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1. Discovering circulating microRNAs as novel biomarkers for severe human diseases 2. Developing broad-spectrum therapeutic microRNAs for human viral diseases 3. Developing a new mass spectrometry (MS)-based technology for detecting and quantifying mosquito-borne flaviviruses 4. Discovering novel broad-spectrum antiviral agents from natural product libraries
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
The four serotypes of dengue virus (DENV-1–4) are viruses of global concern. Although it is a key step in the lifecycle of these viruses, the host-mediated proteolytic maturation of the structural membrane precursor (prM) glycoprotein is an enigmatic molecular event. Maturation of prM is required for DENV infectivity. This proteolysis is thought to be mediated by human furin, a member of the proprotein convertase family of endoproteases that cleaves a wide variety of host cell molecules and is often hijacked by infectious agents to facilitate their lifecycle. DENV prM maturation is enigmatic for three reasons. First, a cleavage sequence that would be poorly processed by furin has been selected in all four serotypes, resulting in a large proportion of uncleaved immature prM on nascent virus particles. Second, it is unknown whether furin is the sole host enzyme responsible for cleaving prM. Third, while this event has been studied in the context of DENV-2, it is unknown whether the other three serotypes behave similarly with regard to prM maturation rate and its dependence on host furin. Research into these biological questions has been hindered by a lack of molecular tools to accurately quantify DENV-1–4 prM maturation.Here, we developed a novel adaptation of multiple reaction monitoring mass spectrometry (MRM-MS) that uses N-terminal acetyl (NTAc) labelling to differentially quantify cleaved M and uncleaved prM. We applied our NTAc-MRM methodology to determine the relative maturation rate of DENV-1–4 derived from cultured human cells and found significant differences among the serotypes. We also found that prM maturation of DENV-1 does not require active furin. Finally, we applied NTAc-MRM to quantify DENV-1–4 prM maturation in the presence of an adenovirus-expressed serine protease inhibitor (serpin), Spn4A, which stoichiometrically inhibits furin-like proteases. We found that the ER-retained form of Spn4A inhibited DENV-1–4 prM maturation, but a constitutively secreted form of Spn4A produced a robust inhibition of the DENV lifecycle, including intracellular vRNA synthesis, which cannot be explained solely in terms of prM maturation. We therefore hypothesize that host cellular targets of furin-like proteases play an important part in the DENV lifecycle.
Cholesterol and lipid levels are maintained through tightly controlled and complex feedback mechanisms that involve regulation of major metabolic genes. Dysregulation of cellular or plasma lipid levels can lead to a wide range of pathologies, including hyperlipidemia, atherosclerosis and other disorders. A number of viruses, including important human viruses of the Flaviviridae family such as hepatitis C virus (HCV) and dengue virus (DENV), utilize and modulate host lipids to support their lifecycles, and the resulting changes in lipid metabolism may contribute to virus-associated pathologies. The overall aim of this thesis was to determine the role of key regulators of host lipid homeostasis, including microRNAs (miR-122, miR-24 and miR-223) and proprotein convertases (SKI-1/S1P and PCSK9) during viral infection and virus-associated disease.To address this aim, we first examined the molecular interplay between three circulating microRNAs known to act as regulators of lipid homeostasis. The data we present in Chapter 2 shows that specific signatures of the three microRNAs were associated with different treatment outcomes in patients with chronic hepatitis C (CHC), indicating that these microRNAs correlate with HCV infection. We then tested the hypothesis that enzymatic regulators of lipid metabolism could also indicate HCV infection, and in Chapter 3 we show that PCSK9 levels were significantly upregulated in patients who achieved a treatment-based viral cure but not in relapsers. These data indicate that changes in PCSK9 concentrations may have an important role in both HCV infection and in host lipid metabolism. In Chapter 4, we tested whether reducing the abundance of lipid droplets via inhibition of SKI-1/S1P with a small molecule PF-429242 suppresses DENV infection. The inhibitor blocked SKI-1/S1P-mediated accumulation of lipid droplets in hepatoma cells and reduced DENV infection, identifying SKI-1/S1P as a potential target for indirect-acting anti-DENV agents. This study on modulators of lipid metabolism during HCV and DENV infections provides new insights into the complex host-virus interactions that associate with virally-induced disease. We hope that our data lay the foundation for understanding disease pathogenesis and support the development of future strategies for Flaviviridae - associated diseases.
The hijacking and manipulation of host cell biosynthetic pathways by human viruses are shared molecular events that are essential for the viral life cycle. Because of increasing evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in host secretory pathway functions, I hypothesized that this multifunctional enzyme could contribute to life cycles of two Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV).The first aim of this project was to investigate whether GAPDH is a host factor that regulates the life cycle steps of HCV in human hepatoma Huh-7.5.1 cells. I used short interfering RNA (siRNA)-mediated silencing of GAPDH both pre- and post-HCV infection in Huh-7.5.1 cells to demonstrate that reducing GAPDH protein abundance inhibits primary HCV infection and production and/or release of infectious HCV virus particles. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the viral infectivity of HCV-infected Huh-7.5.1 cell supernatants, suggesting a predominant role of GAPDH during the post-replication steps of the HCV life cycle. Finally, siRNA-mediated GAPDH suppression in human Huh-7.5.1 cells also significantly inhibited primary DENV-2 infection.In the second aim, I further investigated the diverse functions of GAPDH in human hepatoma cells by performing differential expression profiling of total cellular proteins by quantitative proteomics in two GAPDH knockdown Huh-7-derived cell lines (67D2 and 67b3) and the parental Huh-7 cell line. First, I successfully established GAPDH knockdown Huh-7-derived cell lines using short hairpin RNA (shRNA) lentivirus particles. Second, I demonstrated that the stable shRNA-mediated GAPDH silencing in Huh-7 cells inhibits primary HCV infection and the production of infectious HCV virus particles. Using a quantitative proteomics strategy based on triplex dimethyl labeling and nano-liquid chromatography-tandem mass spectrometry, I determined the cellular proteins deregulated in 67D2 and 67B3 cells. Bio-informatic analysis of the differentially expressed proteins revealed a robust compensatory effect in molecular functions associated with enzymatic activities and “acting binding” in response to the silencing of GAPDH in 67D2 and 67D3 cells.
Over the past twenty years small endogenous non-coding RNAs known as microRNAs have emerged as potent regulators of gene expression during virus infection. During influenza A virus infection the role of miRNAs and their impact on the virus lifecycle is relatively unknown. With seasonal strains that can result in annual epidemics, to newly emerging subtypes that have the potential to cause a worldwide pandemic, influenza A remains a major threat to global human health. Here we aimed to determine the role miRNAs play in the host-pathogen interactions associated with varying pathogenesis during infection with different influenza A virus strains. In Chapter 2, we tested the hypothesis that human cellular miRNA expression would vary between a low pathogenic swine-origin H1N1 influenza A virus strain and a highly pathogenic avian-origin H7N7 influenza A virus strain. Utilizing high throughput microarray analysis, we identified differentially expressed miRNA and mRNA profiles during H1N1 and H7N7 influenza A infection and found strain specific expression patterns that were associated with specific cellular pathways. One of the miRNAs identified in Chapter 2 was miR-24 that targets the proprotein convertase furin, which is responsible for cleaving the hemagglutinin glycoprotein on the surface of highly pathogenic influenza A viruses. In Chapter 3, we hypothesized that synthetic miR-24 could inhibit highly pathogenic H5N1 influenza A infection. Addition of exogenous miR-24 during H5N1 infection resulted in a significant decrease in furin mRNA expression and enzymatic activity as well as reduced infectious virus released and virus spread. In Chapter 4, we hypothesized that novel miRNAs could target the proprotein convertase furin, along with two additional human proprotein convertases: PCSK9 and SKI-1/S1P, that have significant roles during the lifecycles of other enveloped viruses. We identified a novel miRNA, miR-17, that reduced furin mRNA and enzymatic activity. Furthermore, miR-24 was shown to target PCSK9, potentially contributing to the regulation of lipid metabolism. MiRNAs are now recognized as important players during virus infections, especially during the influenza A virus lifecycle. By exploiting the targets of specific miRNAs, we have identified new potential therapeutic options that could be applied to numerous enveloped viruses.
Hepatitis C virus (HCV) utilizes host lipids for every stage of its lifecycle. HCV hijackshost lipid droplets (LDs) to coordinate assembly through the host lipoprotein assembly pathway;this facilitates uptake into hepatocytes through the low density lipoprotein receptor (LDLR).Induction of host lipid metabolism by HCV supports chronic infection and leads to steatosis,exacerbating liver dysfunction in infected patients. One pathway activated by HCV is the sterolregulatory element binding protein (SREBP) pathway which controls lipid metabolism geneexpression. To activate genes in the nucleus, SREBPs must first be cleaved by host subtilisinkexin isozyme-1/site-1 protease (SKI-1/S1P). Proprotein convertase subtilisin/kexin type 9(PCSK9) is one SREBP-regulated protein that post-translationally decreases LDLR expression inthe liver. The overall aim of this thesis was to determine the potential application of these twoimportant regulators of host lipid homeostasis, PCSK9 and SKI-1/S1P, as targets for inhibitingHCV infection.The first hypothesis tested was that inhibiting SKI-1/S1P would block HCV hijacking ofthe SREBP pathway and limit sequestration of host lipids by HCV, blocking virus propagation.To inhibit SKI-1/S1P function, an engineered serine protease inhibitor (serpin) and a smallmolecule inhibitor were employed. Both inhibitors were shown to block SKI-1/S1P cleavage ofSREBPs, reduce LD accumulation in hepatoma cells and inhibit HCV infection. The nexthypothesis explored was that amplifying PCSK9 expression or function in hepatoma cells wouldincrease their resistance to HCV infection through downregulation of LDLR. It was confirmed,using overexpression of wild-type PCSK9 or treating cells with gain-of-function PCSK9, thatPCSK9 can be used to prevent HCV entry into hepatoma cells. Finally, studies are presenteddetailing the discovery and characterization of a non-inhibitory serpin variant with potentantiviral activity against HCV infection. It is hypothesized that inhibition may be related toantiviral functions exhibited by other human serpins or serpin-derived peptides possessingdiverse regulatory properties.Host lipid metabolism is a critical component of the lifecycle of HCV and many otherviruses. These studies confirm that lipid metabolism pathways can be rationally targeted toinhibit viral infection and may lead to the development of novel, indirect-acting therapies againstHCV and related viruses.
West Nile virus (WNV) is the most widely distributed arthropod-borne virus globally. It can cause a potentially fatal infection and has become a public health concern in North America since its introduction in 1999. Currently, there are no vaccines or treatments available for human WNV infections. As such, it is important to understand the virus life cycle, in order to develop effective therapeutics. The WNV protease heterocomplex, NS2B/NS3, is a prime target for antiviral therapy and has become the focus of much research. It is important to understand protease function first, in order to develop effective inhibitors. The overall goal of this thesis was to gain a better understanding into the function of the full-length NS2B/NS3 protease heterocomplex within the intracellular microenvironment. I hypothesized that there are critical residues essential for the interaction between NS2B and NS3 that affect protease activity and protein stability. The first aim of this project was to generate a cell-based fluorescent substrate assay to investigate the protease activity of the full-length NS2B/NS3 protease heterocomplex within the cell. My results demonstrate that the full-length NS2B/NS3 protease heterocomplex functions differently within the context of the cell, compared to what has been previously observed in vitro (Chapter 2). In the second aim, I investigated NS2B function on NS3 protease cis-cleavage and trans-cleavage activity. My results reveal an important dual role the NS2B protein plays in the proper function of the full-length NS2B/NS3 protease heterocomplex (Chapter 3). In the third aim, I utilized the information gathered to rationally design and test a serine protease inhibitor directed against the full-length NS2B/NS3 protease heterocomplex (Chapter 4). Taken together, my results highlight the importance of utilizing cell-based assays to assess protease activity, as this allows for the investigation of NS2B/NS3 protease function in a more physiologically relevant environment. The results presented in this thesis further our understanding of the activity of the full-length WNV NS2B/NS3 protease heterocomplex within the context of the cell. The information gathered gives insight into the regulation of viral protease function that could be utilized in the rational drug design towards the WNV NS2B/NS3 protease heterocomplex.
The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues.This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively.Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development.In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased.The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found.
Master's Student Supervision (2010 - 2018)
Serpins (Serine Protease Inhibitors) are expressed by most organisms and perform a variety of functions. Most serpins inhibit proteases by undergoing a unique conformational change. They are clinically relevant in two ways. First, introduction of single amino acid point mutations transforms the serpins’ labile conformations into pathogenic, inactive polymers causing “serpinopathies”. In particular, human neuroserpin is a brain-specific serpin that, when mutated, causes a debilitating early onset dementia through unknown cellular pathways. Second, serpins are currently under investigation as therapeutic inhibitors of proprotein convertases (PCs). PCs are associated with some bacterial and viral infections as well as cancer. However, no comprehensive investigation into the cellular effects of PC inhibitor expression in mammalian cells has been performed. This thesis details the use of the Drosophila serpin, Spn4A, to address the cellular pathways mediated by serpin polymers or PC inhibition. Spn4A is a neuron-specific, secretory pathway serpin that inhibits Drosophila or human PCs. We hypothesized that Spn4A mutants, encoding homologous disease-causing mutations in human neuroserpin, would form pathogenic polymers and represent an ideal candidate for generating a cell-based and transgenic Drosophila serpinopathy model. Further, we hypothesized that we could evaluate the cellular response to PC inhibition and polymer accumulation by transcriptome profiling of H4 human neuroglioma cells expressing Spn4A wild-type and mutants. We established an expression system using Spn4A and its mutants in H4s. Subsequently, we used microarray analysis to simultaneously address how serpin polymers may induce cytotoxicity as well as the effects of proprotein processing inhibition in neuroglioma cells. We demonstrated that Spn4A mutants formed polymers, were retained in the endoplasmic reticulum, and lacked inhibitory function, but induced few changes on the transcriptome (under 20 genes differentially regulated). To this end, we have developed transgenic Drosophila overexpressing Spn4A variants to further investigate the biological impact of Spn4A mutants. Next, we analyzed the response to the PC inhibitor, Spn4A, and found marked changes in genes related to malignancy. Our genome-wide gene expression studies have provided novel insights into cellular changes in response to polymeric or PC-inhibiting serpins, and establish the foundation for future functional studies.