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      The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

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          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.

          Author Summary

          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.

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          Most cited references 66

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          Structure validation by Calpha geometry: phi,psi and Cbeta deviation.

          Geometrical validation around the Calpha is described, with a new Cbeta measure and updated Ramachandran plot. Deviation of the observed Cbeta atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cbeta deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new phi,psi plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the gamma-turn conformation near +75 degrees,-60 degrees, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the gamma-turn H-bond. Favored and allowed phi,psi regions are also defined for Pro, pre-Pro, and Gly (important because Gly phi,psi angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of alpha-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage). Copyright 2003 Wiley-Liss, Inc.
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            In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, R(merge) values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, R(merge) values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                December 2012
                December 2012
                6 December 2012
                : 8
                : 12
                [1 ]Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
                [2 ]Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [3 ]Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [4 ]Glycosciences Laboratory, Department of Medicine, Imperial College London, London, United Kingdom
                [5 ]Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                University of Michigan, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KR JES YL TSD TS. Performed the experiments: KR JES YL BSB. Analyzed the data: KR JES YL BSB DMR TF TSD TS. Wrote the paper: KR JES YL BSB TF TSD TS.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 17
                This work was supported by US Public Health Service award R01 AI76983, the Elizabeth B. Lamb Center for Pediatric Research, the UK Research Councils' Basic Technology Initiative ‘Glycoarrays’ (GRS/79268), EPSRC Translational Grant (EP/G037604/1), the Wellcome Trust (093378MA), and the National Cancer Institute Alliance of Glycobiologists (U01CA128416). Additional support was provided by US Public Health Service awards P30 CA68485 for the Vanderbilt-Ingram Cancer Center and P60 DK20593 for the Vanderbilt Diabetes Research and Training Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Protein Structure
                Biomacromolecule-Ligand Interactions
                Host-Pathogen Interaction

                Infectious disease & Microbiology


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