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      Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design

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          Abstract

          Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.

          Author Summary

          With the adaptation of chikungunya virus (CHIKV) to transmission by the Aedes albopictus mosquito, a pandemic has occurred resulting in four to six million human infections, and the virus continues to become endemic in new regions, most recently in the Caribbean. CHIKV can cause debilitating polyarthralgia, lasting for weeks to years, and there are currently no licensed vaccines or antiviral therapies available. While an investigational live-attenuated vaccine (LAV) exists, problems with reactogenicity have precluded its licensure. The purpose of the current study was to: i) devise an in vitro passage procedure that reliably generates a panel of CHIKV envelope glycoprotein mutations for screening as vaccine candidates; ii) determine the position of the mutations in the three-dimensional structure of the alphavirus spike complex and their effect on electrostatic potential; iii) determine the attenuation characteristics of each mutation in a murine model of CHIKV musculoskeletal disease; and iv) to identify in vitro assays examining the dependency of infection upon HS that correlate with attenuation and localization in the glycoprotein spike. This approach provides a paradigm for the rational design of future LAVs for CHIKV and other mosquito-borne viruses, by deliberately selecting and combining attenuating processes.

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          Most cited references54

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          Heparan sulphate proteoglycans fine-tune mammalian physiology.

          Heparan sulphate proteoglycans reside on the plasma membrane of all animal cells studied so far and are a major component of extracellular matrices. Studies of model organisms and human diseases have demonstrated their importance in development and normal physiology. A recurrent theme is the electrostatic interaction of the heparan sulphate chains with protein ligands, which affects metabolism, transport, information transfer, support and regulation in all organ systems. The importance of these interactions is exemplified by phenotypic studies of mice and humans bearing mutations in the core proteins or the biosynthetic enzymes responsible for assembling the heparan sulphate chains.
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            Evaluation of comparative protein modeling by MODELLER.

            We evaluate 3D models of human nucleoside diphosphate kinase, mouse cellular retinoic acid binding protein I, and human eosinophil neurotoxin that were calculated by MODELLER, a program for comparative protein modeling by satisfaction of spatial restraints. The models have good stereochemistry and are at least as similar to the crystallographic structures as the closest template structures. The largest errors occur in the regions that were not aligned correctly or where the template structures are not similar to the correct structure. These regions correspond predominantly to exposed loops, insertions of any length, and non-conserved side chains. When a template structure with more than 40% sequence identity to the target protein is available, the model is likely to have about 90% of the mainchain atoms modeled with an rms deviation from the X-ray structure of approximately 1 A, in large part because the templates are likely to be that similar to the X-ray structure of the target. This rms deviation is comparable to the overall differences between refined NMR and X-ray crystallography structures of the same protein.
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              Order out of chaos: assembly of ligand binding sites in heparan sulfate.

              Virtually every cell type in metazoan organisms produces heparan sulfate. These complex polysaccharides provide docking sites for numerous protein ligands and receptors involved in diverse biological processes, including growth control, signal transduction, cell adhesion, hemostasis, and lipid metabolism. The binding sites consist of relatively small tracts of variably sulfated glucosamine and uronic acid residues in specific arrangements. Their formation occurs in a tissue-specific fashion, generated by the action of a large family of enzymes involved in nucleotide sugar metabolism, polymer formation (glycosyltransferases), and chain processing (sulfotransferases and an epimerase). New insights into the specificity and organization of the biosynthetic apparatus have emerged from genetic studies of cultured cells, nematodes, fruit flies, zebrafish, rodents, and humans. This review covers recent developments in the field and provides a resource for investigators interested in the incredible diversity and specificity of this process.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                February 2014
                20 February 2014
                : 8
                : 2
                : e2719
                Affiliations
                [1 ]Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
                [2 ]Department of Microbiology & Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
                [3 ]CEITEC, Masaryk University, Brno, Czech Republic
                [4 ]Department of Diagnostic Medicine & Pathobiology, Biosecurity Research Institute, Kansas State University, Manhattan, Kansas, United States of America
                [5 ]Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
                University of Texas Medical Branch, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CLG JH CS DLV SH WBK KDR. Performed the experiments: CLG JH CS DLV TYS. Analyzed the data: CLG JH CS TYS WBK KDR. Contributed reagents/materials/analysis tools: EG DLV SH. Wrote the paper: CLG JH WBK KDR.

                Article
                PNTD-D-13-01670
                10.1371/journal.pntd.0002719
                3930508
                24587470
                a57fd818-c3a1-4b77-b170-71aa91d41bea
                Copyright @ 2014

                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 October 2013
                : 13 January 2014
                Page count
                Pages: 16
                Funding
                This work was supported by AI081680 (KDR) PNWRCE, AI057156 (KDR) WRCE, and AI083383 (WBK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immune response
                Immunopathology
                Microbiology
                Virology
                Animal models of infection
                Emerging viral diseases
                Viral replication
                Viral structure
                Viral vaccines
                Virulence factors and mechanisms
                Emerging infectious diseases
                Host-pathogen interaction
                Pathogenesis

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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