Structure and Function of a Ganglioside Receptor for Porcine Rotavirus

Abstract

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258–268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. AnN-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (∼2 × 107virions) and 2 × 106 cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 μM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3′- and 6′-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9.84 μM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…The polyomavirus major capsid protein VP1 binds to oligosaccharides terminating in a-2,3-linked sialic acid present on gangliosides (Stehle et al, 1994). Sialogangliosides may also function as initial attachment receptors for rotavirus (Dormitzer et al, 2002;Rolsma et al, 1998). Orthomyxoviruses and paramyxoviruses hemagglutinin (HA) glycoproteins bind sialic acid groups present on glycolipids or glycoproteins (Crennell et al, 2000;Skehel and Wiley, 2000).…”

Differential expression of sialic acid on porcine organs during the maturation process

“…Host range, tissue tropism and target cell specificity demonstrated by a particular virus are determined, at least in part, by a stereochemical fit between viral lectins and complementary carbohydrate receptors on host cell surfaces (Lis and Sharon, 1993). In pigs, the pathogenic effect of viruses, such as influenza virus (Kilbourne et al, 1988), coronavirus (Schultze et al, 1996), rotavirus (Rolsma et al, 1998) and rubulavirus (Reyes-Leyva et al, 1993) depends on a positive organotropism, which is determined by specific interactions between sialic acid-expressing cells and viral adhesion proteins. In general, the infectivity patterns of porcine viruses change with the host maturation stage.…”

Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine

“…known about the mechanisms underlying changes in the expression of this sialic acid during development. Developmental alterations in the appearance of total Neu5Gc have been observed in bovine and rat tissues [12,13] as well as in gangliosides isolated from the small intestine of pig [14,15] and rat [16]. Although evidence for ontogenic changes in hydroxylase activity was reported [13,16], these assays were performed under nonquantitative conditions owing to the omission of exogenous cytochrome b & and cytochrome b & reductase [17], prompting additional investigations.…”

“…For example, enterotoxigenic Escherichia coli with K99 fimbriae infect newborn piglets by binding to Neu5Gc in gangliosides such as Neu5Gcα2 3Galβ1 4Glcβ1 1hceramide [GM3(Neu5Gc]), N-glycolylsialoparagloboside and GM2(Neu5Gc) [20][21][22] attached to intestinal absorptive and mucus-secreting cells [23,24], causing a potentially lethal diarrhoea. Additionally, pig rotavirus, which similarly causes diarrhoea in young pigs, binds to GM3(Neu5Gc) [15]. Another pathogen, pig-transmissible gastroenteritis coronavirus, also recognizes glycoconjugates containing α2,3-bound Neu5Gc [25].…”

“…Another pathogen, pig-transmissible gastroenteritis coronavirus, also recognizes glycoconjugates containing α2,3-bound Neu5Gc [25]. Enhanced levels of Neu5Gc-containing glycoconjugates were found in newborn pigs, which are particularly susceptible to these infections [15]. Studies on the regulation of Neu5Gc expression are, therefore, important for our understanding of the aetiology of these economically relevant pig diseases.…”