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1.
mBio ; 14(5): e0154923, 2023 Oct 31.
Article in English | MEDLINE | ID: mdl-37671888

ABSTRACT

IMPORTANCE: Assessments of viral stability on surfaces or in body fluids under different environmental conditions and/or temperatures are often performed, as they are key to understanding the routes and parameters of viral transmission and to providing clues on the epidemiology of infections. However, for most viruses, the mechanisms of inactivation vs stability of viral particles remain poorly defined. Although they are structurally diverse, with different compositions, sizes, and shapes, enveloped viruses are generally less stable than non-enveloped viruses, pointing out the role of envelopes themselves in virus lability. In this report, we investigated the properties of hepatitis C virus (HCV) particles with regards to their stability. We found that, compared to alternative enveloped viruses such as Dengue virus (DENV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hepatitis delta virus (HDV), and Crimean-Congo hemorrhagic fever virus (CCHFV) that infect the liver, HCV particles are intrinsically labile. We determined the mechanisms that drastically alter their specific infectivity through oxidation of their lipids, and we highlighted that they are protected from lipid oxidation by secreted cellular proteins, which can protect their membrane fusion capacity and overall infectivity.


Subject(s)
Hemorrhagic Fever Virus, Crimean-Congo , Hepatitis C , Humans , Hepacivirus , Hemorrhagic Fever Virus, Crimean-Congo/physiology , Hepatitis C/metabolism
2.
mBio ; 13(2): e0292321, 2022 04 26.
Article in English | MEDLINE | ID: mdl-35258330

ABSTRACT

Nup98, an essential component of the nuclear pore that also participates in annulate lamella pore structures localized in the cytosol, is involved in hepatitis C virus (HCV) assembly. Here, we combined confocal microscopy and biochemical assays to study the interplay between Nup98, core (i.e., the HCV capsid protein), and viral genomes. Our results show that in HCV-infected cells, core protein is necessary and sufficient to induce relocalization of Nup98 from annulate lamellae to lipid droplet-apposed areas in which core/NS5A and HCV genomic RNA [(+)RNA] are clustered to promote viral assembly. Furthermore, we found that Nup98 interacts with HCV RNA and that upon Nup98 downregulation, the viral (+)RNA genome was specifically excluded from areas that contain active translating ribosomes and the core and NS5A proteins. Altogether, these results indicate that Nup98 is recruited by HCV core from annulate lamellae to viral assembly sites to locally increase the concentration of (+)RNA genome, which may favor its encapsidation into nascent virions. IMPORTANCE Nup98 is an essential component of the nuclear pore that also participates in annulate lamella pore structures localized in the cytosol. Nup98 is involved in HCV assembly, though its role remains elusive. Here, we show that Nup98 is retrieved from annulate lamellae during HCV infection. We demonstrate that Nup98 interacts with viral genome within infected cells and that these interactions are essential to maintain viral (+)RNAs in subcellular regions promoting viral replication, assembly, and translation. Importantly, we also show that HCV core nucleocapsid protein is the viral component responsible for the retrieval of Nup98 protein from annulate lamellae, hence allowing an enrichment of Nup98 complexed with viral (+)RNAs in core protein-containing areas. Altogether, our results indicate that Nup98 is recruited from annulate lamellae to viral assembly sites by HCV core protein to promote viral assembly, which highlights a novel virus-induced subversion mechanism of nuclear pore complex components.


Subject(s)
Hepatitis C , Viral Core Proteins , Hepacivirus/genetics , Humans , Nuclear Pore Complex Proteins/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virus Assembly/physiology
3.
Viruses ; 12(4)2020 04 12.
Article in English | MEDLINE | ID: mdl-32290553

ABSTRACT

Hepatitis C virus (HCV) infection is a major public health issue leading to chronic liver diseases. HCV particles are unique owing to their particular lipid composition, namely the incorporation of neutral lipids and apolipoproteins. The mechanism of association between HCV virion components and these lipoproteins factors remains poorly understood as well as its impact in subsequent steps of the viral life cycle, such as entry into cells. It was proposed that the lipoprotein biogenesis pathway is involved in HCV morphogenesis; yet, recent evidence indicated that HCV particles can mature and evolve biochemically in the extracellular medium after egress. In addition, several viral, cellular and blood components have been shown to influence and regulate this specific association. Finally, this specific structure and composition of HCV particles was found to influence entry into cells as well as their stability and sensitivity to neutralizing antibodies. Due to its specific particle composition, studying the association of HCV particles with lipoproteins remains an important goal towards the rational design of a protective vaccine.


Subject(s)
Hepacivirus/physiology , Hepatitis C/metabolism , Hepatitis C/virology , Host-Pathogen Interactions , Lipoproteins/metabolism , Animals , Endoplasmic Reticulum/metabolism , Hepatitis C/immunology , Humans , Lipid Metabolism , Lipoproteins/biosynthesis , Protein Transport , Signal Transduction , Virion , Virus Assembly , Virus Internalization
4.
Nat Commun ; 10(1): 2098, 2019 05 08.
Article in English | MEDLINE | ID: mdl-31068585

ABSTRACT

Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.


Subject(s)
Coinfection/transmission , Hepatitis D/transmission , Hepatitis Delta Virus/physiology , Virus Assembly , Animals , Cell Line, Tumor , Coinfection/virology , Flavivirus/metabolism , Hepacivirus/metabolism , Hepacivirus/pathogenicity , Hepatitis D/virology , Hepatitis Delta Virus/isolation & purification , Hepatitis Delta Virus/pathogenicity , Hepatocytes/transplantation , Hepatocytes/virology , Humans , Mice , Mice, Inbred NOD , Mice, Transgenic , Primary Cell Culture , RNA, Viral/isolation & purification , Ribonucleoproteins/metabolism , Vesiculovirus/metabolism , Viral Envelope Proteins/metabolism , Virion/metabolism
5.
PLoS Pathog ; 13(12): e1006774, 2017 12.
Article in English | MEDLINE | ID: mdl-29253880

ABSTRACT

Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.


Subject(s)
Hepacivirus/physiology , Hepacivirus/pathogenicity , Viral Envelope Proteins/physiology , Viral Proteins/physiology , Amino Acid Sequence , Cell Line , HEK293 Cells , Hepacivirus/genetics , Hepatitis C/etiology , Hepatitis C/virology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Models, Biological , Mutation , Protein Processing, Post-Translational , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Virulence/genetics , Virulence/physiology , Virus Assembly/genetics , Virus Assembly/physiology
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