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      High-Resolution Functional Profiling of Hepatitis C Virus Genome

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          Abstract

          Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3′ non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.

          Author Summary

          Hepatitis C virus (HCV) is a major human health concern that causes fatal liver diseases. Currently no vaccine is available to prevent HCV infection. Though the HCV was identified two decades ago, the virus has only recently been successfully grown in cell culture conditions. The role of HCV protein and regulatory element sub-domains during virus growth is poorly understood. We have developed a mutational analysis method to identify the function of HCV sub-domains at a high resolution. A collection of HCV mutants containing 15-nucleotide random insertions was tested for growth in cell culture. The precise location of the insertions and their effects on virus growth were analyzed by capillary genotyping technology and bioinformatics. Out of the total 2399 HCV mutants identified, 374 mutants grew normally, 111 mutants demonstrated reduced growth, and 1914 mutants failed to grow in cell culture. This mutational analysis method was validated by testing many individual mutant viruses. The present study identified several HCV functional sub-domains required for virus growth, presenting novel therapeutic targets. The HCV mutant viruses identified with the property of reduced growth can be used for designing vaccine candidates.

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          Most cited references63

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          Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.

          A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.
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            The lipid droplet is an important organelle for hepatitis C virus production.

            The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.
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              Robust hepatitis C virus infection in vitro.

              The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                October 2008
                October 2008
                17 October 2008
                : 4
                : 10
                : e1000182
                Affiliations
                [1 ]Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America
                [2 ]Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America
                [3 ]AIDS Institute, University of California, Los Angeles, California, United States of America
                [4 ]Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California, United States of America
                [5 ]Molecular Biology Institute, University of California, Los Angeles, California, United States of America
                University of California San Francisco, United States of America
                Author notes

                Conceived and designed the experiments: VA RS. Performed the experiments: VA RR VK TNH CL. Analyzed the data: VA EYS. Contributed reagents/materials/analysis tools: VF AD. Wrote the paper: VA AD RS.

                Article
                08-PLPA-RA-0520R1
                10.1371/journal.ppat.1000182
                2564836
                18927624
                88c76fe0-331e-44f0-9763-dc7e7cca742c
                Arumugaswami 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
                : 20 May 2008
                : 22 September 2008
                Page count
                Pages: 16
                Categories
                Research Article
                Biotechnology/Applied Microbiology
                Computational Biology/Genomics
                Gastroenterology and Hepatology/Hepatology
                Genetics and Genomics/Functional Genomics
                Infectious Diseases/Viral Infections
                Microbiology/Medical Microbiology
                Oncology/Gastrointestinal Cancers
                Oncology/Oncology Agents
                Virology/Viral Replication and Gene Regulation
                Virology/Viruses and Cancer

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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