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Cell surface ligands for rotavirus: mouse intestinal glycolipids and synthetic carbohydrate analogs
C A Srnka1, M Tiemeyer, J H Gilbert
1Glycomed, Incorporated, Alameda, California 94501.
Abstract:
Rotaviral binding to receptors on epithelial cells in the small intestine is thought to be a key event in the infection process and may be carbohydrate-mediated. Strain SA11 of rotavirus bound in vitro both to glycolipids isolated from mouse small intestine and to authentic glycolipids using thin layer chromatography overlay and microtiter well adsorption assays. Neutral mouse intestinal glycolipids which bound rotavirus were GA1 (Gal beta 1----3GalNAc beta 1---4Glc beta 1----4Glc beta 1----1-ceramide) and pentaosylceramides with terminal N-acetylgalactosamine, while acidic lipids which bound rotavirus included cholesterol 3-sulfate and two compounds termed bands 80 and 81. Digestion with ceramide glycanase suggested that bands 80 and 81 have lactosyl ceramide cores and an unidentified acidic moiety(s). No sialic-acid-containing glycolipids tested were active in viral binding. Band 81, which may have a ganglio core, bound rotavirus with greatest avidity, followed by GA1. Of authentic glycolipids assayed, only GA1 and GA2 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1-ceramide) displayed rotaviral binding. A phosphatidylethanolamide dipalmitoyl-containing neoglycolipid analog of GA2 bound rotavirus with avidity similar to native GA2. Substitution of beta 1----4-linked GlcNAc or beta 1----3-linked GalNAc for terminal GalNAc of GA2 neoglycolipid supported rotaviral binding, while other substitutions abrogated it. These findings suggest that a carbohydrate epitope similar to that of GA2 is sufficient for in vitro rotaviral binding, although binding may be enhanced by galactose and/or an acidic moiety in a secondary epitope.
Insights
Rotavirus binding to intestinal cells involves specific carbohydrates. Glycolipids like GA1 and GA2, and cholesterol 3-sulfate, facilitate rotavirus attachment in vitro, suggesting a carbohydrate epitope similar to GA2 is key for infection.
Area of Science:
- Virology
- Glycobiology
- Cell Biology
Background:
- Rotaviral infection initiates with binding to small intestine epithelial cells.
- This crucial interaction is hypothesized to be mediated by cell surface carbohydrates.
Purpose of the Study:
- To investigate the role of specific glycolipids in rotavirus binding to intestinal cells.
- To identify the carbohydrate structures responsible for rotavirus attachment in vitro.
Main Methods:
- Utilized thin layer chromatography overlay and microtiter well adsorption assays.
- Tested binding of rotavirus (Strain SA11) to isolated mouse intestinal glycolipids and authentic glycolipids.
- Employed ceramide glycanase digestion to analyze unknown glycolipids.
Main Results:
- Neutral glycolipids GA1 and pentaosylceramides with terminal N-acetylgalactosamine bound rotavirus.
- Acidic lipids cholesterol 3-sulfate, and compounds bands 80 and 81 also showed binding.
- Band 81 exhibited the highest avidity, followed by GA1; GA2 also bound rotavirus.
- Neoglycolipid analogs of GA2 demonstrated similar binding avidity, with specific substitutions enhancing or abrogating attachment.
Conclusions:
- A carbohydrate epitope similar to GA2 is sufficient for in vitro rotavirus binding.
- Galactose and/or an acidic moiety may enhance rotaviral binding through secondary epitopes.
- These findings elucidate potential carbohydrate-mediated mechanisms of rotavirus entry.