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      Conservation and Variability of Dengue Virus Proteins: Implications for Vaccine Design

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          Abstract

          Background

          Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes.

          Methodology/Principal Findings

          We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV) proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences) and again in 2007 (12,404 sequences). Forty-four (44) sequences (pan-DENV sequences), mainly those of nonstructural proteins and representing ∼15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 (∼77%) were present in ≥95% of sequences of each DENV type, and 27 (∼61%) were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to ∼5%), as compared to variant frequencies of ∼60 to ∼85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA) supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by studies with HLA-transgenic mice and/or reported to be immunogenic in humans.

          Conclusions/Significance

          Forty-four (44) pan-DENV sequences of at least 9 amino acids were highly conserved and identical in 80% or more of all recorded DENV sequences, and the majority were found to be immune-relevant by their correspondence to known or putative HLA-restricted T-cell determinants. The conservation of these sequences through the entire recorded DENV genetic history supports their possible value for diagnosis, prophylactic and/or therapeutic applications. The combination of bioinformatics and experimental approaches applied herein provides a framework for large-scale and systematic analysis of conserved and variable sequences of other pathogens, in particular, for rapidly mutating viruses, such as influenza A virus and HIV.

          Author Summary

          Dengue viruses (DENVs) circulate in nature as a population of 4 distinct types, each with multiple genotypes and variants, and represent an increasing global public health issue with no prophylactic and therapeutic formulations currently available. Viral genomes contain sites that are evolutionarily stable and therefore highly conserved, presumably because changes in these sites have deleterious effects on viral fitness and survival. The identification and characterization of the historical dynamics of these sites in DENV have relevance to several applications such as diagnosis and drug and vaccine development. In this study, we have identified sequence fragments that were conserved across the majority of available DENV sequences, analyzed their historical dynamics, and evaluated their relevance as candidate vaccine targets, using various bioinformatics-based methods and immune assay in human leukocyte antigen (HLA) transgenic mice. This approach provides a framework for large-scale and systematic analysis of other human pathogens.

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          Most cited references64

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          The Pfam protein families database.

          Pfam is a large collection of protein families and domains. Over the past 2 years the number of families in Pfam has doubled and now stands at 6190 (version 10.0). Methodology improvements for searching the Pfam collection locally as well as via the web are described. Other recent innovations include modelling of discontinuous domains allowing Pfam domain definitions to be closer to those found in structure databases. Pfam is available on the web in the UK (http://www.sanger.ac.uk/Software/Pfam/), the USA (http://pfam.wustl.edu/), France (http://pfam.jouy.inra.fr/) and Sweden (http://Pfam.cgb.ki.se/).
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            Structure of the dengue virus envelope protein after membrane fusion.

            Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three 'fusion loops' at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.
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              Estimation of Entropy and Mutual Information

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                August 2008
                13 August 2008
                : 2
                : 8
                : e272
                Affiliations
                [1 ]Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
                [2 ]Institute of Systems Science, National University of Singapore, Singapore
                [3 ]Department of Medicine, Division of Infectious Diseases, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                [4 ]Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
                [5 ]Product Evaluation and Registration Division, Centre for Drug Administration, Health Sciences Authority, Singapore
                [6 ]Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
                University of California Berkeley, United States of America
                Author notes

                Conceived and designed the experiments: AMK OM EJMN KNS ATH JS JTA. Performed the experiments: AMK OM EJMN. Analyzed the data: AMK OM EJMN KNS ATH GLZ ETM TWT VB JS JTA. Contributed reagents/materials/analysis tools: AMK OM EJMN GLZ JTA. Wrote the paper: AMK OM JS JTA.

                Article
                08-PNTD-RA-0044R2
                10.1371/journal.pntd.0000272
                2491585
                18698358
                5fb646b3-49ba-4b36-8a93-3a5a98abb74a
                Khan et al. 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
                : 5 February 2008
                : 10 July 2008
                Page count
                Pages: 15
                Categories
                Research Article
                Computational Biology/Comparative Sequence Analysis
                Computational Biology/Sequence Motif Analysis
                Evolutionary Biology/Bioinformatics
                Immunology/Antigen Processing and Recognition
                Immunology/Genetics of the Immune System
                Immunology/Immune Response
                Immunology/Immunity to Infections
                Immunology/Leukocyte Activation
                Infectious Diseases/Neglected Tropical Diseases
                Infectious Diseases/Viral Infections
                Microbiology/Immunity to Infections
                Microbiology/Microbial Evolution and Genomics
                Public Health and Epidemiology/Infectious Diseases
                Virology/New Therapies, including Antivirals and Immunotherapy
                Virology/Vaccines

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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