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

Abstract

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.