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Journal ArticleDOI

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment

01 Oct 2013-Journal of Virology (American Society for Microbiology (ASM))-Vol. 87, Iss: 19, pp 10612-10627
TL;DR: The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.
Abstract: Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.
Citations
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Journal ArticleDOI
TL;DR: Currently available cell culture systems for HCV are highlighted, the most prominent host-targeting strategies against hepatitis C are reviewed, and opportunities and risks associated with host- targeting antiviral strategies are critically discussed.
Abstract: Hepatitis C virus (HCV) is a highly variable plus-strand RNA virus of the family Flaviviridae . Viral strains are grouped into six epidemiologically relevant genotypes that differ fr

370 citations

Journal ArticleDOI
TL;DR: Detailed 2D and 3D analyses of CoV ROs are provided and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication.
Abstract: Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

326 citations

Journal ArticleDOI
TL;DR: The current understanding of how Flaviviridae viruses manipulate and usurp cellular pathways in infected cells is discussed, and the importance of these interactions in the context of viral replication and antiviral therapies is discussed.
Abstract: Members of the Flaviviridae virus family comprise a large group of enveloped viruses with a single-strand RNA genome of positive polarity. Several genera belong to this family, including the Hepacivirus genus, of which hepatitis C virus (HCV) is the prototype member, and the Flavivirus genus, which contains both dengue virus and Zika virus. Viruses of these genera differ in many respects, such as the mode of transmission or the course of infection, which is either predominantly persistent in the case of HCV or acutely self-limiting in the case of flaviviruses. Although the fundamental replication strategy of Flaviviridae members is similar, during the past few years, important differences have been discovered, including the way in which these viruses exploit cellular resources to facilitate viral propagation. These differences might be responsible, at least in part, for the various biological properties of these viruses, thus offering the possibility to learn from comparisons. In this Review, we discuss the current understanding of how Flaviviridae viruses manipulate and usurp cellular pathways in infected cells. Specifically, we focus on comparing strategies employed by flaviviruses with those employed by hepaciviruses, and we discuss the importance of these interactions in the context of viral replication and antiviral therapies.

243 citations

Journal ArticleDOI
22 Jul 2014-Viruses
TL;DR: The architecture of these complex membrane rearrangements of members of plus-strand (+) RNA viruses is discussed, and it is hypothesized that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.
Abstract: In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

240 citations


Cites background or result from "Morphological and biochemical chara..."

  • ...Importantly, DMVs contain enzymatically active viral replicase [49] and they originate from ER membranes, similar to what has been found for other members of the family Flaviviridae....

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  • ...Indeed, immunolabeling of purified DMVs revealed an enrichment for viral proteins as well as dsRNA [46,49]....

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Journal ArticleDOI
TL;DR: The molecular mechanisms of viral replicase function, cellular pathways employed during HCV replication factory biogenesis, and viral, as well as cellular, determinants of progeny virus production are reviewed.

229 citations


Cites background from "Morphological and biochemical chara..."

  • ...…of HCV, reduced DMV diameters were observed both by blocking OSBP-mediated cholesterol transport in HCV-infected cells (Wang et al., 2014) and by depletion of cholesterol from purified DMVs (Paul et al., 2013), arguing that cholesterol is an important structural component of HCV-remodeledmembranes....

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  • ..., 2014) and by depletion of cholesterol from purified DMVs (Paul et al., 2013), arguing that cholesterol is an important structural component of HCV-remodeledmembranes....

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  • ...Second, purified DMVs contain enzymatically active viral replicase, suggesting that they are bona fide HCV vRFs (Paul et al., 2013) (Figure 2)....

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References
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Journal ArticleDOI
TL;DR: How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functions of their individual membranes?
Abstract: Throughout the biological world, a 30 A hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?

5,720 citations


"Morphological and biochemical chara..." refers background in this paper

  • ...Such an enrichment of cholesterol in HCVinduced DMVs would result in local cholesterol concentrations that might be similar to that of endosomes or the plasma membrane (64)....

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Journal ArticleDOI
02 Jul 1999-Science
TL;DR: This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies ofHCV and the development of antiviral drugs.
Abstract: An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.

2,982 citations


"Morphological and biochemical chara..." refers background in this paper

  • ...to -5B constitute the minimal viral replicase machinery (10, 11)....

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Journal ArticleDOI
TL;DR: These proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future.

1,520 citations


"Morphological and biochemical chara..." refers background in this paper

  • ...is classified into 7 genotypes and more than 100 subtypes (5)....

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Journal ArticleDOI
08 Dec 2000-Science
TL;DR: This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication and identifies multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro.
Abstract: Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-α rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

1,492 citations


"Morphological and biochemical chara..." refers background in this paper

  • ...to -5B constitute the minimal viral replicase machinery (10, 11)....

    [...]

01 Jan 1999
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