Overexpression of the Turnip Crinkle Virus Replicase Exerts Opposite Effects on the Synthesis of Viral Genomic RNA and a Novel Viral Long Non-Coding RNA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shaoyan Zhang, M.S. Graduate Program in Plant Pathology The Ohio State University 2020 Dissertation Committee: Dr. Feng Qu, Advisor Dr. Maria S. Benitez Ponce Dr. Renukaradhya Gourapura Dr. Scott P. Kenney Dr. Tea Meulia Copyright by Shaoyan Zhang 2020 ABSTRACT Plant viruses routinely inflict serious losses in the yield of food and cash crops, ruining the livelihood of farmers, and leading to hunger and malnutrition in many parts of the globe. Our research focuses on a group of viruses known as positive sense (+) RNA viruses that are the most common disease-causing viruses in crops worldwide. All (+) RNA viruses use single-stranded RNAs as the genome to encode a small number of viral proteins on the genomic RNA. Despite the enormous diversity among the proteins these viruses encode, all (+) RNA viruses encode at least one protein, the viral RNA-dependent RNA polymerase (RdRp), that directs the replication of the viral genome. Existing evidence indicates that the expression of RdRp in virus-infected cells is tightly regulated at low levels, but the consequences of perturbing expression of RdRp have not been well understood. Therefore, our research concentrates on understanding how increasing the RdRp expression levels of a (+) RNA virus could affect the viral RNA replication dynamics. In Chapter one, we review the existing literature about RdRps encoded by different (+) RNA viruses. we first give an overview of the replication process of (+) RNA viruses, then summarize various mechanisms that control the expression levels of RdRp. We also touch on viral derived long noncoding RNAs (lncRNAs), which are often 3’ co-terminal with genomic RNAs of (+) RNA viruses. We introduce turnip crinkle virus (TCV), the i primary model virus used in my thesis research. At the end of Chapter one, we raise the question that how increasing the RdRp expression levels of a (+) RNA virus could affect the viral RNA replication dynamics. We hypothesize that overexpression of the p88 RdRp encoded by TCV compromises TCV replication and upsets the relative accumulation levels of different TCV RNAs. This hypothesis is rigorously tested in Chapters two and three. Previous work in our lab found that p28, the auxiliary replication protein of TCV, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wildtype p28. In Chapter Two we showed that p88, the TCV-encoded RdRp, readthrough product of p28, likewise trans-complemented a p88- defective TCV replicon, but repressed the replication of another TCV replicon encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans- complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans- complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a TCV specific lncRNA. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance. Many positive sense (+) RNA viruses encode long noncoding RNAs (lncRNAs) that play important roles in their infections. A distinguishing feature of these lncRNAs is that they are produced through 5'-to-3' degradation of viral genomic or subgenomic RNAs, by ii exoribonucleases of host cells. In Chapter Three, we further investigated the lncRNA discovered in Chapter Two and found that this TCV-borne lncRNA was produced by a replication-based mechanism. This lncRNA, designated tiny TCV subgenomic RNA (ttsgR), was mapped to the last 283 nucleotides of TCV genomic RNA. It accumulated to high levels in cells of Nicotiana benthamiana plants in which TCV replication took place in the presence of overexpressing the RdRp p88. Moreover, ttsgRNA replicated robustly from templates as short as itself, without the need for any other TCV RNAs, as long as both of the TCV replication proteins, p28 and p88, were provided in trans from nonviral sources. Accordingly, both (+) and (-) sense forms of ttsgR were detected using a strand- specific RT-PCR procedure. ttsgRNA replication did not entail any 5’ RNA secondary structure but required the presence of a G3(A/U)4 motif at the 5’ terminus. Furthermore, it strictly relied on the integrity of the CCC motif at the 3’ terminus. Both of these structural features are shared by TCV genomic and subgenomic RNAs. These findings established that ttsgRNA was the product of a replication-based mechanism, and identified a novel strategy for the biogenesis of lncRNAs associated with (+) RNA viruses. In Chapter four, we extend our RdRp research to a distinct (+) RNA flock house virus (FHV), which infects plants, insects, and also mammalian cells. Unlike TCV, FHV encodes one single replication protein – the RdRp – that directs the entire replication cycle. Our preliminary experiments nevertheless demonstrated that FHV RdRp, when over-expressed in N. benthamiana cells from a non-viral source, was able to complement iii the replication of FHV mutants lacking their own RdRp. More importantly, the same RdRp potently repressed the replication of FHV replicons encoding a functional RdRp. Collectively, my thesis research demonstrated that over-expressing RdRps of (+) RNA viruses appears to have a conserved, repressive effect on the replication of the cognate viruses. These results, combined with findings by others in our lab, are consistent with a working model postulating that viral RdRps and possibly other replication proteins, dynamically regulate the viral replication process depending on the intracellular concentration of these proteins. Manipulating the concentration of these proteins could potentially control the replication of viruses. These findings are expected to provide an important guide for novel virus control strategies. iv DEDICATION To my parents for their unconditional love and support, to my advisor for his patient guidance and fruitful help v ACKNOWLEDGMENTS I cannot begin to express my gratitude to my advisor, Dr. Feng Qu, for his guidance, critiques, and advices throughout my PhD studies. Hundreds of hours of discussions with him cultivated my logical thinking and sense of science. His passion and dedication for science are inspiring and set a standard that I will always strive to observe. My deep and heartfelt thanks to my committee members Drs. Maria Soledad Benitez Ponce, Renukaradhya Gourapura, Scott Kenney, and Tea Meulia. It has been my privilege to have had the opportunity to be guided by all of you! Although your own research differs from my focus, you understood my research with a surprising depth, and are always willing to spend time to help and guide me. I am also very grateful to Dr. Monica Lewandowski for her wise suggestions whenever I run into questions and troubles. I’m fortunate to work with previous and current members in Qu’s Lab. I am deeply appreciative of Junping Han, Xiuchun Zhang, Xiaofeng Zhang, Qin Guo, Rong Sun, Xiaolong Yao, Fides Zaulda, Limin Zheng, Camila Perdoncini Carvalho and Tu Huynh for their friendship and collaborations. The assembly line we developed to scale up Nicotiana benthamiana planting will be a legend in our Lab. A steadily growing environment is vital for research that like mine uses plants heavily, I am fortunate to have found the greenhouse and culture rooms are usually well maintained, and for that I am thankful for Bob James for maintaining greenhouse and vi keeping greenhouse supplies handy. Thanks to Lee Wilson for his always readiness to solve any mechanical problem in the greenhouse, culture room and other matters. Thanks to MCIC for technical support in sequencing and confocal imaging. Thanks to the Labs of Drs. Lucy Stewart, Peg Redinbaugh, Sally Miller for generous equipment sharing. Thanks to department for tuition assistance. It wouldn’t have been easy for a foreign student to study and live abroad, if not for the wise counsel, generous help and support of Mark Jones, Jane Todd, Kristen Willie, Jody Whitter, Andrea Kaszas, Maria Elena, Fiorella Carter, Santosh Dhakal, Sankar Renu, Hugo Pantigoso, Luis Huezo, Kaylee South, Chengsong Hu, Yi Han, Pia Golborne, Lu Zhao, Zhenyu Li, Pailing Liu, Yiyun Lin, Timothy Frey, Ram Khadka, Wanderson Moraes, Cecilia Freitas, Hong Hanh Tran, Deogracious Massawe, Brian Hodge, Seyed Mousavi, DeeMarie Troyer-Marty, Sizo Mlotshwa, Wirat Pipatpongpinyo, Mingde Liu, Ke Li, Yixuan Hou, Su, and Daowen Hou. I absolutely treasure our friendship and I am so grateful to have them in my life. And finally, I cannot begin to express my love and gratitude to my mom and dad, who gave me the best education they can ever afford and care for me more than themselves. To my little sister, who always makes me laugh. vii VITA June, 2011…………………...………… B.S. Agriculture, Hainan University, China June, 2014……………. M.S., Molecular Plant Pathology, Hainan University, China July, 2015……………………...… Visiting Scholar, Department of Plant Pathology, Food Animal Health Research Program, The Ohio State University, U.S.A August, 2015 to present................................................. Graduate Research Associate, Department of Plant Pathology, The Ohio State University, U.S.A PUBLICATIONS Guo, Q., Zhang, S., Sun, R., Yao, X., Zhang, X.-F., Tatineni, S., Meulia, T., and Qu, F. 2020. Superinfection exclusion by p28 of turnip crinkle virus is separable from its replication function. Molecular Plant-Microbe Interactions 33:364-375. Sun, R., Zhang, S., Zheng, L., and Qu, F. 2020. Translation-independent roles of RNA secondary structures within the replication protein coding region of turnip crinkle virus. Viruses 12:350. viii Zhang, S., Sun, R., Guo, Q., Zhang, X.-F., and Qu, F.