Irene T. Schulze

Effects of Glycosylation on the Properties and Functions of Influenza Virus Hemagglutinin

The influenza virus A hemagglutinin (HA) is a trimeric glycoprotein that contains 3-9 N-linked glycosylation sequons per subunit, depending on the strain. The location of these sites is determined by the nucleotide sequence of the HA gene, and, since the viral genome is replicated by an error-prone RNA polymerase, mutations, which add or remove glycosylation sites, occur at a high frequency. Mutations that are not lethal to the virus add to the structural diversity of the virus population. Factors that determine the glycosylation of the HA are reviewed herein, as are the effects of host-specific glycosylation on receptor binding, fusion activity, and antigenic properties of the virus. Effects of host-specific glycosylation and selection on virulence and on vaccine efficacy and surveillance are discussed. In addition, inadequacies in our understanding of HA glycosylation and its effects on host range are emphasized.

The Glycosylation of the Influenza A Virus Hemagglutinin by Mammalian Cells A SITE-SPECIFIC STUDY

We have characterized the glycans at individual sites on the hemagglutinin of three influenza A variants to obtain information on the role of cell-specific glycosylation in determining the receptor binding properties of this virus. The variants differ in whether they have a glycosylation site at residue 129 on the tip of the hemagglutinin and whether amino acid 184 (near to the receptor binding site) is His or Asn. We found that all sites on each variant are glycosylated in Madin-Darby bovine kidney cells, that the glycosylation is site-specific, and that the glycans at the same site in each variant are highly similar. One site that is buried in the hemagglutinin trimer contains only oligomannose glycans. The remaining sites carry complex glycans of increasing size as the distance of the site from the viral membrane decreases. Most of these complex glycans are terminated with α-galactose residues, a consequence in bovine cells of the removal of terminal sialic acids by the viral neuraminidase. Although the glycans at residue 129 are among the smallest on the molecule, they are large enough to reach the receptor binding pocket on their own and adjacent monomers. The results suggest that the reduction in receptor binding observed with Madin-Darby bovine kidney cell-grown virus is due to the combined effect of large complex glycans at the tip of the hemagglutinin and a His to Asn substitution close to the receptor binding pocket.

Structure of the Influenza Virion

Publisher Summary This chapter discusses the influenza virus, which is presently available from biochemical and biophysical studies and from the electron microscopy. It attempts to design a model of the particle based on this information. Although much is known about these viruses, the available information is incomplete in many respects. For example, detailed information about a specific portion of the virion or about a specific biochemical process may be available, but from one virus strain only. Attempts to formulate a detailed model are also frustrated by the limitations of the experimental procedures themselves. Procedures employed to prepare a sample for viewing in the electron microscope may alter the structure of the virus. Disrupting the virion and separating its components for biochemical and biophysical analyses provides further opportunities for artifacts. However, these procedures also provide complementary information about the structure of the virion. Facts about the structure can be derived from the evidence at hand only, when a number of seemingly independent observations converge on and mutually support a conclusion. The structure proposed in the chapter is, therefore, intended to be a working model, based on the present state of knowledge. It is put forth at this time as much to provoke questions as to answer them.

Plaque assay of dengue and other group B arthropod-borne viruses under methyl cellulose overlay media.

Abstract KB cell monolayers, infected with dengue-2 virus and maintained under liquid medium, give rise to persistently virus-yielding cultures regardless of the input dose of virus or of the intensity of initial cytopathic changes. Persistently infected cultures continuously yield 0.3–0.4 LD50 per cell for periods of at least 3 years. Conditions are defined under which maximal cytopathic effects (CPE) of dengue-2 virus are obtained in KB cell cultures. Based on the finding that agar prevents CPE and demonstrable viral multiplication, a plaque assay procedure for dengue-2 virus has been developed which involves the use of methylcellulose overlay medium. As used, this method gives highly reproducible results with an average efficiency equivalent to that of the LD50 titration in mice. Preliminary tests with dengue-1, yellow fever, Japanese B, Saint Louis, and Murray Valley encephalitis viruses suggest the general usefulness of the methylcellulose overlay technique for group B arborviruses.

Reversible inhibition of type 2 dengue virus by agar polysaccharide

Abstract Infectivity and hemagglutinating activity of dengue-2 virus are inhibited by a sulfated polysaccharide from agar gel. Inhibition is due to a reversible reaction between virus particles and the inhibitor. Virus-inhibitor complex formation can be demonstrated by sedimenting the inhibitor with the virus particles. Dilution of virus-inhibitor mixtures results in recovery of infectious particles so that pretreatment of virus with inhibitor does not increase inhibition over that which can be accounted for by the amount of inhibitor in the medium at the time the virus is plated. Under conditions of inhibitor excess, the fraction of virus inhibited by a given concentration of inhibitor is constant, i.e., independent of the total virus present. The ratio of virus to inhibitor in the complex has been determined from hemagglutination inhibition tests to be approximately 2 to 1. Presence of inhibitor in culture medium throughout a virus growth cycle does not alter the actual virus yield, but does bind free virus after its liberation from the cell. No evidence for interaction of inhibitor with cells (either KB cells or goose erythrocytes) has been obtained. Comparative studies using other group B arboviruses indicate that these viruses differ in sensitivity to the inhibitor.

Inhibition of Infectious and Hemagglutinating Properties of Type 2 Dengue Virus by Aqueous Agar Extracts.

Abstract Aqueous agar extract (AE), added to liquid or methylcellulose overlay medium, inhibits cytopathic effects or plaque formation by dengue-2 virus. Free virus mixed with AE undergoes reduction in (1) intracerebral pathogenicity for mice, (2) capacity to form plaques in KB cell monolayers, (3) hemagglutinating capacity. Various lines of evidence are presented which suggest that the inhibitory effect of agar overlay media and of AE are due to direct combination of the inhibitory substance with free virus particles. The inhibitor has been partially purified by a method involving coprecipitation with bovine serum albumin and deproteinization of the dissociated precipitate with phenol. It has been characterized as a sulfated polysaccharide.

Effects of sialylation of influenza virions on their interactions with host cells and erythrocytes

Abstract Influenza virions can be sialylated by incubating purified virus with sialyltransferase and CMP-sialic acid. Sialic acid residues are added only to the virion hemagglutinin. Both portions of the hemagglutinin subunit have terminal galactose residues which accept sialic acid, with the HA 1 region being preferentially sialylated early in the reaction. The rate of sialylation depends on the source of the virus; virions derived from chick embryo fibroblasts are sialylated at approximately 20 times the rate of virus from an established line of bovine kidney cells. Sialylation destroys the hemagglutinating activity of the particles but leaves the neuraminidase activity unchanged. The ability of these preparations to form plaques on MDBK cells is either enhanced or unchanged depending on the amount of sialic acid added to the virions. Maximum enhancement is observed when approximately 1250 sialic acid residues are added per virion. Addition of more residues decreased the amount of enhancement. Heavily sialylated virus shows the same infectivity as unsialylated virus although it has an isoelectric point below pH 4.0; that of unsialylated virus is 6.0 to 6.5. In an attempt to determine the basis for the loss of hemagglutinating activity and the enhancement of infectivity, we have measured the ability of sialylated and control virions to bind to erythrocytes and to host cells under conditions of either virus or cell excess. Identical results are obtained with sialylated and control virus at all virus to cell ratios tested, indicating that the changes induced by sialylation do not reflect changes in the ability of virus to bind to cellular receptors. The results suggest that the formation of infectious units by aggregation of randomly defective particles along with changes in step(s) following binding are responsible for the observed alterations in biological activities.

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.

Viral interference-mediated selection of a plaque-type variant of influenza virus

Abstract Two variants of the A 0 /WSN strain of influenza virus that can be distinguished by plaque morphology on MDBK cells have been isolated. The two variants, designated F and C, produce identical plaques on chick embryo fibroblasts (CEF). They also have identical growth rates in CEF and are indistinguishable in density, size, polypeptide composition, RNA-dependent RNA polymerase activity, hemagglutinating activity, neuraminidase activity and infectivity. Although the two variants are also highly similar when grown in MDBK cells, cocultivation in these cells results in a rapid disappearance of the F variant due to a unidirectional interference. C virions that arise by mutation during the replication of F in MDBK cells interfere with the synthesis of F virions and thereby eliminate them from the progeny. Since this interference does not operate in CEF, double infection results in concomitant replication of the two variants. Thus, in one host but not in the other the newly arising variant establishes conditions by which its replication is favored over that of its predecessor.

Characterization of host cell binding variants of influenza virus by monoclonal antibodies

We have previously shown that a plaque-type mutant of influenza virus A/WSN has a growth advantage in MDBK cells because its hemagglutinin (HA) has a greater affinity for host cell receptors than does the HA of the parent virus. We show here that the mutant is also less sensitive than the parent to neutralization by antibodies to epitopes in at least two regions on the HA. WSN-specific monoclonal antibodies which had higher radioimmunoassay (RIA) titers against the parent than the mutant virus also had higher plaque inhibition (PI) and hemagglutination inhibition (HI) titers. In contrast, cross-reacting antibodies bound equally well to the parent and mutant viruses as judged by RIA but those which bound to the Cb region of the HA exhibited higher PI and HI titers against the parent virus. The results suggest that preferential neutralization of the parental virus by antibodies can contribute to the selective advantage of mutants which have increased affinity for cellular receptors.