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      Induction of Endoplasmic Reticulum-Derived Replication-Competent Membrane Structures by West Nile Virus Non-Structural Protein 4B

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          Abstract

          Replication of flaviviruses (family Flaviviridae) occurs in specialized virus-induced membrane structures (IMS). The cellular composition of these IMS varies for different flaviviruses implying different organelle origins for IMS biogenesis. The role of flavivirus non-structural (NS) proteins for the alteration of IMS remains controversial. In this report, we demonstrate that West Nile virus strain New York 99 (WNV NY99) remodels the endoplasmic reticulum (ER) membrane to generate specialized IMS. Within these structures, we observed an element of the cis-Golgi, viral double-stranded RNA, and viral-envelope, NS1, NS4A and NS4B proteins using confocal immunofluorescence microscopy. Biochemical analysis and microscopy revealed that NS4B lacking the 2K-signal peptide associates with the ER membrane where it initiates IMS formation in WNV-infected cells. Co-transfection studies indicated that NS4A and NS4B always remain co-localized in the IMS and are associated with the same membrane fractions, suggesting that these proteins function cooperatively in virus replication and may be an ideal target for antiviral drug discovery.

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          Most cited references 48

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          Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites

          Summary Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.
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            Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex.

            Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.
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              SOSUI: classification and secondary structure prediction system for membrane proteins.

              The system SOSUI for the discrimination of membrane proteins and soluble ones together with the prediction of transmembrane helices was developed, in which the accuracy of the classification of proteins was 99% and the corresponding value for the transmembrane helix prediction was 97%. The system SOSUI is available through internet access: http://www.tuat.ac.jp/mitaku/sosui/. sosui@biophys.bio.tuat. ac.jp.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                20 January 2014
                : 9
                : 1
                Affiliations
                [1 ]Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America
                [2 ]Department of Pediatrics, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America
                [3 ]Pacific Center for Emerging Infectious Diseases Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America
                The Pirbright Institute, United Kingdom
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: PHK VRN. Performed the experiments: PHK JFK. Analyzed the data: PHK VRN JFK RY. Contributed reagents/materials/analysis tools: PHK VRN RY. Wrote the paper: PHK VRN JFK RY.

                Article
                PONE-D-13-09922
                10.1371/journal.pone.0084040
                3896337

                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: 13
                Funding
                This study was partially supported by grants from the Hawaii Community Foundation (43055), Centers of Biomedical Research Excellence (P20GM103516), National Institute of General Medical Sciences, National Institutes of Health (NIH), and Institutional Funds. Also Research Centers in Minority Institutions Program (G12MD007601). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Molecular Cell Biology
                Cellular Structures
                Subcellular Organelles
                Medicine
                Infectious Diseases
                Tropical Diseases (Non-Neglected)
                Flavivirus
                Viral Diseases
                West Nile fever
                Neurology
                Infectious Diseases of the Nervous System
                Encephalitis

                Uncategorized

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