Identifying Host Factors Important for Hantavirus Entry
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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Identifying Host Factors Important for Hantavirus Entry Josiah Petersen University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Virology Commons Recommended Citation Petersen, Josiah, "Identifying Host Factors Important for Hantavirus Entry" (2013). Publicly Accessible Penn Dissertations. 909. https://repository.upenn.edu/edissertations/909 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/909 For more information, please contact [email protected]. Identifying Host Factors Important for Hantavirus Entry Abstract Hantaviruses are a genus of rodent-borne viruses within the family Bunyaviridae. Humans are a dead end host, wherein Hantavirus infection can cause severe, potentially fatal clinical disease. The specific mechanisms of Hantavirus infection are poorly understood. In particular, the host proteins and entry pathways that mediate Hantavirus entry are not yet known. We set out to identify these factors using two separate, but potentially complementary methods, focusing our efforts on a representative Hantavirus - Andes Virus (ANDV). First, using a high-throughput, luciferase-based assay with ANDV pseudotypes, we screened a siRNA library of ~9000 genes to identify host factors involved in ANDV infection. We ultimately identified 9 genes important for Hantavirus entry including SREBF2, a transcriptional regulator of cholesterol uptake. At the same time, a separate genetic screen performed by a collaborator independently identified SREBF2 and other members of the same cholesterol regulatory pathway (SCAP, S1P, S2P). Further experiments using siRNAs and small molecule inhibitors with both pseudotypes and live ANDV, identified an important role for cholesterol in ANDV internalization. Second, we attempted to use the highly characterized NCI-60 panel of cell lines to identify genes whose expression is correlated with ANDV entry. We infected a panel of 32 cell lines with ANDV pseudovirions in a FACS-based assay to measure relative permissiveness and then correlated permissiveness with gene expression using cDNA microarray data for these cell lines. Using this analysis we identified a list of 33 genes that potentially play a role in ANDV entry. Although initial follow-up experiments have failed to validate a subset of these genes, further experiments will be needed to fully test the entire list of candidate genes. Further analysis and experiments combining this list of genes with the cholesterol-dependence of ANDV entry may lead to further identification of specific pathways and factors utilized by Hantaviruses for infection and possibly potential targets for therapy. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Robert W. Doms Keywords Hantavirus, host factor, siRNA screen, virus entry Subject Categories Microbiology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/909 IDENTIFYING HOST FACTORS IMPORTANT FOR HANTAVIRUS ENTRY Josiah E. Petersen A DISSERTATION In Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment for the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation Signature ________________________________________ Robert W. Doms, Chair and Pathologist-in-Chief, Children’s Hospital of Philadelphia Graduate Group Chairperson Signature ________________________________________ Daniel S. Kessler, Associate Professor, Department of Cell Developmental Biology Dissertation Committee: Jeffrey M. Bergelson, Professor of Pediatrics Ronald G. Collman, Professor of Medicine Sara Cherry, Associate Professor Susan R. Ross, Professor of Microbiology Jianxin You, Assistant Professor of Microbiology Acknowledgements I would first like to thank my mentor Bob Doms for his guidance and support. He has provided not only the resources, but also the continual encouragement and the scientific environment that have allowed me to take risks, to fail, to learn, and to succeed. I could not be more grateful for his mentorship and to have experienced the enthusiasm he has for science and care he gives his students. I would especially like to thank Sara Cherry who has been my second thesis mentor in many ways. This project would not be anywhere without her knowledge, guidance, and patience. More than that, she has encouraged and guided me throughout this entire process, providing an invaluable source of ideas, advice, and constructive criticism that have helped me to grow as a scientist and a person. I am thankful to the members of my thesis committee: Ron Collman, Jianxin You, Jeff Bergelson, Susan Ross for their useful suggestions and discussions. I would like thank members of the Doms Lab, both past and present, including Caroline Agrawal- Gamse, Jess Harrison, Chuka Didigu, Zahra Parker, Amber Riblett, Craig WIlen, Lauren Banks, Emily Bruce, Fang-Hua Lee, Meg Laakso, and Val Hardy. I would especially like to thank Meda Higa and Jason Wojcechowskyj, with whom I worked closely on all Bunyavirus-related projects. I would like to thank Paul Bates and his lab, particularly Ken Briley and Mary Jane Drake, with whom we collaborated extensively. I am forever grateful to my parents, brother, and sister for all of the love and support I could need. Lastly, thank you to the close friends that I have made during my time both at W&M and in Philadelphia for their enthusiastic support of all my endeavors, both inside and outside of the lab. ii ABSTRACT IDENTIFYING HOST FACTORS IMPORTANT FOR HANTAVIRUS ENTRY Josiah Enoch Petersen Robert W. Doms Hantaviruses are a genus of rodent-borne viruses within the family Bunyaviridae. Humans are a dead end host, wherein Hantavirus infection can cause severe, potentially fatal clinical disease. The specific mechanisms of Hantavirus infection are poorly understood. In particular, the host proteins and entry pathways that mediate Hantavirus entry are not yet known. We set out to identify these factors using two separate, but potentially complementary methods, focusing our efforts on a representative Hantavirus - Andes Virus (ANDV). First, using a high-throughput, luciferase-based assay with ANDV pseudotypes, we screened a siRNA library of ~9000 genes to identify host factors involved in ANDV infection. We ultimately identified 9 genes important for Hantavirus entry including SREBF2, a transcriptional regulator of cholesterol uptake. At the same time, a separate genetic screen performed by a collaborator independently identified SREBF2 and other members of the same cholesterol regulatory pathway (SCAP, S1P, S2P). Further experiments using siRNAs and small molecule inhibitors with both pseudotypes and live ANDV, identified an important role for cholesterol in ANDV internalization. Second, we attempted to use the highly characterized NCI-60 panel of cell lines to identify genes whose expression is correlated with ANDV entry. We infected a panel of 32 cell lines with ANDV pseudovirions in a FACS-based assay to measure relative permissiveness and then correlated permissiveness with gene expression iii using cDNA microarray data for these cell lines. Using this analysis we identified a list of 33 genes that potentially play a role in ANDV entry. Although initial follow-up experiments have failed to validate a subset of these genes, further experiments will be needed to fully test the entire list of candidate genes. Further analysis and experiments combining this list of genes with the cholesterol-dependence of ANDV entry may lead to further identification of specific pathways and factors utilized by Hantaviruses for infection and possibly potential targets for therapy. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………... ii ABSTRACT……………………………………..……………………………….. iii TABLE OF CONTENTS……………………………………………………....... v LIST OF TABLES…………………………………………………….................. vi LIST OF ILLUSTRATIONS……………………………………………………. vii CHAPTER 1: Introduction……………………………………………………... 1 Hantavirus Background…………………………………………………. 2 General Virus Entry……………………………………………………... 6 Hantavirus Entry Factors……………………………………………….. 9 References…………………………………………………….................... 14 CHAPTER 2: siRNA Screen to Identify ANDV Host Factors……………….. 24 Abstract. ……………………………………………………..................... 25 Introduction……………………………………………………................ 26 Results……………………………………………………......................... 28 Discussion…………………………………………………….................... 58 Materials and Methods…………………………………………………... 62 Acknowledgements……………………………………………………..... 70 References…………………………………………………….................... 71 CHAPTER 3: NCI60 Comparative Genomics Analysis……………………… 75 Abstract……………………………………………………....................... 76 Introduction……………………………………………………................ 78 Results……………………………………………………......................... 86 Discussion…………………………………………………….................... 107 Materials and Methods………………………………………………….. 109 Acknowledgements……………………………………………………..... 109 References…………………………………………………….................... 114 CHAPTER 4: Summary and Future Directions………………………………. 117 Summary……………………………………………………...................... 118 Future Directions……………………………………………………........ 119 References…………………………………………………….................... 129 APPENDIX A: Genes correlated with VSV-VSV ∆G infection..……………… 133 APPENDIX B: Genes correlated