Luận án tiến sĩ: Chức năng protein IE2 86 kDa của Cytomegalovirus người

UNIVERSITY OF CALIFORNIA, SAN DIEGO Functional Roles of the Human Cytomegalovirus IE2 86 kDa Protein in HCMV-Infected Cells A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Elizabeth A. White Committee in Charge: Professor Deborah H. Spector, Chair Professor Michael David Professor Daniel J. Donoghue Professor Lorraine Pillus Professor Matthew D.

Weitzman 2007 UMI Number: 3244736 Copyright 2007 by White, Elizabeth A. All rights reserved. UMI Microform 3244736 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 Copyright Elizabeth A. White, 2007 All rights reserved.

The dissertation of Elizabeth A. White is approved, and it is acceptable in quality and form for publication on microfilm: Chair University of California, San Diego 2007 iii DEDICATION For the people who have mattered the most: Mom, Dad, Catherine, and Tony. You have all my love and gratitude. iv TABLE OF CONTENTS Signature Page…….

iv Table of Contents. v List of Figures. xiii Abstract of the Dissertation. 1 HCMV Regulatory Factors.

7 Interactions Between HCMV and the Host Cell.19 Bacterial Artificial Chromosome-Mediated Mutagenesis of the HCMV Genome .23 Goal of the Work.31 Small Internal Deletions in the Human Cytomegalovirus IE2 Gene Result in Non-Viable Recombinant Viruses with Differential Defects in Viral Gene Expression.33 Materials and Methods .75 The IE2 60 kDa and 40 kDa Proteins are Dispensable for Human Cytomegalovirus Replication, but are Required for Efficient Delayed Early and Late Gene Expression and Production of Infectious Virus.77 Materials and Methods .113 Exon 3 of the Human Cytomegalovirus Major Immediate Early Region is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation.115 Materials and Methods .172 Alteration of the IE1 72 3’ UTR Leads to Up-Regulation of RNA and Protein Expression Consistent With a Defective microRNA-Target Interaction .174 Materials and Methods .205 vi LIST OF FIGURES Figure 1.1 Major events in HCMV replication.2 Predominant immediate-early loci in the HCMV genome.3 Major IE region mutants relevant to the dissertation.1 Construction of the HCMV IE2 86 deletion mutant BACs.2 Increased IE1 and IE2 transcription at IE times in cells electroporated with the IE2 86 mutant BACs.3 IE1 72 and IE2 86 expression in wild-type- and IE2 86 mutant BAC- electroporated cells at 1 day postelectroporation.4 IE1 72 and IE2 86 expression in wild-type- and IE2 86 mutant BAC- electroporated cells at 9 days postelectroporation.5 Reduced UL112-113 transcription in IE2 86 mutant BAC-electroporated cells.6 UL112-113 and UL44 expression in IE2 86∆356-356 BAC-electroporated cells.7 UL89 and R160461 transcription is increased at IE times in mutant BAC- electroporated cells.8 pp28 expression in mutant BAC-electroporated cells.1 Construction of the IE2 40 and IE2 60 deletion mutant viruses.2 Major immediate-early protein expression is altered following infection with IE2 ∆40 and IE2 ∆60 viruses.3 Deletion of IE2 40 and IE2 60 reduces virus production following high- multiplicity infection.4 IE1 72 and IE2 86 RNA levels are altered in deletion mutant virus-infected cells.5 Early viral protein expression is not altered following infection with IE2 ∆40 and IE2 ∆60 viruses.6 UL84 and pp65 protein expression is reduced following infection with IE2 ∆40 and IE2 ∆60 viruses.7 UL83, but not UL84, RNA levels are altered in deletion mutant virus- infected cells.1 Construction of the IE ∆30-77 mutant BAC.2 IE ∆30-77 virus replicates with a kinetic defect not rescued by wild-type IE1 72 protein provided in trans.3 Early viral gene expression is delayed following low-multiplicity infection of HFF with IE ∆30-77 recombinant virus.4 The kinetics of viral DNA replication in IE ∆30-77 virus-infected cells follow the increase in viral early gene expression.5 IE and early protein expression is altered in IE ∆30-77 virus-infected cells.6 IE1 72 protein expression is not maintained and viral early protein expression is delayed during high multiplicity infection with IE ∆30-77 virus.7 IE1 72 and UL44 proteins are not efficiently expressed at the same time in IE ∆30-77 virus-infected cells.8 Viral gene expression is delayed in IE ∆30-77 virus-infected cells.9 Altered expression of cell cycle regulatory proteins in IE ∆30-77 virus- infected cells.10 PML is not dispersed following infection with IE ∆30-77 virus.1 Cells transfected with the IE2 86 truncation mutant construct express increased IE and late transcripts, but not viral early genes.2 Cells infected with the IE2 86 truncation mutant virus overexpress IE1 72 protein, but do not express early or late viral proteins or stabilize APC substrates.3 The IE2 86∆88-135 and IE2 86∆88-290 viruses are viable and express early and late viral genes.4 Cells infected with the IE ∆30-77 mutant virus express elevated levels of p53 protein and continue to express p21 protein.1 The HCMV major IE region contains a predicted target site for the HCMV miRNA UL112-1.2 Changes to the UL123 3’ UTR result in increased IE1 72 protein expression, but not increased IE2 86 protein expression.3 Changes to the UL123 3’ UTR result in increased UL123 transcript levels.4 Changes to the UL123 3’ UTR result in decreased production of infectious virus.5 Eliminating the expression of IE2 60 does not alter levels of IE1 72 protein.195 ix ACKNOWLEDGEMENTS This work could never have been completed without the help of many people, both in the Spector lab and elsewhere. I am incredibly lucky to have had a constant source of encouragement and enthusiasm, an insightful critic of scientific results, and a formidable role model in my advisor, Deborah Spector. I am sure that the opportunities, experiences, and training I received in her lab have been truly unique and worthwhile. Thank you also to my thesis committee members.

My work has been significantly strengthened by your suggestions. I am constantly thankful for the help and support I have received from my fellow Spector lab members. To Veronica Sanchez, Chuck Clark, and Chris Morello, who have taught me how to do almost everything I can in the lab, thank you for your help, friendship, suggestions, and moral support. Special thanks go in particular to Chuck, without whom the construction of the HCMV recombinants that form the basis of this dissertation would never have been possible, and to Ronnie, who has never been too busy to offer suggestions and help with experiments and results.

In the latter part of my time in the lab, I have benefited tremendously from working with Rebecca Sanders and Christia Del Rosario, whose continuing work on the IE2 project is very promising. Thank you also to the many past and present members of the lab with whom I did not work directly, in particular Jeff Mahr, Ming Ye, Anokhi Kapasi, and Karen Tran. I have truly learned from everyone who has come through the lab. I have also been very fortunate to work with Finn Grey and Jay Nelson of Oregon Health and x Science University, and several results that have come out of our collaboration are described in the appendix.

My work in the lab has been possible only because of the support I have received from my family. Thank you to my parents: my mother, who will always be my first biology teacher, and my father, who is my best example of what it means to be a scientist. I am truly grateful for their support of my decision to attend UCSD and for their encouragement ever since. Thank you to my sister, Catherine, for your friendship, laughter, and for always believing in me.

Finally, my very deepest appreciation goes to Tony, for his constant and unwavering support during my last three years in graduate school. I am thankful every day for your love and friendship, for the experiences we have had together, and for your presence in my life, and I will always owe you a tremendous debt of gratitude. The text of Chapter 2, in full, is a reprint of the material as it appears in Journal of Virology, 78:1817-1830, 2004. Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression.

The dissertation author was the primary investigator and author of this paper. The text of Chapter 3, in full, is a reprint of the material as it was accepted for publication in Journal of Virology, December 2006. The IE2 60 kDa and 40 kDa proteins are dispensable for human cytomegalovirus replication, but are required for efficient delayed early and xi late gene expression and production of infectious virus. The dissertation author was the primary investigator and author of this paper.

The text of Chapter 4, in full, is a reprint of the material as it appears in Journal of Virology, 79:7438-52, 2005. Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation. The dissertation author was the primary investigator and author of this paper. xii VITA 1999-2001 Undergraduate Research Assistant, Dartmouth College 2001 Bachelor of Arts, Dartmouth College 2001-2007 Graduate Student Research Assistant, University of California, San Diego 2007 Doctor of Philosophy, University of California, San Diego PUBLICATIONS Ozkan, M.

(2001) Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria. (2001) Absorbance of opaque microstructures in optically diffuse media. (2004) Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression. (2005) Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation.

Early viral gene expression and function. In: Human herpesviruses: biology, therapy, and immunoprophylaxis. Cambridge University Press. The IE2 60 kDa and 40 kDa proteins are dispensable for human cytomegalovirus replication, but are required for efficient delayed early and late gene expression and production of infectious virus.

Accepted for publication in Journal of Virology, December 2006. xiii FIELD OF STUDY Major Field: Biological Sciences Studies in Molecular Virology Professor Deborah H. Spector xiv ABSTRACT OF THE DISSERTATION Functional Roles of the Human Cytomegalovirus IE2 86 kDa Protein in HCMV-Infected Cells by Elizabeth A. White Doctor of Philosophy in Biology University of California, San Diego, 2007 Professor Deborah H.

Spector, Chair The human cytomegalovirus (HCMV) IE2 86 kDa protein is an essential viral regulatory factor that has been shown in transient transfection and in vitro assays to transactivate early viral promoters and to interact with many viral and cellular proteins. To understand the functions provided by this protein in the HCMV-infected cell, we have constructed and characterized a family of recombinant viruses xv containing changes to the IE2 gene and other parts of the HCMV major immediate early (IE) region. The study of these HCMV mutants has allowed us to confirm that several of the predicted functions of IE2 86 are relevant in the virus-infected cell and has identified new functions for the protein. Introducing small deletions into the C-terminus of IE2 86 resulted in viruses that do not support early gene expression, replicate, or repress the major IE promoter.

Surprisingly, these constructs also mediated up-regulation of several delayed early and late viral genes, suggesting that IE2 functions are required for the proper regulation of late viral gene expression. By constructing several viruses that do not express the IE2 40 and IE2 60 kDa proteins, which are present in infected cells at late times post infection, we continued to investigate the regulation of late gene expression by IE2. Again, we found that the IE2 40 and IE2 60 proteins are required for proper late gene expression and for repression of the major IE promoter. Interactions between the virus and the host cell are also crucial for proper HCMV replication, and a recombinant virus with a deletion in exon 3 of the major IE region demonstrated that IE2 86 is important not only for transactivation of viral early promoters, but also for dysregulation of the host cell cycle.