Hepatitis C Virus Selectively Alters the Intracellular Localization of Desmosterol

Villareal, Valerie A.; Fu, Dan; Costello, Deirdre A.; Xie, X. Sunney; Yang, Priscilla L.

doi:10.1021/acschembio.6b00324

Cited by 20 publications

(24 citation statements)

References 25 publications

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“…With SRS imaging, they were able to reveal heterogeneous partitioning of free cholesterol, cholesterol ester and triglyceride into LDs (Figure 3C and D). In another study 37 , d 6 -desmosterol was found accumulated in LDs when the cells were infected by hepatitis C virus.…”

Section: Lipid Metabolismmentioning

confidence: 87%

Applications of vibrational tags in biological imaging by Raman microscopy

Zhao

Shen

et al. 2017

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As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman microscopy has made big impacts to many disciplines of science. While the label-free imaging has been the prevailing strategy in Raman microscopy, recent development and applications of vibrational/Raman tags, particularly when coupled with stimulated Raman Scattering (SRS) microscopy, have generated intense excitement in biomedical imaging. SRS imaging of vibrational tags has enabled researchers to study a wide range of small biomolecules with high specificity, sensitivity and multiplex capability, at single live cell level, tissue level or even in vivo. As reviewed in the article, this platform has facilitated imaging distribution and dynamics of small molecules such as glucose, lipids, amino acids, nucleic acids, and drugs that are otherwise difficult to do with other means. As both the vibrational tags and Raman instrumental development progress rapidly and synergistically, we anticipate that the technique will shed light onto an even broader spectrum of biomedical problems.

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Section: Lipid Metabolismmentioning

confidence: 87%

Applications of vibrational tags in biological imaging by Raman microscopy

Zhao

Shen

et al. 2017

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“…Desmosterol has been detected in replicase-containing membranes by LC-MS analysis ( Figure 2) and in NS5Aassociated lipid droplets by stimulated Raman scattering microscopy 11 . 11 . Pharmacological inhibition of DHCR7 leads to depletion of intracellular desmosterol and a profound reduction in both HCV proteins and genomic RNA that is rescued by the addition of exogenous desmosterol.…”

Section: Figure 1 the Membranous Web Is A Specialized Membranous Enmentioning

confidence: 99%

Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection

2016

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Hepatitis C virus (HCV) uniquely affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization. These effects are important for productive viral replication since inhibition of desmosterol synthesis has an antiviral effect that can be rescued by the addition of exogenous desmosterol. Here, we use subgenomic replicons to show that desmosterol has a major effect on replication of HCV JFH1 RNA. Fluorescence recovery after photobleaching (FRAP) experiments performed with synthetic supported lipid bilayers demonstrate that substitution of desmosterol for cholesterol significantly increases lipid bilayer fluidity, especially in the presence of saturated phospholipids and ceramides. We demonstrate using LC-MS that desmosterol is abundant in the membranes upon which genome replication takes place and that supported lipid bilayers derived from these specialized membranes also exhibit significantly higher fluidity compared to negative control membranes isolated from cells lacking HCV. Together, these data suggest a model in which the fluidity-promoting effects of desmosterol on lipid bilayers play a crucial role in the extensive membrane remodeling that takes place in the endoplasmic reticulum during HCV infection. We anticipate that the supported lipid bilayer system described can provide a useful model system in which to interrogate the effects of lipid structure and composition on the biophysical properties of lipid membranes as well as their function in viral processes such as genome replication.

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“…For example, many RNA viruses, including HCV, are known to remodel sections of the host ER membrane to form specialized replication compartments ( 37 – 41 ). In our previous work, we have documented specific HCV-induced changes to host lipid metabolism, including a large-fold increase in desmosterol ( 15 , 16 ). Enrichment of desmosterol in the membrane where viral RNA replication occurs is, moreover, correlated with an increase in the diffusivity of the membranes where RNA replication occurs ( 17 ).…”

Section: Discussionmentioning

confidence: 99%

“…Huh7.5–SGR cells stably replicating the HCV JFH1 (2a) subgenomic replicon were generated by electroporation of Huh7.5 cells with in vitro transcribed replicon RNA as previously described ( 18 ). Huh7.5–SGR cells were maintained in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, 10% fetal bovine serum (Invitrogen), and G418 sulfate (750 µg/ml final concentration) ( 15 , 16 , 18 ).…”

Section: Methodsmentioning

confidence: 99%

“…Both proteomic and lipidomic profiling by targeted liquid chromatography - mass spectrometry (LC-MS) have shown that HCV perturbs multiple host lipid biosynthetic pathways ( 14 ). Additionally, unbiased lipidomic MS profiling by our laboratory previously discovered that HCV causes a 10-fold increase in intracellular desmosterol, an immediate precursor to cholesterol, without affecting the abundance of cholesterol ( 15 – 17 ). We demonstrated that the HCV-induced accumulation of desmosterol is functionally important for HCV replication, as evidenced by strong reduction of accumulated HCV RNA when desmosterol is depleted from cells and restoration of HCV RNA levels upon addition of exogenous desmosterol but not cholesterol ( 15 , 17 ).…”

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

Tallorin

Villareal

Hsia

et al. 2020

Journal of Biological Chemistry

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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-localize. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

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Hepatitis C Virus Selectively Alters the Intracellular Localization of Desmosterol

Cited by 20 publications

References 25 publications

Applications of vibrational tags in biological imaging by Raman microscopy

Applications of vibrational tags in biological imaging by Raman microscopy

Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection

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

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