Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

Neufeldt, Christopher J.; Joyce, Michael; Levin, Aviad; Steenbergen, Renske D.M.; Pang, Daniel; Shields, Justin; Tyrrell, D. Lorne; Wozniak, Richard W.

doi:10.1371/journal.ppat.1003744

Cited by 59 publications

(101 citation statements)

References 68 publications

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“…Accordingly, several HCV proteins, including Core, contain functional nuclear localization signal and nuclear export signal sequences that are required to bind cargo proteins and cross the NPC (17). This hypothesis would be in agreement with the model proposed by Neufeldt et al (16), which suggests the insertion of NPC in the cytoplasm of infected cells to compartmentalize virus replication/assembly. To date, viruses have been described to target the NPC, to cross the nuclear envelope, and/or to control host transcription.…”

Section: Discussionsupporting

confidence: 89%

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Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Lussignol

Kopp

Molloy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virushost interactions underlying HCV infection. Hepatitis C virus (HCV) is a positive-sense ssRNA virus of the Flaviviridae family. This bloodborne pathogen causes chronic liver infection that develops into cirrhosis and hepatocellular carcinoma and is the leading indication for liver transplantation. Over 185 million people are chronically infected with HCV (2). Despite great advances in the ability to study this virus in vitro, significant gaps remain in our understanding of the infectious particle and the virus-host interactions required for HCV propagation.The protein composition of HCV is not known. Although the capsid protein, Core, and the E1 and E2 envelope glycoproteins are thought to be the major constituents of the virion, it remains to be determined if nonstructural viral proteins are packaged as well. A growing body of literature suggests that cellular proteins are important components of HCV. Indeed, this virus closely associates with LDL and very-LDL components, forming a chimeric lipoviral particle (LVP). Biochemical and ultrastructural studies demonstrated that infectious HCV particles are coated with endogenous apolipoproteins that play key roles in viral attachment and entry, explaining the higher infectivity of lipoprotein-associated HCV (2).The heterogeneous size and appearance of extracellular HCV, ranging from 40 to >100 nm in diameter, suggests that the set of associated proteins (both viral and cellular), as well as their stoichiometry, might vary across the virion population. Additionally, the specific infectivity of HCV changes according to the cell system/host that the virus is produced in, highlighting a stro...

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Section: Discussionsupporting

confidence: 89%

“…5). A previous study identified Nup98 as an important host protein for HCV infection (16). Here, we show that Nup98 is copurified with extracellular HCV virions ( Table 2), physically interacts with Core ( Fig.…”

Section: Discussionsupporting

confidence: 65%

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Lussignol

Kopp

Molloy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…9,47,48 Therefore, the observed topological alteration or modification of these structures by HCV infection may be determinant at the onset of an efficient infection. Indeed, a fraction of the viral proteins localize to mitochondria-associated endoplasmic reticulum membranes, [37][38][39] and we have shown that mitochondria-associated endoplasmic reticulum membranes-resident host factors are determinant at the onset of HCV infection. 39 Thus, it is conceivable that mitochondria-associated endoplasmic reticulum membranes are preferential sites for the initial establishment of HCV replicase complexes as it has previously been proposed.…”

Section: Reversion Of Hcv-induced Ultrastructural Alterations By Dirementioning

confidence: 89%

Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection

Pérez-Berná

Rodríguez²,

Chichón³

et al. 2016

ACS Nano

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Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.

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“…Indeed, many proteomic and functional RNAi genetic screens revealed that modulation of nuclear pore complexes (NPCs), nuclear transport receptors and the RAN‐GTPase system have profound effects on viral replication . For instance, interactions between viral proteins, nucleoporins (Nups), RAN, importin‐α (IMP‐α), importin‐β (IMP‐β) and exportin (EXP) have been observed with hepatitis C virus (HCV), picornavirus, and human immuno‐deficiency virus (HIV)‐1 . In HCV infection, viral proteins (core, NS2, NS3 and NS5A) harbor a nuclear localization signal (NLS) and/or nuclear export signal (NES) suggesting that nuclear‐cytoplasmic shuttling is important for it virus life cycle that is mostly within the cytoplasm .…”

Section: Introductionmentioning

confidence: 99%

Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

Gagné

Tremblay

Park

et al. 2017

Traffic

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In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon-β (IFNB1) induction. Using a comprehensive microscopy-based high-content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A-interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF-κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown-a hub protein highly targeted by several viruses-decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus-mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF-κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad-spectrum antiviral therapies.

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Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

Cited by 59 publications

References 68 publications

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection

Importin β1 targeting by hepatitis C virus NS3/4A protein restricts IRF3 and NF‐κB signaling of IFNB1 antiviral response

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