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      Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

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          Abstract

          Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

          Author Summary

          Viruses are obligate intracellular pathogens, and all of them share one crucial step in their life cycle—the attachment to their host cell via cellular receptors, which are usually proteins or carbohydrates. This step is decisive for the selection of target cells and virus entry. In this study, we investigated murine polyomavirus (MuPyV), which attaches to host gangliosides with its major capsid protein, VP1. We have solved the crystal structures of VP1 in complex with previously known interaction partners as well as with the ganglioside GT1a, which we have identified as a novel functional receptor for MuPyV. Earlier studies have shown that different strains with singular amino acid exchanges in the receptor binding pocket of VP1 display altered pathogenicity and viral spread. Our investigations show that, while these exchanges do not abolish binding or significantly alter interaction modes to our investigated carbohydrates, they have subtle effects on glycan affinity. The combination of receptor specificity, abundance, and affinity reveals a much more intricate regulation of pathogenicity than previously believed. Our results exemplify how delicate changes to the receptor binding pocket of MuPyV VP1 are able to drastically alter virus behavior. This system provides a unique example to study how the first step in the life cycle of a virus can dictate its biological properties.

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          Most cited references41

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          Optimal description of a protein structure in terms of multiple groups undergoing TLS motion.

          A single protein crystal structure contains information about dynamic properties of the protein as well as providing a static view of one three-dimensional conformation. This additional information is to be found in the distribution of observed electron density about the mean position of each atom. It is general practice to account for this by refining a separate atomic displacement parameter (ADP) for each atomic center. However, these same displacements are often described well by simpler models based on TLS (translation/libration/screw) rigid-body motion of large groups of atoms, for example interdomain hinge motion. A procedure, TLSMD, has been developed that analyzes the distribution of ADPs in a previously refined protein crystal structure in order to generate optimal multi-group TLS descriptions of the constituent protein chains. TLSMD is applicable to crystal structures at any resolution. The models generated by TLSMD analysis can significantly improve the standard crystallographic residuals R and R(free) and can reveal intrinsic dynamic properties of the protein.
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            Receptor binding by an H7N9 influenza virus from humans.

            Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.
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              Structural basis for sialic acid-mediated self-recognition by complement factor H.

              The serum protein complement factor H (FH) ensures downregulation of the complement alternative pathway, a branch of innate immunity, upon interaction with specific glycans on host cell surfaces. Using ligand-based NMR, we screened a comprehensive set of sialylated glycans for binding to FH and solved the crystal structure of a ternary complex formed by the two C-terminal domains of FH, a sialylated trisaccharide and the complement C3b thioester-containing domain. Key residues in the sialic acid binding site are conserved from mice to men, and residues linked to atypical hemolytic uremic syndrome cluster within this binding site, suggesting a possible role for sialic acid as a host marker also in other mammals and a critical role in human renal complement homeostasis. Unexpectedly, the FH sialic acid binding site is structurally homologous to the binding sites of two evolutionarily unrelated proteins. The crystal structure also advances our understanding of bacterial immune evasion strategies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                16 October 2015
                October 2015
                : 11
                : 10
                : e1005104
                Affiliations
                [1 ]Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
                [2 ]Department of Molecular, Cellular, and Developmental Biology, and the BioFrontiers Institute, University of Colorado, Boulder, Colorado, United States of America
                [3 ]Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                Penn State University School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MHCB AML UN TS SDO RLG. Performed the experiments: MHCB AML SDO. Analyzed the data: MHCB AML UN TS SDO RLG. Wrote the paper: MHCB AML UN TS SDO RLG.

                [¤]

                Current Address: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK

                Article
                PPATHOGENS-D-15-00669
                10.1371/journal.ppat.1005104
                4608799
                26474293
                a3a5910e-ce05-460b-8fe1-b91d9af66f95
                Copyright @ 2015

                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

                History
                : 18 March 2015
                : 22 July 2015
                Page count
                Figures: 7, Tables: 2, Pages: 22
                Funding
                MHCB, AML, UN, and TS are grateful for financial support from the Deutsche Forschungsgemeinschaft SFB 685 ( www.sfb685.de). SDO and RLG acknowledge the National Institutes of Health R01-CA37667 ( www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All relevant data are within the paper and its Supporting Information files except for the protein sequences of MuPyV RA VP1 and PTA VP1, which are available from UniProt under the accession numbers P49302 and Q76TX8, respectively, as well as the PDB entries of the constructs used in this publication (PDB IDs 5CPU, 5CPW, 5CPX, 5CPY, 5CPZ, and 5CQ0). Please also refer to Table 2 in the manuscript.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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