The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication

2006 ◽  
Vol 84 (1) ◽  
pp. 67-79 ◽  
Author(s):  
Selena M Sagan ◽  
Yanouchka Rouleau ◽  
Cynthia Leggiadro ◽  
Lubica Supekova ◽  
Peter G Schultz ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Host Cell ◽  
Molecular Mechanisms ◽  
Cholesterol Metabolism ◽  
Cell Structure ◽  
Metabolic Inhibitors ◽  
General Effect ◽  
Cellular Lipid

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid–cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as β-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.Key words: hepatitis C virus, lipid metabolism, fluorescence microscopy, electron microscopy, membranous web, statins.

scholarly journals Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

2011 ◽  
Vol 85 (13) ◽  
pp. 6106-6116 ◽  
Author(s):  
K. P. Romano ◽  
J. M. Laine ◽  
L. M. Deveau ◽  
H. Cao ◽  
F. Massi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Host Cell ◽  
Molecular Mechanisms ◽  
Substrate Recognition ◽  
Cell Substrate

scholarly journals Hepatitis C Virus Infection Induces Hepatic Expression of NF-κB-Inducing Kinase and Lipogenesis by Downregulating miR-122

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Brianna Lowey ◽  
Laura Hertz ◽  
Stephan Chiu ◽  
Kristin Valdez ◽  
Qisheng Li ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Human Hepatocytes ◽  
Hcv Infection ◽  
Pivotal Role ◽  
Cellular Lipid

ABSTRACT Hepatitis C virus (HCV) harnesses host dependencies to infect human hepatocytes. We previously identified a pivotal role of IκB kinase α (IKK-α) in regulating cellular lipogenesis and HCV assembly. In this study, we defined and characterized NF-κB-inducing kinase (NIK) as an IKK-α upstream serine/threonine kinase in IKK-α-mediated proviral effects and the mechanism whereby HCV exploits this innate pathway to its advantage. We manipulated NIK expression in Huh7.5.1 cells through loss- and gain-of-function approaches and examined the effects on IKK-α activation, cellular lipid metabolism, and viral assembly. We demonstrated that NIK interacts with IKK-α to form a kinase complex in association with the stress granules, in which IKK-α is phosphorylated upon HCV infection. Depletion of NIK significantly diminished cytosolic lipid droplet content and impaired HCV particle production. NIK overexpression enhanced HCV assembly, and this process was abrogated in cells deprived of IKK-α, suggesting that NIK acts upstream of IKK-α. NIK abundance was increased in HCV-infected hepatocytes, liver tissues from Alb-uPA/Scid mice engrafted with human hepatocytes, and chronic hepatitis C patients. NIK mRNA contains an miR-122 seed sequence binding site in the 3′ untranslated region (UTR). miR-122 mimic and hairpin inhibitor directly affected NIK levels. In our hepatic models, miR-122 levels were significantly reduced by HCV infection. We demonstrated that HNF4A, a known transcriptional regulator of pri-miR-122, was downregulated by HCV infection. NIK represents a bona fide target of miR-122 whose transcription is downregulated by HCV through reduced HNF4A expression. This effect, together with the sequestering of miR-122 by HCV replication, results in “derepression” of NIK expression to deregulate lipid metabolism. IMPORTANCE Chronic hepatitis C virus (HCV) infection is a major global public health problem. Infection often leads to severe liver injury that may progress to cirrhosis, hepatocellular carcinoma, and death. HCV coopts cellular machineries for propagation and triggers pathological processes in the liver. We previously identified a pivotal role of IKK-α in regulating cellular lipid metabolism and HCV assembly. In this study, we characterized NIK as acting upstream of IKK-α and characterized how HCV exploits this innate pathway to its advantage. Through extensive mechanistic studies, we demonstrated that NIK is a direct target of miR-122, which is regulated at the transcription level by HNF4A, a hepatocyte-specific transcription factor. We show in HCV infection that NIK expression is increased while both HNF4A and miR-122 levels are decreased. NIK represents an important host dependency that links HCV assembly, hepatic lipogenesis, and miRNA biology.

scholarly journals Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

2020 ◽  
Vol 295 (35) ◽  
pp. 12426-12436 ◽  
Author(s):  
Lorillee Tallorin ◽  
Valerie A. Villareal ◽  
Chih-Yun Hsia ◽  
Mary A. Rodgers ◽  
Dominique J. Burri ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Rna Replication ◽  
Hcv Rna ◽  
Genome Replication ◽  
Subgenomic Replicon ◽  
Lipid Environment

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

scholarly journals Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease

2012 ◽  
Vol 56 (4) ◽  
pp. 1907-1915 ◽  
Author(s):  
Christoph Welsch ◽  
Sabine Schweizer ◽  
Tetsuro Shimakami ◽  
Francisco S. Domingues ◽  
Seungtaek Kim ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Interaction Network ◽  
Rna Replication ◽  
Viral Fitness ◽  
Dynamics Simulation ◽  
Structural Flexibility ◽  
Residue Interaction

ABSTRACTDrug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC50) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.

scholarly journals Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

1998 ◽  
Vol 18 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Michael Gale ◽  
Collin M. Blakely ◽  
Bart Kwieciszewski ◽  
Seng-Lai Tan ◽  
Michelle Dossett ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Functional Assay ◽  
Binding Region ◽  
Dimerization Domain ◽  
Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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