Biomedical subjects

C M Deom

Publications and source records attributed to C M Deom.

21 records · Page 2Linked to original sources

PubMed1986

Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin.

During serial passage in Madin-Darby bovine kidney (MDBK) cells, a substrain of influenza virus A/WSN is lost from the population and is replaced by a mutant virus with altered host cell binding properties. This selection does not occur during growth in chicken embryo fibroblasts (CEF). It occurs during growth in MDBK cells because the parental virus produced by these cells has a dramatically reduced affinity for cellular receptors [Crecelius, D.M., Deom, C. M. & Schulze, I. T. (1984) Virology 139, 164-177]. We have now compared the hemagglutinin (HA) subunits, HA1 and HA2, of the parent and mutant viruses by NaDodSO4/PAGE and have found that when the viruses are grown in either host cell the HA1 subunit of the mutant is smaller than that of the parent virus. The nonglycosylated HAs, made in the presence of tunicamycin, have the same apparent molecular weight, indicating that the HA1 subunit of the mutant virus contains less carbohydrate than that of the parent. This reduction in carbohydrate content was observed with 11 independently derived mutants that had been selected by growth in MDBK cells. The nucleotide sequence of the HA gene of the parent and mutant viruses indicates that there are five potential glycosylation sites on the parent HA1 subunit and four on the mutant and that the mutation responsible for this difference is a single base change that eliminates the glycosylation site at amino acid 125 of the parent HA1 subunit. Treatment of the parent and mutant HAs from both cell sources with endo-beta-N-acetylglucosaminidases F and H showed that the HA1 of the parent virus has four complex and one high-mannose oligosaccharides, whereas that of the mutant virus has three complex and one high-mannose oligosaccharides. Thus, all of the potential sites on both HA1 subunits are glycosylated. We conclude that the oligosaccharide attached to amino acid 125 of the parent HA by MDBK cells can reduce the affinity of the virus for cellular receptors and that the mutant virus has a higher affinity than the parent because the mutant HA is not glycosylated at that site. Since amino acid 125 of the parent HA is glycosylated by both CEF and MDBK cells, we further conclude that the host-determined structure of the oligosaccharide at that site affects the affinity of the parent virus for cellular receptors and, thereby, determines whether the mutant virus will have a growth advantage.

Amino Acid Sequence↗

PubMed1985

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties.

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.

Animals↗

PubMed1984

Biological properties of a hemagglutinin mutant of influenza virus selected by host cells.

Chick embryo fibroblast (CEF)-grown stocks of the WSN strain of influenza A(HINI) contain two variants which were designated F and C for fuzzy and clear plaque morphology on Madin-Darby bovine kidney (MDBK) cells. During growth in MDBK cells plaque-isolated F virus was completely replaced by C virus (L. Noronha-Blob and I.T. Schulze (1976), Virology 69, 314-322). The parental (F) and the mutant (C) viruses contain hemagglutinins which differ in their ability to bind to host cells. In addition, the host cells from which the purified viruses are obtained affect their binding properties. Thus, as compared to MDBK-grown F virus (FBK), MDBK-grown C virus (CBK) produced high amounts of mRNA and high virus yields in MDBK cells. CBK had greater affinity for SA alpha 2,3Gal and SA alpha 2,6Gal linkages on derivatized human erythrocytes than did FBK, independent of whether neuraminidase was present on the virions. CBK was also resistant to components of calf serum which inhibited FBK hemagglutination at 37 degrees. As compared to FBK, CBK had increased ability to bind to both MDBK cells and CEF at 37 degrees in the presence or absence of an inhibitor of neuraminidase. In addition, when cells with virus bound at 0 degrees were transferred to 37 degrees, CBK remained cell associated whereas about 80% of FBK dissociated from both cells. Thus, mutation from F to C increased the ability of the virus to associate with MDBK cell receptors. Studies carried out with F and C viruses from both cells indicated that the expression of the mutation depended in part on the host cells in which the virus was grown and in part on the cells used to measure the binding properties. A model relating these observations to selection of HA variants in nature is presented.

Animals↗