University of Alberta Functional Studies of HIV-1 Vpr Protein and Developrnent of hu-PBL-SCIDheige Mouse Mode1 for the Studies of HIV-1 Infection in vivo Chengsheng Zhang @ A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of DOCTOR OF PAILOSOPHY Department of Medical Microbiology and Immunology Edmonton, Alberta Spring, 1998 Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. nie Wellington Ottawa ON K 1A 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 à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distxibuer ou copies of this thesis in microforni, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownershp 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 fkom it Ni la thése 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 Infection of human CD4+ lymphocytes with human irnmunodeficiency virus type 1 (HTV-1) ofien leads to cell death and persistent infection rarely occurs in tissue culture. In this study, several chronically infected ce11 lines were esrablished. Mutations with HTV-lvpr gene occurred and no Vpr expression was detected in ail the chronically infected ce11 lines, suggesting that Vpr may play a critical role in preventing the establishment of penistently infected ce11 lines. A conditional expression system has been used to charactenze the effects of HIV-1 Vpr, HIV-2ISN Vpr and Vpx in the fission yeast Schizosaccharornyces pombr (S. pombe ). Vpr or Vpx could inhibit cell proliferation and induce ce11 cycle arrest at G 11s and G2/M phases in S. pombe. In addition, Vpr or Vpx were found to be cytotoxic in S. pornbe. HZV-1 Vpr-induced growth inhibition and cytotoxicity were mapped to the C-terminal region of Vpr. Funher studies showed that p34kdc2 kinase activity was apparently suppressed in yeast cells expressing Vpr or Vpx, indicating that Vpr and Vpx rnay induce ce11 cycle amest through a p34Icdc2 pathway. We have also shown that three p34kdc-7 mutants including cdc2-3w, cdc2- l w, and cdc2- lw, Amilcl were susceptible to Vpr-induced ce11 cycle arrest, suggesting that Vpr rnight not act directly on the Cdc25, Weel or Mikl. HIV- 1 NCp7 was able to slow down the ce11 cycle progression in S. pornbe. Two Vpr-resistant mutant yeast strains were genented by EMS mutagenesis. We have atternpted to identify Vpr-interacting proteins by the screening of yeast genomic DNA Libraries. However, we were unable to identify any target protein. We have developed a hu-PBL-SCIDheige mouse model. High levels of human irnrnunoglobulins and lymphocytes were detected in the reconstituted mice. These rnice could be infected by cell-Free HIV-1 viruses. Reversal of CD4lCD8 ratio was obsemed in most of the HN- 1 infected rnice. In addition, this mouse model was used to study anti-HIV-1 protective irnmunity in vivo by which we have provided evidence suggesting that some multiply exposed, HIV- 1 seronegative individuals have protective immunity to HIV- 1 infection. Acknowiedgments 1 would like to take this opportunity to extend my sincere appreciation to ail the individuals who have provided me help and support during my Ph.D training program. First, I would like to express my deepest gratitude to my mentor, Dr. Lung-Ji Chang, who has provided me ail the available facilities and reagents for my research, dedicated supervision, encouragement, and continuous support, which made it possible for the completion of my Ph.D program. I am also very grateful to my CO-supervisor, Dr. Larry Guilbert, who has provided dedicated and excellent supervision during the final stage of my Ph.D program when my supervisor Dr. Chang was on his sabbatical year at the University of Flonda. 1 want to extend my special thanks to my former supervisory cornmittee member, Dr. Colin Rasmussen, who has introduced me the yeast expression system and allowed me to work on the yeast project in his laboratory. 1 also want to thank Our collaborator. Dr. Stan Houston for identiQing the high-risk, seronegative individuals and collecting the blood sarnples. 1 would like to thank al1 the members of my supervisory and final defense committee, Drs. Hanne Ostergaard, Manijeh Pasdar, Dennis Kunimoto. and Dr. James Srniley for the tirne and efforts they have spent on discussing my thesis research projects, providing ideas and suggestions, and giving critical readings of my thesis. A special thank goes to Dr. Harris Goldstein for making a tirne-table from his busy schedule to serve as my extemal examiner on my thesis and final defense. 1 would like to thank Drs. John EIIiott and Peter Dickie for their continuous help, support, and discussion on my research projects. Dr. Elliott has also provided me the plasrnid FE14 for the construction of pCAl plasmid. 1 would also like to thank dl my colleagues at the Glaxo Heritage Research Institute at the University of Alberta, especially Mrs. Vicki Urlacher, Mr.s Anne Hudson, Mrs. Fei Zhong, Dr. Yan Cui, Mrs. Deborah Robinson, Mr. Carlos Chen, and Dr. Alexy Atrazhev for their help and fnendship. 1 am also very grateful to Dr. Marissa Wohllebe at the general office of MM1 for her continuous help and suppon throughout my Ph.D program. 1 am indebted to rny father Mr. Xinzhen Zhang and my mother Mn. Giuying Qiu: my brothers and sisters Mrs. Ximei Zhang, Mr. Huosheng Zhang, Mr. Kai Zhang, Mr. Xuan Zhang, and Mrs. Xiying Zhang; my father-in-law Mi Dewu Wang, and my mother-in-law Mrs. Fengzhi Hou for their love, support, and encouragement. Finally, special thanks to my wife Liping Wang, my son Jeffery Yufeng Zhang, and my lovely daughter Jennifer Shijia Zhang for their love, support, and patience. In particular, my wife Liping who has shared most of the responsibilities on taking care of our two children and doing the house work. During my Ph.D training program, 1have been supported by the University of Alberta Ph.D scholarship, Alberta Provincial Government Scholarship, and a full-time studentship From the Alberta Heritage Foundation for Medical Research for which 1 am very grateful. TABLE OF CONTENTS Chapters Page Introduction HIV- 1 and lentiviruses ...................................................................... 1 Genome structure of HIV and SiV........................................................ 1 ... Life cycle of HIV- 1 repiication .............................................................-7 HIV-1 infection in vivo..................................................................... 3 Acute HIV- 1 infection ...................................................................... 4 Persistent HIV- 1 infection and Long-Term Non-progressors .......................... 5 High-risk, multiply exposed, HN- 1 seronegative individuals ........................ -5 HIV-1 infection of human CD4+ cells in vitro ........................................... 6 Tropism of HIV- 1........................................................................... 7 Acute HIV- 1 infection ........................................................................ 8 Persistent HIV- 1 infection .................................................................... 9 HIV-1 Vpr ................................................................................... 10 Possible roles of Vpr in viral infection and replication .............................. 10 . Virion-association. of Vpr ................................................................... 10 Nuclear localization of Vpr and its related functions.................................. Il Effect of Vpr on the transcription of LTR .............................................. 11 Effect of Vpr on viral infection and replication............ ... .................. 11 Vpr and ce11 cycle progression ............................................................ 12 Ce11 cycle regulation in eukaryotic cells.................................................. 12 Ce11 cycle state and HIV- 1 replication .................................................... 15 Effect of Vpr on host ce11 proliferation and ce11 cycle progression ................... 16 Potential rote of Vpr in the pathogenesis of HIV- 1 infection in vivo............... 16 Animal modets of AIDS ................................................................... 17 SIV-induced simian AIDS mode1............................. .... .................... 17 FIV-induced FAIDS mode1....................... .. .................................... 18 HIV- 1 infected Chimpanzee mode1...................................................... 18 1 1 murine models .................................................................... -19 SCID mice .............................................................................. 19 SCID-hu mouse model ................................................................... -21 hu-PBL-SCID mouse mode1................................. .. 33 Rationai, hypothesis, and objectives .................................................... 14 Figures and Tables ........................................................................