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UCLA Electronic Theses and Dissertations UCLA UCLA Electronic Theses and Dissertations Title High-Throughput Genetics in Virus Research: Application and Insight Permalink https://escholarship.org/uc/item/2zk3w6n2 Author Wu, Nicholas Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles High-Throughput Genetics in Virus Research: Application and Insight A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular Biology by Nicholas Ching Hai Wu 2015 ABSTRACT OF THE DISSERTATION High-Throughput Genetics in Virus Research: Application and Insight by Nicholas Ching Hai Wu Doctor of Philosophy in Molecular Biology University of California, Los Angeles, 2015 Professor Ren Sun, Chair Traditional genetics, which includes forward and reverse genetics, has been employed extensively to study influenza virus. Although traditional genetics is powerful, it has a limited throughput which only focuses on the linkage of one mutation with one phenotype at a time. In my thesis research, a high-throughput genetics platform is being developed to examine the phenotypic outcomes of all point mutations in a viral gene or genome in parallel. The underlying concept is to randomly mutagenize every nucleotide of an entire genome, monitor enrichment or diminishment of all point mutations under specified growth conditions, and perform massive deep-sequencing to determine which mutations contribute to negative, neutral, or positive outcomes under the given conditions. Using this high-throughput genetics platform, the fitness effects of individual point mutations was profiled across influenza A virus hemagglutinin gene. This technique was further applied to identify novel functional residues and interferon-sensitive mutation. The high-throughput genetics platform can potentially be adapted to study any microbes that can be genetically manipulated. My thesis also describes a novel experimental approach, tag linkage sequencing, to monitor viral quasis- ii pecies. Tag linkage sequencing utilizes a molecular tag to identify short sequencing reads that are from the same original DNA template. This allows the reconstruction of individual viral genomes within a viral quasispecies from deep sequencing data. This approach was employed to investi- gate the genetic content of a clinical sample from a patient infected with human immunodeficiency virus (HIV). iii The dissertation of Nicholas Ching Hai Wu is approved. Eleazar Eskin Matteo Pellegrini James O. Lloyd-Smith Ren Sun, Committee Chair University of California, Los Angeles 2015 iv This dissertation is dedicated to my wife, Janice Han Yuen Lee, for her love, support and encouragement. v TABLE OF CONTENTS ABSTRACT....................................................................................ii DEDICATIONS. .v TABLE OF CONTENTS. .vi ACKNOWLEDGEMENTS . vii VITA...........................................................................................ix PUBLICATIONS. ix CHAPTER 1. INTRODUCTION TO HIGH-THROUGHPUT GENETICS . 1 BIBLIOGRAPHY FOR CHAPTER 1 . .7 CHAPTER 2. HIGH-THROUGHPUT PROFILING OF INFLUENZA A VIRUS HEMAGGLUTININ GENE AT SINGLE-NUCLEOTIDE RESOLUTION . 13 BIBLIOGRAPHY FOR CHAPTER 2 . 32 CHAPTER 3. FUNCTIONAL CONSTRAINT PROFILING OF A VIRAL PROTEIN REVEALS DIS- CONCORDANCE OF EVOLUTIONARY CONSERVATION AND FUNCTIONALITY . 37 BIBLIOGRAPHY FOR CHAPTER 3 . 64 CHAPTER 4. HIGH-THROUGHPUT IDENTIFICATION OF LOSS-OF-FUNCTION MUTATIONS FOR ANTI-INTERFERON ACTIVITY IN INFLUENZA A VIRUS NS SEGMENT . 71 BIBLIOGRAPHY FOR CHAPTER 4 . 92 CHAPTER 5. HIV-1 QUASISPECIES DELINEATION BY TAG LINKAGE DEEP SEQUENCING 97 BIBLIOGRAPHY FOR CHAPTER 5 . 121 CHAPTER 6 PERSPECTIVES . 127 BIBLIOGRAPHY FOR CHAPTER 6 . 133 vi ACKNOWLEDGEMENTS First and foremost, I would like to thank God for all the blessings, opportunities, and people that I have met in my life. I would also like to express my sincere gratitude to my dearest parents for their unconditional love and support throughout the years, and allowing me to realize my own potential. I would like to thank my PhD advisor and mentor, Dr. Ren Sun, for providing me with an excellent atmosphere for doing research, and the freedom he has given me to pursue scientific questions of my own interest. I would not have been able to complete my dissertation without his critical guidance. His motivation, enthusiasm, and immense knowledge have truly inspired me. I am also very grateful to my committee members, Dr. Eleazar Eskin, Dr. Matteo Pellegrini, Dr. David Eisenberg, and Dr. James Lloyd-Smith. Thank you to Dr. Eleazar Eskin for providing me the opportunity to collaborate with him. I truly enjoyed this experience and have learned a lot about algorithm development. Thank you to Dr. Matteo Pellegrini for helpful advice on transcriptome analysis. Thank you to Dr. David Eisenberg for generously granting me the opportunity to rotate with him in my first year and let me utilize his computational resouce for research. Thank you to Dr. James Lloyd-Smith for all the useful comments on my research and helping me to develop my background in evolutionary biology. I would like to thank all the past and present Sun lab members who have created such a dy- namic and joyful research environment. Thank you to Dr. Arthur Young, Dr. Anders Olson, Dr. Laith Al-Mawsawi, Dr. Jun Feng, Dr. Hangfei Qi, Dr. Jiaying Feng, Dr. Roland Remenyi, Dr. Danyang Gong, Dr. Shuai Le, Dr. Xiaojuan Zheng, Yong Hoon Kim, Yushen Du, Harding Luan, Nguyen Nguyen, Kevin Tran, and Tian-Hao Zhang for all the technical and intellectual supports. A special thank you to Dr. Arthur Young for teaching me all the experimental techniques and pro- viding important intellectual input into my research. I would also like to thank all my collaborators. Thank you to Dr. Martha Lewis, Dr. Otto Yang, and Dr. Justin De La Cruz for providing HIV clinical samples for my viral quasispecies project. Thank you to Dr. Alexander Zelikovsky, Dr. Serghei Mangul, Dr. Nicholas Mancuso, and Alex Artyomenko for the wonderful collaboration on algorithm development. Thank you to Dr. Claude Loverdo and Dr. Ruain Ke for the discussion and insight on viral evolution. Thank you to Dr. Stanley Nelson, Traci Toy, Dr. Xinmin Li, Jamie Zhou, and vii Dr. Janice Yoshizawa for their help on performing Illumina sequencing. Thank you to Sugandha Dandekar and Hemani Wijersuriya for their help on performing 454 sequencing. Thank you Dr. Lin Jiang for his help and advice on protein modeling. Lastly, I would also like to thank my family and friends for supporting and encouraging me through- out my life. Without them, I may never have gotten to where I am today. Chapter 2 is adopted from the following paper: Nicholas C. Wu*, Arthur P. Young*, Laith Q. Al- Mawsawi, C. Anders Olson, Jun Feng, Hangfei Qi, Shu-Hwa Chen, I-Hsuan Lu, Chung-Yen Lin, Harding H. Luan, Nguyen Nguyen, Stanley F. Nelson, Xinmin Li, Ting-Ting Wu, and Ren Sun. (2014) High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide res- olution. Scientific Reports 4: 4942. *equal contributors Chapter 3 is adopted from the following manuscript: Nicholas C. Wu, C. Anders Olson, Yushen Du, Shuai Le, Kevin Tran, Danyang Gong, Laith Q. Al-Mawsawi, Hangfei Qi, Ting-Ting Wu, and Ren Sun. Functional constraint profiling of PA influenza virus polymerase subunit at single-amino acid resolution. Chapter 4 is adopted from the following paper: Nicholas C. Wu, Arthur P. Young, Laith Q. Al- Mawsawi, C. Anders Olson, Jun Feng, Hangfei Qi, Harding H. Luan, Xinmin Li, Ting-Ting Wu, and Ren Sun. (2014) High-throughput identification of amino acid residues essential for anti-interferon function in influenza A virus NS segment. Journal of Virology 88(17): 10157-10164. Chapter 5 is adopted from the following paper: Nicholas C. Wu, Justin De La Cruz, Laith Q. Al- Mawsawi, C. Anders Olson, Hangfei Qi, Harding H. Luan, Nguyen Nguyen, Yushen Du, Shuai Le, Ting-Ting Wu, Xinmin Li, Martha J. Lewis, Otto O. Yang, and Ren Sun. (2014) HIV-1 quasispecies delineation by tag linkage deep sequencing. PLoS One 9(5): e97505. viii VITA 2010 B.S., Chemistry - Biochemistry Specialization University of Virginia Charlottesville, Virginia 2013 Advancement to Candidacy for Ph.D. University of California, Los Angeles Los Angeles, California PUBLICATIONS 1. Laith Q. Al-Mawsawi, Nicholas C. Wu, C. Anders Olson, Vivian Cai Shi, Hangfei Qi, Xiaojuan Zheng, Ting-Ting Wu, and Ren Sun. (2014) High-throughput profiling of point mutations across the HIV-1 genome. Retrovirology 11(1): 124. 2. C. Anders Olson, Nicholas C. Wu, and Ren Sun. (2014) A comprehensive biophysical de- scription of pairwise epistasis throughout an entire protein domain. Current Biology 24(22): 2643-2651. (Featured Article) 3. Roland Remenyi*, Hangfei Qi*, Sheng-Yao Su, Zugen Chen, Nicholas C. Wu, Vaithilingaraja Arumugaswami, Shawna Truong, Virginia Chu, Tamar Stokelman, Hung-Hao Lo, C. Anders Olson, Ting-Ting Wu, Shu-hwa Chen, Chungyen Lin, and Ren Sun. (2014) A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins. mBio 5(5): e01469-14. *equal contributors 4. Danyang Gong, Nicholas C. Wu, Yafang Xie, Jun Feng, Leming Tong, Kevin F. Brulois, Hard- ing Luan, Yushen Du, Jae U. Jung, Cun-Yu Wang, Mo Kwan Kang, No-Hee Park, Ren Sun, and Ting-Ting Wu. (2014) Kaposi’s sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication. Journal of Virology 88(19): 11369-11382. ix 5. Nicholas C. Wu, Arthur P. Young, Laith Q. Al-Mawsawi, C. Anders Olson, Jun Feng, Hangfei Qi, Harding H. Luan, Xinmin Li, Ting-Ting Wu, and Ren Sun. (2014) High-throughput iden- tification of amino acid residues essential for anti-interferon function in influenza A virus NS segment. Journal of Virology 88(17): 10157-10164. 6. Serghei Mangul*, Nicholas C. Wu*, Nicholas Mancuso, Alex Zelikovsky, Ren Sun, and Eleazar Eskin. (2014) Accurate viral population assembly from ultra-deep sequencing data. Bioinformatics 30 (12): i329-i337. *equal contributors 7. Laith Q. Al-Mawsawi*, Nicholas C. Wu*, Justin De La Cruz, Vivian Cai Shi, Ting-Ting Wu, Martha J.
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