross-reactive CSF-1, we have expressed the entire v-fms gene as well as a nontransforming deletion mutant (SC2) in chicken embryo cells (CEC). For this purpose the avian retroviral vectors pDS3 and pREP, based on Rous sarcoma virus, were used to isolate recombinant virus particles. CEC infected with virus that carried the entire v-fms gene expressed high amounts of gp140v-fms, comparable to those in SM-FeSV transformed NRK cells. However, these CEC remained flat, retained their fibronectin network, and did not produce enhanced levels of plasminogen activator. The cells grew faster than control CEC for more than 8 weeks but failed to form colonies in soft agar. Within 2 days after addition of CSF-1 to the growth medium, a transformed cell phenotype was induced, as judged by loss of the fibronectin network, again with a growth rate fourfold faster than that of the parental cells and with colony formation in soft agar. Moreover, human CSF-1 caused a rapid tyrosine phosphorylation of v-fms molecules detectable within 5 min after addition of the growth factor. In contrast, CSF-1 had none of the above effects on cells that expressed the SC2 v-fms deletion mutant.

Signals for Membrane-Associated Transport in Eukaryotic Cells

The signals that target proteins of eukaryotic cells from the site of synthesis to their final destination have attracted much attention since Blobel and Dobberstein (1975a,b) proposed the classic signal hypothesis. Today, however, the picture is more complex than originally anticipated, and the sorting machinery of eukaryotic cells is far from being understood at the molecular level.