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UNIVERSITY OF CALIFORNIA RIVERSIDE Transcriptome Analyses of Ascovirus Genome Expression in Lepidopteran Larvae and Host Responses A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Microbiology by Heba Ahmed Hamed Mohamed Zaghloul June 2018 Dissertation Committee: Dr. Brian A. Federici, Chairperson Dr. Ayala L.N. Rao Dr. Adler R. Dillman Copyright by Heba Ahmed Hamed Mohamed Zaghloul 2018 The Dissertation of Heba Ahmed Hamed Mohamed Zaghloul is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENT I would like to thank my major professor Dr. Brian Federici for his guidance and support over the past five years. I have learned much from his research experience and wisdom. He constantly encouraged me during my graduate studies, research, and this dissertation. My discussions with him were always fruitful. His wide knowledge in different areas of science is impressive. I will always be proud that I have been one of his students. Also, I would like to extend my thanks to my dissertation committee members, Dr. Ayala L. N. Rao and Dr. Adler R. Dillman. Your guidance, discussion and feedback were of great importance to my completion of this work. Moreover, I would like to thank present and previous Federici lab members for their help and guidance with mastering different methods and techniques as I carried out my research. I especially thank Robert Hice and Peter Arensburger for their continuous support, guidance, discussions and feedback over the years that helped me develop several skills. In addition, I extend my gratitude to Dennis Bideshi, Hyun-Woo Park and Stephanie Nanneman. I also thank the Fulbright Program, sponsored by the U.S. Department of State’s Bureau of Educational and Cultural Affairs, administered by AMIDEAST in Egypt, my home country, for their financial support for my PhD at UCR from 2013-2017. Being a Fulbrighter is a great experience that I will always remember and appreciate. I hope to be a good representative for the Fulbright program in the future and inspire other Egyptian students to conduct their research and education in U.S. universities, and act as a bridge iv between the two countries through education. In addition, I extend my gratitude to Mr. & Mrs. Johnson for supporting UCR graduate students with the S. Sue Johnson Endowed Graduate Fellowship Award, of which I was delighted to be the recipient during the 2016-2017 academic year. Furthermore, I would like to acknowledge the financial support for my dissertation research through the Graduate Dean’s Dissertation Research Grant in 2017. The text of chapter 2 in my dissertation, in large part, is a reprint of my second chapter that appeared in the published paper, Zaghloul HAH, Hice R, Arensburger P, Federici BA. 2017, “Transcriptome analysis of the Spodoptera frugiperda ascovirus in vivo provides insights into how its apoptosis inhibitors and caspase promote increased synthesis of viral vesicles and virion progeny. Journal of Virology 91:e00874-17. https://doi.org/10.1128/ JVI.00874-17.” The co-authors on this paper provided important assistance and guidance for this dissertation chapter. Moreover, I also extend my acknowledgement to Dr. Yves Bigot, INRA, France, for constructive critical comments on this study. Finally, I would like to thank all my friends in USA and Egypt for their continuous encouragement and support. Last but not least, I would like to thank all my family members especially my parents, sister and brothers. Without your unconditional love, support and encouragement, I would not be who I am today. v DEDICATION I dedicate this work to my beloved family. vi ABSTRACT OF THE DISSERTATION Transcriptome Analyses of Ascovirus Genome Expression in Lepidopteran Larvae and Host Responses by Heba Ahmed Hamed Mohamed Zaghloul Doctor of Philosophy, Graduate Program in Microbiology University of California, Riverside, June 2018 Dr. Brian A. Federici, Chairperson Ascoviruses, family Ascoviridae, are large, enveloped ds DNA viruses that mainly attack lepidopteran larvae of the family Noctuidae causing a chronic, but fatal disease. Their cytopathology differs from other viruses by destroying the nucleus, some species using a caspase, followed by cleavage of the cell into numerous viral vesicles, where most virions are produced. Cell cleavage resembles apoptosis, but differs markedly because mitochondria are not destroyed but rescued by ascoviruses, providing energy for replication and membrane synthesis for viral vesicles and virion envelopes. Ascovirus transmission is also unique in that virions are vectored to hosts on the ovipositor of parasitic wasps. In my research, I used molecular techniques to determine genomic expression patterns underlying ascovirus cytopathology in larvae for two ascoviruses, the Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV). Specifically, through in vivo transcriptome studies, including RNA-Sequencing, qRT- PCR, manual genome curating and bioinformatic analysis, I determined three temporal vi i classes for SfAV and TnAV genes; early, late, and very late. In SfAV, three proteins that inhibit apoptosis were synthesized early, whereas the caspase was synthesized very late, which correlates with apoptotic-like events resulting in vesicle formation. I identified 15 SfAV bicistronic and tricistronic messages, and similar transcripts in TnAV, indicating multi-cistronic transcripts are common in ascoviruses, and may regulate transcription or translation. Analyses of viral vesicle transcripts from Trichoplusia ni hemolymph revealed much higher viral genome expression than in the fat body, epidermis and tracheal matrix, showing larval blood is important for virus reproduction. Host responses to SfAV infection in Spodoptera frugiperda larvae showed mitochondrial gene expression was similar to controls or upregulated slightly during vesicle formation. Interestingly, few cytoskeleton genes known to encode motor proteins were upregulated despite mitochondrial involvement in vesicle formation. Analysis of antimicrobial peptides genes showed several where highly upregulated, as much as 32-fold, for example, moricin and gloverin, whereas innate immunity genes of the Toll, melanization, and phagocytosis pathways were activated only moderately by infection. Phenoloxidase and Toll negative regulators increased 15-fold and 3-fold, respectively. Therefore, conserving mitochondria while balancing immunity gene inducers and repressors may explain the chronic nature of ascovirus diseases. viii TABLE OF CONTENTS CHAPTER I. Literature Review and Research Objectives (Ascoviruses) 1.1 Introduction…………………………………………………………………….. 1 1.2 Ascovirus historical background………………………………………………… 2 1.3 Ascovirus classification………………………………………………………….. 4 1.4 Ascovirus virion structure……………………………………………………….. 5 1.5 Ascovirus virion structural proteins……………………………………………... 7 1.6 Ascovirus genome organization...……………………………………………….. 7 1.7 Gross pathology………………………………………………………………….. 9 1.8 Histopathology and tissue tropism………………………………………………. 10 1.9 Ascovirus cytopathology…...……………………………………………………. 10 1.10 Ascovirus transmission…………………………………………………………. 14 1.10.1 Transmission in nature...………………………………………………… 16 1.10.2 Experimental transmission in the laboratory...………………………….. 17 1.11 Ascovirus strategies for evading host innate immunity and host responses…… 17 1.12 Significance of ascoviruses…………………….………………………………. 22 1.13 Research objectives…………………………………………………………….. 23 1.13.1 Determine the SfAV-1a temporal gene expression profile for conserved core genes and those for putative membrane-associated proteins….. 24 1.13.2 Determine the SfAV-1a-infected host response especially for the mitochondrial, cytoskeleton and innate immunity genes……………………… 25 1.13.3 Comparison of the vesicle-specific transcription patterns in blood versus the fat body, epidermis and tracheal matrix during TnAV host infection 25 1.14 References……………………………………………………………………... 27 ix CHAPTER II. Transcriptome Analysis of the Spodoptera frugiperda Ascovirus In Vivo Provides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny 2.1 Abstract ………………………………………………………………………….. 33 2.2 Introduction ……………………………………………………………………... 34 2.3 Materials and methods...………………………………………………………… 37 2.3.1 Virus strain and host infection…………………………………………….... 37 2.3.2 Total RNA isolation………………………………………………………… 38 2.3.3 Purification and DNase treatment of RNA…………………………………. 38 2.3.4 Enrichment of mRNA, RNA-Seq library preparation, and sequencing…….. 39 2.3.5 SfAV-1a ORFs reannotation based on the RNA-Seq data………………….. 39 2.3.6 Bioinformatic analysis, RPKMs, and read mapping………………………... 40 2.3.7 Promoter motif signal detection…………………………………………….. 41 2.3.8 RT-qPCR validation of the RNA-Seq data ………………………………… 41 2.3.9 Validation of the bicistronic and tricistronic messages…………………....... 42 2.3.10 IRESPred for IRES prediction in intergenic regions…………………….... 43 2.3.11 Accession number(s)……………………………………………………..... 43 2.4 Results……………………………………………………………………............. 43 2.4.1 Editing and reannotation of SfAV-1a genes based on RNA-Seq data……… 43 2.4.2 Cluster analysis of SfAV-1a expressed core and TMD genes……………… 45 2.4.3 Identification of SfAV-1a bicistronic and tricistronic messages………….... 50 2.4.4 Validation of the SfAV-1a bicistronic and tricistronic messages…………... 52 2.4.5 Validation of the RNA-Seq data by RT-qPCR analysis……………………. 52 2.4.6 SfAV-1a promoter