Yanouchka Rouleau

Hepatitis C virus induced up‐regulation of microRNA‐27: A novel mechanism for hepatic steatosis

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR‐27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR‐27 expression. Furthermore, we establish that miR‐27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti‐Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR‐27bs repression of peroxisome proliferator‐activated receptor (PPAR)‐α and angiopoietin‐like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR‐α agonist, bezafibrate, is able to reverse the miR‐27b‐induced lipid accumulation in Huh7 cells. This miR‐27b‐mediated repression of PPAR‐α signaling represents a novel mechanism of HCV‐induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR‐27b expression levels and hepatic lipid accumulation in HCV‐infected SCID‐beige/Alb‐uPa mice. Conclusion: Collectively, our results highlight HCVs up‐regulation of miR‐27 expression as a novel mechanism contributing to the development of hepatic steatosis. (Hepatology 2014;58:98–108)

Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism.

Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naïve MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.

Nanoscale aggregation of cellular beta2-adrenergic receptors measured by plasmonic interactions of functionalized nanoparticles.

Adrenergic signaling that controls the contraction of cardiac myocyte cells and the beating of the mammalian heart is initiated by ligand binding to adrenergic receptors contained in nanoscale multiprotein complexes at the cellular membrane. Here we demonstrate that the surface-enhanced Raman scattering (SERS) of functionalized silver nanoparticles can be used to report on the receptor aggregation state of specifically label beta(2)-adrenergic receptors on mouse cardiac myocyte cells. Furthermore, multimodal imaging including Raman, Rayleigh scattering, scanning electron microscopy, and luminescence imaging was combined to fully characterize the beta(2)-adrenergic receptor-mediated aggregation of silver nanoparticles on the membrane of cardiac myocytes. Scanning electron microscopy analysis reveals distinct SERS active clusters of between 10 and 70 nanoparticles per signaling domain from ultra-high-resolution images of beta(2)-adrenergic receptor clusters on the cellular membrane. These techniques can be generally applied to study the aggregation of other cell surface receptors and explore their distribution on cell surfaces.

Modulation of Fatty Acid Synthase Enzyme Activity and Expression during Hepatitis C Virus Replication

The hepatitis C virus (HCV) induces alterations of host cells to facilitate its life cycle. Fatty acid synthase (FASN) is a multidomain enzyme that plays a key role in the biosynthesis of fatty acids and is upregulated during HCV infection. Herein, we applied activity-based protein profiling (ABPP) that allows for the identification of differentially active enzymes in complex proteomic samples, to study the changes in activity of FASN during HCV replication. For this purpose, we used an activity-based probe based on the FASN inhibitor Orlistat, and observed an increase in the activity of FASN in the presence of a subgenomic and a genomic HCV replicon as well as in chimeric SCID/Alb-uPA mice infected with HCV genotype 1a. To study the molecular basis for this increase in FASN activity, we overexpressed individual HCV proteins in Huh7 cells and observed increased expression and activity of FASN in the presence of core and NS4B, as measured by western blots and ABPP, respectively. Triglyceride levels were also elevated in accordance with FASN expression and activity. Lastly, immunofluorescence and ABPP imaging analyses demonstrated that while the abundance and activity of FASN increases significantly in the presence of HCV, its localization does not change. Together these data suggest that the HCV-induced production of fatty acids and neutral lipids is provided by an increase in FASN abundance and activity that is sufficient to allow HCV propagation without transporting FASN to the replication complexes.

Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus

Background Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees. Results Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol. Conclusion Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

Nanoscale organization of β2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize beta(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(2)AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the beta(2)AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of beta(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

Copper-catalysed cycloaddition reactions of nitrones and alkynes for bioorthogonal labelling of living cells

An adapted biocompatible version of the Kinugasa reaction, the copper-catalysed alkyne-nitrone cycloaddition followed by rearrangement (CuANCR), was developed for live-cell labelling. CuANCR labelling was demonstrated for both mammalian and bacterial cells. A method for metabolic incorporation of the nitrone group is also described.

Bleomycin is a Potent Small‐Molecule Inhibitor of Hepatitis C Virus Replication

The bleomycins (BLMs; 1) are a family of antitumor antibiotics isolated from Streptomyces verticillus. They are known to bind to and degrade double-stranded (ds) DNA in a sequence-selective manner. BLM-mediated DNA degradation occurs at 5’GT-3’ and 5’-GC-3’ sequences in cell-free and cellular systems, including in mammalian cells. More recently, BLMs have also been shown to cleave RNA in a site-selective manner. Examples include tRNA, mRNA, rRNA, and RNA–DNA duplexes. Interestingly, BLM-mediated cleavage of RNA appears to be conACHTUNGTRENNUNGtrolled by RNA tertiary structure rather than primary sequence and, with tRNA, cleavage occurs at sites containing both single-stranded and dsRNA. The specific targeting of ternary RNA structure implies that BLM might act selectively against other exogenous RNAs such as RNA viruses. Here we have examined the ability of BLM to target subgenomic hepatitis C virus (HCV) replicon RNA that is replication-competent in Huh7 human hepatoma cell lines. We have demonstrated that BLM targets HCV RNA elements faster than cellular DNA and RNA and that BLM can, therefore, act as a chemical probe for the rapid inhibition of replicating HCV RNA in cell culture. HCV is a positive-strand RNA virus of the Flaviviridae family with an ~9.6-kb genome (Figure 1B). This RNA genome codes for at least ten mature viral proteins. HCV genomic RNA contains many unique structural elements, such as stem loops (SL), hairpins, and bulges, which are formed from short stretches of complementary sequences. SLs exist in the 5’-untranslated region (5’-UTR), which contains a structurally unique internal ribosomal-entry site (IRES), and in the 3’-UTR. There are also two SLs in the core part of the genome and six that dominate the NS5B region. In addition, NS5B protein mediates the replication of HCV’s genomic RNA and produces negativestrand and dsRNA intermediates that resemble A-DNA helices. All of these HCV RNA structures are potential targets for BLMs. Since BLMs have been shown to display high selectivity for the destruction of RNAs with particular tertiary structures, and cytoplasmic RNAs are more easily targeted by BLM than is genomic DNA, which is localized in the nucleus, it should be possible to use BLMs to target pathogenic RNA selectively over genomic DNA. BLM might target the tertiary structure of single-stranded genomic viral RNA or dsRNA intermediates for replication. The dsRNA intermediates represent potential targets for BLM because it might intercalate among unique structural elements in replicating pathogenic dsRNA and initiate its destruction. In order to determine BLM activity against HCV RNA, we first examined the effects of blenoxane (BLM A2 and BLM B2) towards subgenomic HCV replicons, which are well-established cellular models for HCV replication. We utilized Huh-7 cells harboring a tricistronic subgenomic HCV replicon that was constructed from HCV genomic RNA of the genotype 1b and derived from the clone pFK-I389neo/luc/NS3-3’/5.1 previously described (Figure 1B). In this replicon, the neomycin-resistance gene product is translated via the HCV internal ribosomal entry site (IRES), and translation of the firefly luciferase gene product and nonstructural HCV proteins is controlled by the encephalomyocarditis virus (EMCV) IRES. We evaluated BLM activity by measuring the firefly luciferase signal (see Supporting Information for full details) after 24 and 48 h of exposure. BLM caused a concentration-dependent reduction in the luciferase signal, and we determined the IC50 to be 1.5 mm against the HCV replicon (Figure 1C, D). To assess if BLM affected cellular DNA, a cytotoxicity assay, which measured lactate dehydrogenase release following lysis of treated cells, was performed (Figure 1E). The assay showed the effects of BLM on cellular DNA only at concentrations 10 mm and only after 48 h of exposure; this suggests that there is poor cellular uptake through the nuclear membrane in Huh-7 cells. To assess if BLM affected cellular RNA, total protein levels in lysates of BLM-treated cells were quantified. No effects on cellular protein levels were ACHTUNGTRENNUNGobserved at concentrations of BLM sufficient to eliminate HCV ACHTUNGTRENNUNGreplicon replication (Figure 1D); this suggests that host cell tRNAs and rRNAs were not affected. Taken together, these data support the hypothesis that BLM selectively targets HCV replicon RNA over cellular DNA and RNA in Huh-7 cells. BLM may show selectivity for cleavage of HCV RNA over the cellular DNA since it shows low penetration through the nuclear membrane. Therefore, BLM will act faster on HCV RNA located in the cytoplasm than on DNA located in the nucleus. Furthermore, unlike for DNA, there is no mechanism by which RNA can repair itself. Therefore, BLM-mediated damage to viral HCV RNA is irreversible, leading to immediate HCV inhibition. Although the subgenomic replicon used in our studies lacks the RNA coding for the structural HCV proteins, it does contain the 5’-UTR, the NS3-NS5B region of the genome, and the 3’UTR. It also possesses a great deal of secondary and tertiary RNA structure, as is found within the genomic RNA. Therefore, it is not unexpected that BLM targets subgenomic HCV RNA. There could be a few mechanisms by which BLM inhibits viral replication. One possibility is that BLM intercalates into dsRNA that is formed during replication. Another possibility is that BLM cleaves RNA by strand scission in the shallow minor groove of the NS5B region, most likely at the junction of the singleand double-stranded regions. However, similar HCV inhibition would be observed if cleavage were to occur in the EMCV IRES of the replicon, which regulates translation of the HCV proteins. To assess if BLM could affect the EMCV IRES, we [a] B. Rakić, M. Br lotte, Y. Rouleau, S. B langer, Prof. Dr. J. P. Pezacki The Steacie Institute for Molecular Sciences National Research Council of Canada 100 Sussex Drive, Ottawa, K1A 0R6 (Canada) Fax: (+1)613-952-0068 E-mail : john.pezacki@nrc-cnrc.gc.ca Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.