University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 The Cellular Mirna, Mir-190, is Upregulated in Type I Ebv Latency by Ebers and Modulates Cellular Mrnas involved in Cell Survival and Viral Reactivation Elizabeth Mary Cramer University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, Molecular Biology Commons, and the Virology Commons Recommended Citation Cramer, Elizabeth Mary, "The Cellular Mirna, Mir-190, is Upregulated in Type I Ebv Latency by Ebers and Modulates Cellular Mrnas involved in Cell Survival and Viral Reactivation" (2015). Publicly Accessible Penn Dissertations. 1670. https://repository.upenn.edu/edissertations/1670 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1670 For more information, please contact [email protected]. The Cellular Mirna, Mir-190, is Upregulated in Type I Ebv Latency by Ebers and Modulates Cellular Mrnas involved in Cell Survival and Viral Reactivation Abstract Epstein-Barr Virus (EBV) is a highly prevalent human pathogen infecting over 90% of the population. Much of the success of the virus is attributed to its ability to maintain latency through different programs in host cells. MicroRNAs (miRNA) are small, non-coding RNAs capable of post-transcriptionally regulating mRNA expression. A microarray comparison of EBV type I latency and type III latency infected cells yielded evidence of differential cellular microRNA expression. I hypothesized that one of these differentially upregulated type I latency miRNAs, miR-190, is important in maintenance of latency I, and miR-190 upregulation is due to viral gene expression. Lentiviral overexpression systems were used to overexpress miR-190 and a microarray of gene expression revealed candidate miR-190 targets, including: TP53INP1 and NR4A3. The modulation of these targets by miR-190 was confirmed through evaluating mRNA and protein level changes in the presence or absence of miR-190. In the case of TP53INP1, a 3’UTR target site was identified through mutagenesis. The effect of miR-190 expression was evaluated for markers of cell cycle and cell death by flow cytometry, western blot and RT-PCR. Measures of viral reactivation were lowered in the presence of miR-190 after induction by anti-IgG stimulation. I also observed upregulation of miR-190/Talin2 promoter activity or miR-190 expression in the presence of EBERs, Epstein-Barr encoded RNAs. Interestingly, a panel of type I latency cell lines had higher EBER1 expression compared to their type III latency counterparts. Work by others has indicated that EBERs activate the double-stranded RNA (dsRNA) sensor, retinoic acid-inducible gene 1 (RIG-I). Transiently expressed, constitutively activated RIG-I induced miR-190 expression and promoter activity. Knockdown of RIG-I in the type I latency cells yielded lowered miR-190 expression levels. To investigate how miR-190 is upregulated I generated miR-190/ Talin2 promoter reporters that lacked YinYang1 (YY1) and Nuclear factor-κB (NF-kB) binding motifs. In the presence of EBERs, promoters with these deleted binding motifs had lowered activation compared to the full miR-190/TLN2 promoter. This work describes a mechanism by which EBERs upregulate a cellular miRNA, miR-190, which aids in type I latency preservation by preventing apoptosis, promoting cell cycle and maintaining virus in its latent state. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Yan Yuan Keywords EBERs, EBV, miRNA, Type I latency Subject Categories Cell Biology | Molecular Biology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1670 THE CELLULAR MIRNA, MIR-190, IS UPREGULATED IN TYPE I EBV LATENCY BY EBERS AND MODULATES CELLULAR MRNAS INVOLVED IN CELL SURVIVAL AND VIRAL REACTIVATION Elizabeth M. Cramer A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation _____________________ Yan Yuan, Ph.D. Professor of Microbiology Graduate Group Chairperson ________________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell &Developmental Biology Dissertation Committee Paul Lieberman, Ph.D., Hilary Koprowski, M.D., Endowed Professor Robert Doms, M.D., Ph.D., Professor of Pathology and Laboratory Medicine Roselyn Eisenberg, Ph.D., Professor of Microbiology Andrei Thomas-Tikhonenko, Ph.D., Professor of Pathology & Lab Medicine THE CELLULAR MIRNA, MIR-190, IS UPREGULATED IN TYPE I EBV LATENCY BY EBERS AND MODULATES CELLULAR MRNAS INVOLVED IN CELL SURVIVAL AND VIRAL REACTIVATION COPYRIGHT 2015 Elizabeth M. Cramer DEDICATION This thesis is dedicated to my family. They have supported me through my many, many years of graduate school. If I had been told in my first year that I would be married and have a one year old when I defended my thesis I would not have believed it. My husband, Steve, has been my rock. Commuting to New York City while living in Philadelphia is a crazy thing to do, but Steve did just that so we could be together. And for a blessed period of time we both lived and worked in Philadelphia. Now it is my turn to make a move for Steve. Our daughter, Laura, is the best thing that ever happened to us. I want her to know that all things are possible with the love of family and lots and lots of support (both moral and financial). I cannot wait for our next adventures as a family! I owe a great deal to my parents as well. Geoffrey and Lorett Cramer are strong supporters of my graduate school education. When the going gets tough they encourage me to work harder and persevere, which is important in grad school and life. My father is always ready to spend time in Philadelphia and encourages me to relax, live my life and not just stress about what’s going on in lab. My mother has never hesitated to help me in any way possible—with childcare, planning a wedding or deciding what step to take next in life. She also knows when I need some babying myself, but most of the time she encourages me to toughen up. She is the strongest woman I know, a great mom/Bashie and the best grad school cheerleader ever! I love you all and I couldn’t have done it without you! iii ACKNOWLEDGMENT I would like to thank my thesis advisor, Dr. Yan Yuan for his scientific and professional guidance. He made my transition to a new lab in my third year of grad school a smooth one. He also understands the importance of family. He provides a great example of what it looks like to both work hard and be devoted to family. I would also like to thank my thesis committee, Drs. Paul Lieberman, Roselyn Eisenberg, Robert Doms and Andrei Thomas-Tikhonenko for their helpful suggestions and guidance. My lab-mates have been very helpful in the production of the data for this thesis. Dr. Yan Wang and Dr. Ying Shao did the initial Sav I and Sav III microarray comparisons and both contributed to my training in the lab. Dr. Ronghua Meng constructed the EBERs mutant BACMIDs. I am lucky to have these individuals as both colleagues and friends. I would also like to thank Dr. Carolina Pombo and Dr. Christa Heyward for their help editing this thesis. Their guidance through this process has been invaluable. Finally, I would like to thank all the members of my extended family— particularly my mother-in-law, Virginia Nadel. She provided childcare and support over the past year giving me the time to work in the lab and on my thesis. iv ABSTRACT THE CELLULAR MIRNA, MIR-190, IS UPREGULATED IN TYPE I EBV LATENCY BY EBERS AND MODULATES CELLULAR MRNAS INVOLVED IN CELL SURVIVAL AND VIRAL REACTIVATION Elizabeth M. Cramer Yan Yuan Epstein-Barr Virus (EBV) is a highly prevalent human pathogen infecting over 90% of the population. Much of the success of the virus is attributed to its ability to maintain latency through different programs in host cells. MicroRNAs (miRNA) are small, non- coding RNAs capable of post-transcriptionally regulating mRNA expression. A microarray comparison of EBV type I latency and type III latency infected cells yielded evidence of differential cellular microRNA expression. I hypothesized that one of these differentially upregulated type I latency miRNAs, miR-190, is important in maintenance of latency I, and miR-190 upregulation is due to viral gene expression. Lentiviral overexpression systems were used to overexpress miR-190 and a microarray of gene expression revealed candidate miR-190 targets, including: TP53INP1 and NR4A3. The modulation of these targets by miR-190 was confirmed through evaluating mRNA and protein level changes in the presence or absence of miR-190. In the case of TP53INP1, a 3’UTR target site was identified through mutagenesis. The effect of miR-190 expression was evaluated for markers of cell cycle and cell death by flow cytometry, western blot and RT-PCR. Measures of viral reactivation were lowered in the presence of miR-190 after induction by anti-IgG stimulation. I also observed upregulation of miR-190/Talin2 v promoter activity or miR-190 expression in the presence of EBERs, Epstein-Barr encoded RNAs. Interestingly, a panel of type I latency cell lines had higher EBER1 expression compared to their type III latency counterparts. Work by others has indicated that EBERs activate the double-stranded RNA (dsRNA) sensor, retinoic acid-inducible gene 1 (RIG-I). Transiently expressed, constitutively activated RIG-I induced miR-190 expression and promoter activity. Knockdown of RIG-I in the type I latency cells yielded lowered miR-190 expression levels. To investigate how miR-190 is upregulated I generated miR-190/Talin2 promoter reporters that lacked YinYang1 (YY1) and Nuclear factor-κB (NF-kB) binding motifs.