Ragunath Singaravelu

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)

Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

BackgroundHepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.ResultsThe PS4≡ probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled in situ compared to the in vitro labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4≡ targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation.ConclusionsThese findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of in situ ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.

Hepatitis C virus and microRNAs: miRed in a host of possibilities

It is well-established that the host microRNA (miRNA) milieu has a significant influence on the etiology of disease. In the context of viruses, such as hepatitis C virus (HCV), microRNAs have been shown to influence viral life cycles both directly, through interactions with the viral genome, and indirectly, through regulation of critical virus-associated host pathways. Several miRNA profiling studies have demonstrated that HCV infection aberrantly regulates a significant number of human miRNA. However, the biological relevance of these modulations remains poorly understood. In this review, we summarize recent research that has shed light on the pro-viral and anti-viral roles of HCV-induced changes in human miRNA expression and their significance in the development of HCV related sequelae and response to therapy.

Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes

Hepatitis C virus (HCV) replication is dependent on the formation of specialized membrane structures; however, the host factor requirements for the formation of these HCV complexes remain unclear. Herein, we demonstrate that inhibition of stearoyl-CoA desaturase 1 (SCD-1) halts the biosynthesis of unsaturated fatty acids, such as oleic acid, and negatively modulates HCV replication. Unsaturated fatty acids play key roles in membrane curvature and fluidity. Mechanistically, we demonstrate that SCD-1 inhibition disrupts the integrity of membranous HCV replication complexes and renders HCV RNA susceptible to nuclease-mediated degradation. Our work establishes a novel function for unsaturated fatty acids in HCV replication.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells.

Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEBs role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEBs role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathways role in LD dynamics and the VLDL pathway.

microRNA-33 Regulates Macrophage Autophagy in Atherosclerosis

Objective— Defective autophagy in macrophages leads to pathological processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. microRNA-33 (miR-33) is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis. Approach and Results— Using coherent anti-Stokes Raman scattering microscopy, we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression of autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1 (ATP-binding cassette transporter A1)-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis, and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr−/− mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques and promotes apoptotic cell clearance to reduce plaque necrosis. Conclusions— Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.

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.

The role of microRNAs in metabolic interactions between viruses and their hosts.

Productive viral infection requires changes to the cellular metabolic landscape in order to obtain the building blocks and create the microenvironments necessary for the viral life cycle. In mammals, these alterations of metabolic pathways have been shown to be mediated in part by host and virus-encoded microRNAs. To counteract virally-induced changes in the cellular metabolic profile, the interferon-regulated antiviral response restricts viral access to key metabolites by altering cellular metabolism, mediated through induction of specific microRNAs regulating key lipid biosynthetic processes. In this review, we examine recent studies demonstrating the important role of microRNAs in the regulation of metabolic flux during viral infection.

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.