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Proprotein Convertases and Serine Protease Inhibitors: Developing Novel Indirect-Acting Antiviral Strategies Against Hepatitis C Virus

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Proprotein Convertases and Serine Protease Inhibitors: Developing Novel Indirect-Acting Antiviral Strategies Against Hepatitis C Virus PROPROTEIN CONVERTASES AND SERINE PROTEASE INHIBITORS: DEVELOPING NOVEL INDIRECT-ACTING ANTIVIRAL STRATEGIES AGAINST HEPATITIS C VIRUS by Andrea D. Olmstead B.Sc., The University of Saskatchewan, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2011 © Andrea D. Olmstead, 2011 Abstract Hepatitis C virus (HCV) utilizes host lipids for every stage of its lifecycle. HCV hijacks host 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 sterol regulatory element binding protein (SREBP) pathway which controls lipid metabolism gene expression. To activate genes in the nucleus, SREBPs must first be cleaved by host subtilisin kexin 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 in the liver. The overall aim of this thesis was to determine the potential application of these two important regulators of host lipid homeostasis, PCSK9 and SKI-1/S1P, as targets for inhibiting HCV infection. The first hypothesis tested was that inhibiting SKI-1/S1P would block HCV hijacking of the 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 small molecule inhibitor were employed. Both inhibitors were shown to block SKI-1/S1P cleavage of SREBPs, reduce LD accumulation in hepatoma cells and inhibit HCV infection. The next hypothesis explored was that amplifying PCSK9 expression or function in hepatoma cells would increase 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, that PCSK9 can be used to prevent HCV entry into hepatoma cells. Finally, studies are presented detailing the discovery and characterization of a non-inhibitory serpin variant with potent antiviral activity against HCV infection. It is hypothesized that inhibition may be related to antiviral functions exhibited by other human serpins or serpin-derived peptides possessing diverse regulatory properties. Host lipid metabolism is a critical component of the lifecycle of HCV and many other viruses. These studies confirm that lipid metabolism pathways can be rationally targeted to inhibit viral infection and may lead to the development of novel, indirect-acting therapies against HCV and related viruses. ii Preface A version of the research presented in Chapter 2 has been conditionally accepted pending minor revisions to the journal PLoS Pathogens (Olmstead, A.D., Knecht, W., Lazarov, I., Dixit, S. B., Jean, F. (2011) Human subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) is a key host factor for robust HCV infection). In the last year of my PhD, I supervised a co-op student, Ina Lazarov who provided technical assistance for Figure 2.3 C and D and performed the western blots for Figure 2.4 E. The serpin model presented in Figure 1.6, Figure 2.1 and Figure 4.1 was generated with the program pyMOL by Dr. Surjit Dixit (Zymeworks Inc., Vancouver, BC, Canada). Dr. Wolfgang Knecht (Lead Generation - Target Production, AstraZeneca R&D, Mölndal, Sweden) contributed to this work by generating and purifying the recombinant protein SKI-1/S1P used in Figure 2.2 and Figure 4.2. A fellow PhD graduate student, Steven McArthur, performed curve fitting analysis presented in Figure 2.7 A and Figure 3.6 A and stick models generated in pyMOL which are presented in Figure 4.8. All reagents provided by external research groups are indicated in the materials and methods. I designed all the experiments described in this thesis together with my supervisor Dr. François Jean. I conducted all of the research, analyzed the data, and wrote the first drafts of the manuscripts presented in Chapters 2, which was then revised together with Dr. Jean. This work was funded by a CIHR operating grant (F. Jean). Graduate student funding was also provided by the Canadian Institute for Health Research (CIHR-UBC translational research in infectious diseases scholarship and CIHR masters award) and the Michael Smith Foundation for Health Research (MSFHR junior graduate student award). iii Table of Contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations ..................................................................................................................... xi Acknowledgements ...................................................................................................................... xiv Dedication ..................................................................................................................................... xv Chapter 1: Introduction .................................................................................................................. 1 1.1 Hepatitis C virus ........................................................................................................... 1 1.1.1 Emergence and characterization ............................................................................ 1 1.1.2 HCV pathogenesis ................................................................................................. 2 1.1.3 Current antiviral strategies .................................................................................... 3 1.2 The liver and host lipid metabolism ............................................................................. 4 1.2.1 Lipoproteins .......................................................................................................... 4 1.2.2 Low density lipoprotein receptor .......................................................................... 5 1.2.3 Sterol regulatory element binding protein pathway .............................................. 6 1.3 Hepatitis C virus molecular biology ............................................................................. 6 1.3.1 HCV proteins......................................................................................................... 7 1.3.2 The HCV lifecycle ................................................................................................ 8 1.3.3 HCV and host lipid metabolism ............................................................................ 9 1.3.4 HCV hijacking of the SREBP pathway............................................................... 10 1.4 Proprotein convertases regulating lipid homeostasis .................................................. 10 1.4.1 Subtilisin kexin isozyme-1/site-1 protease .......................................................... 11 1.4.2 Proprotein convertase subtilisin/kexin type 9 ..................................................... 11 1.4.3 Host proprotein convertases as antiviral targets .................................................. 12 1.5 Serine protease inhibitors (serpins) ............................................................................ 13 1.5.1 Serpin structure ................................................................................................... 14 1.5.2 Mechanism of action ........................................................................................... 14 iv 1.5.3 Carboxy-terminal serpin activities ...................................................................... 15 1.5.4 The Drosophila melanogaster secretory pathway serpin, Spn4A....................... 17 1.6 Research hypothesis and rationale .............................................................................. 17 1.6.1 Aim 1 ................................................................................................................... 18 1.6.2 Aim 2 ................................................................................................................... 19 1.6.3 Aim 3 ................................................................................................................... 19 Chapter 2: Identification and characterization of the human subtilisin kexin isozyme-1/site-1 protease as a key host factor for hepatitis C virus infection ......................................................... 32 2.1 Introduction ................................................................................................................ 32 2.2 Materials and methods ...............................................................................................
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