Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.
Receptor utilization plays an important role in viral disease. Viruses must recognize a receptor or sometimes multiple receptors to infect a cell. Mammalian orthoreoviruses (reoviruses) serve as useful models for studies of viral receptor binding and pathogenesis. The reovirus experimental system allows manipulation of both the virus and the host to define mechanisms of viral attachment and disease. Like many viruses, reoviruses engage carbohydrate molecules on the cell-surface, but the oligosaccharide sequences bound and the function of glycan binding in infection were not known prior to this study. We used glycan array screening to determine that serotype 1 reoviruses bind ganglioside GM2 and found that this interaction is required for efficient infection of some types of cells. To better understand how reovirus engages GM2, we determined the structure of the reovirus attachment protein σ1 in complex with the GM2 glycan and defined residues that are required for functional receptor binding. Reoviruses are being tested in clinical trials for efficacy in the treatment of cancer. Cancer cells commonly have altered glycan profiles. Therefore, understanding how reoviruses engage cell-surface glycans might lead to improvements in oncolytic therapy.