Rochester Institute of Technology RIT Scholar Works Theses 12-14-2018 Transcriptome-Wide Analysis of VSV Strains with Varying Ability to Block NF-κB Thomas M. Russell [email protected] Follow this and additional works at: https://scholarworks.rit.edu/theses Recommended Citation Russell, Thomas M., "Transcriptome-Wide Analysis of VSV Strains with Varying Ability to Block NF-κB" (2018). Thesis. Rochester Institute of Technology. Accessed from This Thesis is brought to you for free and open access by RIT Scholar Works. It has been accepted for inclusion in Theses by an authorized administrator of RIT Scholar Works. For more information, please contact [email protected]. Transcriptome-Wide Analysis of VSV Strains with Varying Ability to Block NF-κB by Thomas M. Russell A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Bioinformatics Thomas H. Gosnell School of Life Sciences College of Science Rochester Institute of Technology Rochester, NY December 14, 2018 1 Rochester Institute of Technology Thomas H. Gosnell School of Life Sciences Bioinformatics Program To: Head, Thomas H. Gosnell School of Life Sciences The undersigned state that Thomas Russell, a candidate for the Master of Science degree in Bioinformatics, has submitted his thesis and has satisfactorily defended it. This completes the requirements for the Master of Science degree in Bioinformatics at Rochester Institute of Technology. Thesis committee members: Name Date _________________________________________ _________________ Maureen C. Ferran, Ph.D. Thesis Advisor _________________________________________ _________________ Feng Cui, Ph.D. _________________________________________ _________________ Matthew Morris, Ph.D. _________________________________________ _________________ Julie A. Thomas, Ph.D. _________________________________________ _________________ 2 Abstract Vesicular stomatitis virus (VSV) is a negative sense, single-stranded RNA (ssRNA) virus that is currently studied for its uses as a vaccine viral vector and its potential in oncolytic therapy. The M protein of VSV is the active participant in the virus’s abrogation of the host innate immune response. This activity is thought to be due to the M protein’s ability to downregulate overall host transcription by blocking nuclear transport. A VSV mutant containing a D52G mutation in the M protein (22-20) has been shown to block nuclear transport while being defective as a suppressor of NF-κB activation. It is thus believed that while previous M protein mutant viruses are defective in both host transcription suppression and NF-κB activation, 22-20 is deficient in only one of these activities. This study aims to identify differentially expressed genes (DEGs) and pathways through transcriptome analysis of host response to VSV mutants. Our research leads us to believe that 22-20 does in fact down regulate overall host transcription, while being inefficient in suppressing NF-κB mediated immune response pathways. We also discovered important DEGs in the apoptosis pathway, including the antiapoptotic protein Mcl1, where downregulated by 22-20 to a greater degree than another mutant, 22-25. This combination of factors, deficiency in NF-κB suppression and increased apoptotic activation, could make 22-20 an interesting candidate for oncolytic research. 3 TABLE OF CONTENTS ABSTRACT ............................................................................................................................................ 3 INTRODUCTION .................................................................................................................................... 7 INTRODUCTION TO THE VIRUS ..................................................................................................................... 7 THE INFECTIOUS CYCLE OF VSV .................................................................................................................... 9 TYPE I INTERFERON RESPONSE .................................................................................................................. 12 INDUCTION OF AN IFN RESPONSE .............................................................................................................. 12 VIRAL EVASION OF THE IFN RESPONSE ........................................................................................................ 14 VSV AND THE IFN RESPONSE ................................................................................................................... 15 PROJECT FOUNDATION ............................................................................................................................ 16 BROADER IMPACTS ................................................................................................................................. 20 MATERIALS AND METHODS ............................................................................................................... 21 CELLS, VIRUSES, AND INFECTIONS ............................................................................................................... 21 RNA ISOLATION .................................................................................................................................... 21 RNA-SEQ ............................................................................................................................................. 21 DATA QC AND ANALYSIS ......................................................................................................................... 22 INTERNAL RNA-SEQ WORKFLOW .............................................................................................................. 23 DIFFERENTIAL EXPRESSION AND BIOLOGICAL PATHWAY ANALYSIS .................................................................... 23 RESULTS ............................................................................................................................................. 24 MULTIDIMENSIONAL SCALING ................................................................................................................... 24 4 HEATMAP ANALYSIS ............................................................................................................................... 25 VENN DIAGRAM COMPARISON ................................................................................................................. 26 KEGG AND GENE ONTOLOGY ANALYSIS ...................................................................................................... 27 DISCUSSION ....................................................................................................................................... 52 FUTURE WORK ...................................................................................................................................... 57 CONCLUSION ..................................................................................................................................... 57 REFERENCES ....................................................................................................................................... 58 Abbreviations and Acronyms Antigen presenting cells ............................................................................................................... 12 cDNA: Complimentary DNA .......................................................................................................... 21 CEC: Chicken-embryo cells ........................................................................................................... 17 DEG(s): Differentially expressed gene(s) ...................................................................................... 19 dsRNA: double stranded RNA ....................................................................................................... 12 EBOV: Ebola virus ......................................................................................................................... 14 EMEM: Eagle’s Minimum Essential Medium ................................................................................ 21 FDR: False discovery rate .............................................................................................................. 27 G: Glycoprotein .............................................................................................................................. 7 GFF: General feature format ........................................................................................................ 22 GO: Gene Ontology ...................................................................................................................... 48 HS: Horse Serum ........................................................................................................................... 21 5 IFN: Interferon .............................................................................................................................. 12 IRF: Interferon regulating factor ................................................................................................... 13 ISG: Interferon stimulated genes .................................................................................................. 14 IκBs: Inhibitors of κB ..................................................................................................................... 13 L: Large polymerase protein, catalytic ............................................................................................ 7 logFC: Log fold change .................................................................................................................