Design and Generation of a Recombinant Hepatitis B-Based Viral Vector System for Use in Cellular Receptor Studies BY Robert G. Garces A thesis submitted in conformity with the requirements for the degree Master of Science Graduate Department of Medical Biophysics University of Toronto O Copyright by Robert G. Garces 2001. National library Bibliothèque nationale ofCanada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. nie Wellington Ottawa ON KI A ON4 Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seil reproduire' prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur foxmat électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract The hepatitis B vir~~.~rem- a prevdent global problem despite the availability of recombinant vaccines. One of the greatest challenges facing hepatitis B researchers is the unavailahiiity of an in vitro hepatitis B vins replication system. As a resutt, mitial viral entry steps as well as the cellular receptor for HBV remain undehed. In this study, construction and genmtion of recombuiant hepatitis B viral particles carrying a reporter gene has been attempted. DiBerent immortalized cell lines were tested for their ability to dnve hepatitis B promoters. The enhancecl green fluorescent protein (EGFP) was placed under control of the ciiffirent hepatitis B promoter regions and transfected into Chang Liver, Chinese Hamster Ovary (CHO), HeLa, Hepatoblastoma G2 (Hep G2), Human Hepaîoma 7 @uH-7), Ost-7, and SB cells. Onty Hep G2 and HUEZ-7 cells were capable of driving EGFP expression under d HBV promoters. Recombinant bacdoWuses car- replication-defectke HBV genomes and HBV packaging genes were generated to deliver HBV DNA into Hep G2 and HuH-7 ce&. The ability of these ceil lùies to secrete replication-defective HBV particles was codkmed using specific PCR reactiom and electron microscopy. Further development of this viral systern wodd eventually provide us a usefhi tool to help elucidate the early stages of the hepatitis B vinis replication cycle. This, in turn, will Merthe development of drugs and therapies for those infected with the hepatitis B virus. First and foremost, I would like to thank my parents who have given their unwavering support throughout my lifetime. Since bir-they have shaped my life with their care, love, and support even through the most trying of times. 1 am eternally gratefid for aiI they bave done for me, 1 wouid particularly iike to thank my supervisor, mentor, and fiîend, Chris Richardson, who has opened my eyes Merin the world of science and We. His advice, ideas, and anecdotes have made working under his supervision very mernorable and enjoyable. 1 would also like to thank Dr. GilPrivé and Dr. Dwayne Barber for their support and helpfbi suggestions during my project. Special tbanks to my brother, De& and my love, Linda, for all their encouragement and support over the years. In closhg, 1 would also like to thank Farida Sarangi Jimgyu Diao, and Jason Davis for their assistance with my project and providing advice, both in science and in We. Thanks should also go out to the entire Richardson lab, both past and present, for all the memorable moments gone by. 1. Background and Introduction . .. .. .. 1 1.1. A brief history of modem studies on the hepatitis B virus 1 1.2. The hepatitis B virus WV)structure 2 1.3. The hepatitis B genome 4 1-4. The hepatitis B vins Wecycle 7 1 -4.1. Vira2 attachmenf 7 1 -4.2. Viral entry, uncoating and nuclear transport 9 1 -4.3. Genorne repair and transmption 9 1.4.4. Nucleocapsid assembly 10 1-4.5. Eü3 V DNA replication 11 1.4.6. Budding and secretion of HBVparticZes 13 1 -5.Hepatitis B Proteins 14 1-5. 1. Hepatitis B core and e proteins 14 1 -5-2. Hepatitis B sm-jiace proteins 17 1 -5-3. Hepatitis B poljmerase protein 22 1 -5.4. Hepatr-tis B Xprotein 22 1.6. Hepatitis B epidemiology 25 1.7. Hepatitis B viral expression systems 27 1.8. Use ofbaculovinis as a DNA delivery system 28 1 -9. General research objectives 28 2.MaterialsaodMethods ...................................................30 2.1. Cell lines and virus 30 2.2. Generation of recombinant baculoviruses carrying distinct hepatitis B viral proteins 30 2.3. Bacdovirus Plaque Assay 32 2.4. DNA Sequencing 32 2.5, Anti'bodies 32 2-6. SDS-polyaqIamide gel eletrophoresis and inununo blot andysis 33 2.7. ~ite-s&xinc mutagenesis of hepatitis B virus genorne and prornoter constmct generation 34 2.8. Mutagenized HBV DNA transfections into various cell lines 37 2.9. Fluorescent analysis of transfected ce& 38 2.10. Generation of hepatitis B virus replication defective genome and packaging vectors 38 2.1 1. Transfection of hepatitis B virus replication defective genome and packaging vectors 43 2.12. Generation of recombinant baculovinis carrying the hepatitis B virus repfication- defative genome and packagbg vectors 43 2.13. Amplification and preparation of recombinant bacdovirus stocks 44 2.14. Pseudo-infection optïmization usmg recombinant baculovinis on Hep G2 cells 45 2.15. Flow cytometry analysis of pseudo-infected cell lines 46 2.16. Harvesting HBV particles fiom baculovkus infections of SB, Hep G2 and HuH-7 cells 47 2.17. Harvesting hepatitis B particles ftom transfected HuH-7 cells 47 2.1 8. Sucrose gradient dyskof secreted particles 48 2.19. DNA analysis of concentrated media fiom transfected HuH-7 cells 49 2.20. Southem blot analysis of Hep G2 ceh transfected with defectne genome and packaging vectors 51 2.21. Embedding and sectioning of SB, Hep G2 and HuH-7 for electron microsopic dysis 54 2.22. Tmmunogold labehg of thin-sectimed embedded specimens 56 2.23. Tmmunogold labelhg of concentrated particles nom concentrated cell culture media 56 2.24. Neg&e staining of prepared grids 57 2.25. Electron microscope analysis 58 3.Results ..-.....--.................----.......-.........1.....-....1...,59 3.1. Acknowledgements 59 3.2. Site-specific mutagenesis can be used to analyze hepatitis B promoter strengths in different immortalized cell hes 59 3.3. Generation of hepatitis B Wa1 proteins ushg the bacdovirus expression system 62 3.4. Generation of CMV-promoter driven hepatitis B genomic transcript constmcts in bacdovirus expression vectors and creation of recombinant baculovinises 65 3.5. Hepatitis B protein expression in Hep G2 and HuH-7 cells 66 3.6. Cornparison between Superfeçtm transfections and recombinant baculovirus infections in CHO, Chang Liver, and Hep G2 cek 69 3.7. Effects of varying the mdtiplicity of infection of recombinant baculovkus on EGFP expression 72 3.8. Effects of varying inoculation times of recombinant bacdovirus on EGFP expression 72 3.9. Secretion of hepatitis B Surface proteins following incubation with recombinant baculoMus in Hep G2 cells 74 3.1 0. Secretion of hepatitis B surface proteins after transfection of HBV-bacdoviral vectors in HuH-7 cells 76 3.12. PoZymerase chai.reacfon analysis of media fiom cells infected with hepatitis B Wal genome carryïng recombinant kculov8uç 81 3.13. Electron mîcroscopic anasis of hepatitis B sub-viral particles found in thin sections ofrecornbmaat baculovjrus infected cells 83 3.14. Electron microscope analysis of hepatitis B sub-viral particles found in the media of recombinant bacdovnuS hfiected tek 85 3.15. Eiectron microscope analysis of hepatitis B subviral particles foimd in the media of recombinant baculovird-vector transfected HuH-7 celis 87 Section 1 Figure 1.1. Hepatitis B virus particle types Figure 1.2. Hepatitis B virus genome Figure 1.3. Hepatitis B virus He cycle Figure 1-4. Hepatitis B genome replication Figure 1-5. ORF C and the hepatitis B wre protein domains Figure 1.6. ORF S and the hepatitis B &e protein domains Figure 1-7: Hepatitis B surfàce protein folding models Figure 1.8. ORF P and the hepatitis B po1ymerase protein domains Figure 1.9. ORF X and the hepatitis B X protein domains Figure 1.10. Global dishiution of chronic hepatitis B carriers Section 2 Figure 2.1. Regions of the hepatitis B genome altered by site-directed mutagenesis 35 Figure 2-2.HBV-Bacdovirus constnict design 39 Figure 2.3. HBV-Bacdoviral vector system 41 42 -- - Section 3 Figure 3.1. Expression of EGFP by endogenous hepatitis B viral promoters in 60 - 61 various cell lines Figure 3.2. SB - HBV protein expression Figure 3-3. HepG2 - HBV protem expression Figure 3.4. HuH-7 - HBV protein expression Figure 3-5. Cornparison between SuperFectTMtransfection & recombinant bacdovinis Xection Figure 3.6. MOI and inoculation the variation Figure 3 -7.Secreted samples fiom BacHBV infected HepG2 ce& Figure 3-8. Secreted samples fiom tradected HuH-7 ce& Figure 3.9. Southem blot andysis of DNA samples harvested fiom BacHBV vector transfwted Ha-7 cek Figure 3.10. Detection of DNA using PCR in concentrated media ffom HuH-7 transfected with various HBV constructs Figure 3.11. Electron microscope analysis of SB cells infected with recombinant baculovinis Figure 3.12. Electron microscope analysis of particles derived fiom SBcelis infectecl with recombinant baculovinis Figure 3.13.