Neda Nasheri

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)

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.

An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19

MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19s utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1fmol and a linear dynamic range from 1pmol to 1fmol.

Activity-based protein profiling of host–virus interactions

Virologists have benefited from large-scale profiling methods to discover new host–virus interactions and to learn about the mechanisms of pathogenesis. One such technique, referred to as activity-based protein profiling (ABPP), uses active site-directed probes to monitor the functional state of enzymes, taking into account post-translational interactions and modifications. ABPP gives insight into the catalytic activity of enzyme families that does not necessarily correlate with protein abundance. ABPP has been used to investigate several viruses and their interactions with their hosts. Differential enzymatic activity induced by viruses has been monitored using ABPP. In this review, we present recent advances and trends involving the use of ABPP methods in understanding host–virus interactions and in identifying novel targets for diagnostic and therapeutic applications.

The Efficacy of siRNAs against Hepatitis C Virus Is Strongly Influenced by Structure and Target Site Accessibility

Hepatitis C virus (HCV) is a global health problem. Designing therapeutic agents that target HCVs RNA genome remains challenging. HCV genomic RNA is large and highly structured with long-range genome-scale ordered RNA structures. Predicting the secondary- and tertiary-structure elements that reveal the accessibility of target sites within HCV RNA is difficult because of the abundance of long-range interactions. Target site accessibility remains a significant barrier to the design of effective therapeutics such as small interfering RNAs (siRNAs) against different strains of HCV. Here we developed two methods that interrogate the folding of HCV RNA, an approach involving viral RNA microarrays (VRMs) and an HCV viral RNA-coated magnetic bead-based (VRB) assay. VRMs and VRBs were used to determine target site accessibility for siRNAs designed against the HCV genome. Both methods predicted potency of siRNAs in cell-culture models for HCV replication that are not easily predicted by informatic means.

A New Chemical Probe for Phosphatidylinositol Kinase Activity

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive “clickable” probe, PIK‐BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK‐BPyne to a wortmannin activity‐based probe also known to target PIKs. We found that PIK‐BPyne can act as an effective in situ activity‐based probe, and for the first time, report changes in PI4K‐IIIβ activity induced by the hepatitis C virus. These results establish the utility of PIK‐BPyne for activity‐based protein profiling studies of PIK function in native biological systems.

Enhanced specificity of the viral suppressor of RNA silencing protein p19 toward sequestering of human microRNA-122.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.

Systems biology methods help develop a better understanding of hepatitis C virus–induced liver injury

R ecently, systems biology methods have risen to the forefront of techniques for elucidating the molecular details of disease progression. There has been a transition from an emphasis on individual genes to more comprehensive analyses, as technologies continue to evolve for quantitative high-throughput molecular detection. This has led to a variety of studies that have integrated different ‘‘omics’’ approaches to identify key factors in complex host-pathogen molecular networks that are potentially clinically relevant targets for therapy. These multidisciplinary strategies continue to enhance our understanding of disease progression and identify prognostic markers; specifically, the identification of key factors linked to disease progression susceptibility and therapy response. To this end, several studies have been performed aiming to link hepatitis C virus (HCV) disease progression and/ or therapy outcome to both genetic and proteomic markers. Genome-wide association and profiling studies have identified human single nucleotide polymorphisms and several interferon stimulated genes, respectively, with varying predictive values. In the current issue of HEPATOLOGY, Katze and colleagues have published seminal studies using systems biology methods to gain a better understanding of how HCV reinfection in liver transplant patients can lead to the rapid development of liver disease. HCV infection remains the leading cause of orthotopic liver transplantation (OLT). For those suffering from chronic HCV infection, OLT remains one of the last recourses. Viremia recurrence is essentially universal in the graft posttransplant; however, the rate of disease progression varies, with 5%-30% of patients developing cirrhosis 5 years posttransplant due to accelerated rates of fibrosis. The accelerated requirement for retransplantation (RT) in this subset poses an additional economic burden, with patient and graft survival rates post-RT being lower than after primary OLT. Serial liver biopsy remains the best way of monitoring disease progression. However, this technique is invasive and risks the possibility of misdiagnosis. Reliable and noninvasive prognostic markers of HCV-associated disease progression are currently being evaluated, with the goal to improve allograft survival. Thus far, significant factors influencing disease progression that have been identified include HCV RNA levels preand post-OLT, viral genetics, donor age, and donor/recipient genetics. There has been limited work, however, investigating molecular signatures that predict clinical disease progression prior to histological evidence of fibrosis. One study, examining gene expression of recurrent HCV-infected biopsies 1 year post-OLT, reported an increase in myofibroblast (MF) and MF-like cell markers and a decrease in retinoidrelated proteins. These observations suggest decreased hepatic stellate cell (HSC) quiescence is correlated with rapid fibrosis progression. Another proteomics study linked an up-regulation of genes associated with oxidative stress and mitochondrial dysfunction to later stages of fibrosis (Batts-Ludwig stage 3-4). These studies emphasized a correlation between oxidative stress, HSC activation, and fibrosis. A more recent study by Mas et al. examined gene expression in biopsies at the time of HCV recurrence to develop a prognostic signature, which, based on nine differentially expressed genes, was capable of distinguishing mild and severe fibrosis at 3 years post-OLT. This report highlighted the potential for identifying predictors of fibrosis in gene expression early after OLT. In this issue of HEPATOLOGY, alternate avenues for diagnosing liver fibrosis, along with characterizing prognostic signatures indicative of rapid disease progression, are explored. These two companion studies utilized systems biology approaches to characterize early molecular signatures that correlated with rapid progression in both liver disease and fibrosis in HCVinfected transplant patients. These reports make use of the novel singular value decomposition initialized multidimensional scaling (SVD-MDS) analysis to separate Abbreviations: DAA, directed anti-viral agent; DEG, differentially expressed gene; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDAC, histone deacetylase; HSC, hepatic stellate cells; MF, myofibroblast; OLT, orthotopic liver transplantation; RT, retransplantation; SVD-MDS, singular value decomposition initialized multidimensional scaling. Address reprint requests to: John Paul Pezacki, Ph.D., Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6. E-mail: John.Pezacki@nrc.ca; fax: 613-941-8447. CopyrightVC 2012 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.25727 Potential conflict of interest: Nothing to report.

Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe

To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome.

Activity-based profiling of the proteasome pathway during hepatitis C virus infection.

Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin‐dependent and ubiquitin‐independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity‐based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.