Author
Takaji Wakita
Other affiliations: University of Tokyo, Institute of Medical Science
Bio: Takaji Wakita is an academic researcher from National Institutes of Health. The author has contributed to research in topics: Hepatitis C virus & Virus. The author has an hindex of 74, co-authored 470 publications receiving 24434 citations. Previous affiliations of Takaji Wakita include University of Tokyo & Institute of Medical Science.
Papers published on a yearly basis
Papers
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TL;DR: It is shown that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7) and provides a powerful tool for studying the viral life cycle and developing antiviral strategies.
Abstract: Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15–1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture–generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.
2,809 citations
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TL;DR: 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 is reported.
Abstract: 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.
1,766 citations
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TL;DR: A novel function of LDs is revealed in the assembly of infectious HCV and a new perspective on how viruses usurp cellular functions is provided.
Abstract: 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.
1,197 citations
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TL;DR: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection in a transient replication assay, providing a powerful new tool for researching HCV.
619 citations
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TL;DR: Postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins and the HCV clone JFH-1, and data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.
Abstract: Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.
608 citations
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28 Jul 2005
TL;DR: PfPMP1)与感染红细胞、树突状组胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作�ly.
Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1(PfPMP1)与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员,通过启动转录不同的var基因变异体为抗原变异提供了分子基础。
18,940 citations
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TL;DR: It is found that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection.
Abstract: The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.
3,508 citations
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Icahn School of Medicine at Mount Sinai1, University of Michigan2, Pasteur Institute3, University of California, San Francisco4, European Bioinformatics Institute5, University of California, San Diego6, University of North Carolina at Chapel Hill7, Fred Hutchinson Cancer Research Center8, Gladstone Institutes9, Buck Institute for Research on Aging10
TL;DR: A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
Abstract: A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
3,319 citations
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TL;DR: The current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagic for cell survival and homeostasis are presented.
Abstract: Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases. We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis. We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.
3,249 citations
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TL;DR: This document has been approved by the AASLD, the Infectious Diseases Society of America, and the American College of Gastroenterology.
3,013 citations