A Respiratory Syncytial Virus Replicon That Is Non-Cytotoxic and Capable of Long-Term Foreign Gene Expression DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Olga Malykhina The Integrated Biomedical Science Graduate Program The Ohio State University 2011 Dissertation Committee: Mark Peeples Douglas McCarty Michael Oglesbee Jianrong Li Copyright by Olga Malykhina 2011 Abstract Respiratory syncytial virus (RSV) infection of most cultured cell lines causes cell- cell fusion and death. Cell fusion is caused by the fusion (F) glycoprotein and is clearly cytopathic, but other aspects of RSV infection may also contribute to cytopathology. To investigate this possibility, we generated an RSV replicon that lacks all three of its glycoprotein genes and so cannot cause cell-cell fusion or virus spread. This replicon includes a green fluorescent protein gene and an antibiotic resistance gene to enable detection and selection of replicon-containing cells. Adaptive mutations in the RSV replicon were not required for replicon maintenance. Cells containing the replicon could be cloned and passaged many times in the absence of antibiotic selection, with 99% or more of the cells retaining the replicon after each cell division. Transient expression of the F and G (attachment) glycoproteins supported the production of virions that could transfer the replicon into most cell lines tested. Since the RSV replicon is not toxic to these cultured cells and does not affect their rate of cell division, none of the 8 internal viral proteins, the viral RNA transcripts, or the host response to these molecules or their activities are cytopathic. However, the level of replicon genome and gene expression is controlled in some manner, well below that of complete virus and, as such, might avoid cytotoxicity. RSV replicons could be useful for cytoplasmic gene expression in vitro and in vivo, and for screening compounds active against the viral polymerase. ii Dedication To: Elena Malykhina Serguei Malykine My Pulie iii Acknowledgments I would like to thank Mark Peeples for his guidance throughout my scientific career. Thank you everyone in Peeples’ lab for assisting me in my research through contribution of ideas and help with experiments. Specifically I would like to thank Mark Yednak for starting the replicon project, Charles Rice for the BHK-SR19-T7 cells, Russell and Joan Durbin for help with the IFN assay, Cynthia McAllister for help with flow sorting, Barb Newton and Steven Kwilas for excellent technical assistance, Beth McNally and Emilio Flano for their help with the qPCR, Rachel Fearns for the full-length RSV cDNA clone, D53/BsiWI, and Asuncion Mejias, Blerta Dimo, and Yannis Ioannidis for the help with microarrays. Additionally, I would like to thank Koi and Heather for the fun times we had in the lab together. =) This work was supported by Apath, LLC, and by grants AI047213 and HL051818 from the National Institutes of Health. PLC was supported by the NIAID, NIH Intramural Research Program. iv Vita Bachelor of Science 1998-2002 Major: Biology Minor: Chemistry North Park University Doctor of Philosophy 2004-present Integrated Biomedical Science The Ohio State University Publication Olga Malykhina, Mark A. Yednak, Peter L. Collins, Paul D. Olivo and Mark E. Peeples. 2011. A Respiratory Syncytial Virus Replicon That Is Non-Cytotoxic and Capable of Long-Term Foreign Gene Expression. J Virol, 85:4792-4801. Fields of Study Major Field: Integrated Biomedical Science Minor Field: Molecular Virology and Gene Therapy v Table of Contents Abstract…………………………………………………………………………………...ii Dedication………………………………………………………………………………..iii Acknowledgments………………………………………………………………………iv Vita………………………………………………………………………………………..v List of Illustrations……………………………………………………………………..viii Abbreviations…………………………………………………………………………....xi Chapter 1: Introduction…………………………………………………………………1 Classification…………………………………………………………………….1 The Virion………………………………………………………………………...2 The Viral Proteins……………………………………………………………….2 The F Protein…………………………………………………………….3 The G Protein……………………………………………………………5 The SH Protein…………………………………………………………..7 The M Protein……………………………………………………………8 Replication Complex Proteins………………………………………………9 The M2-1 Protein………………………………………………………11 The M2-2 Protein………………………………………………………12 The NS1 and NS2 Proteins…………………………………………..13 RNA……………………………………………………………………………..14 The RSV Life Cycle……………………………………………………………16 vi Vaccines………………………………………………………………………..20 Passive Immunization…………………………………………………24 Antiviral Agents………………………………………………………...25 Reverse Genetics……………………………………………………………...26 Infection of immortalized cells in culture.……………………………………29 Infection of experimental animals……………………………………………30 Chapter 2: The RSV Replicon………………………………………………………..32 Introduction……………………………………………………………………..32 Materials and Methods………………………………………………….…….36 Results………………………………………………………………………….44 Discussion………………………………………………………………………71 Chapter 3: Application of the RSV Replicon……………………………………..…78 Replicons that also express viral glycoprotein genes………………….…..79 Virion formation by the RSV replicon………………………………………..80 Identifying a viral mechanism(s) that controls the replication of the RSV replicon……………………………………………………………………83 Identifying a cellular mechanism(s) that controls the replication of the RSV replicon……………………………………………………………………84 Replicon-Containing Cell Line for Drug Screening……………………….105 vii List of Illustrations List of Figures Chapter 1: Figure 1.1: RSV life cycle……………………………………………………………..19 Figure 1.2: Launching recombinant green fluorescent protein expressing RSV (rgRSV) from cDNA……………………………………………………………………28 Chapter 2: Figure 2.1: Launch of the RSV Replicon from cDNA………………………………35 Figure 2.2: Derivation of the MP295 replicon cDNA……………………………….46 Figure 2.3: RT-PCR analysis of viral RNA from replicon-containing cells………48 Figure 2.4: Fluorescent photomicrograph of BHK-SR19-T7 cells containing the RSV replicon, as indicated by the expression of GFP……………………………..52 Figure 2.5: Replicon stability in cloned cells passaged with and without blastisidin……………………………………………………………………………….57 Figure 2.6: Replicon-containing cells from a clonal BHK-SR19-T7 population and clear cells that appeared in the culture during passage…………………………...60 Figure 2.7: Diagram showing the position of mutations in Rep295.2 and Rep295.5 genomes……………………………………………………………………63 Figure 2.8: Growth rate of replicon-containing cells compared to parental cells……………………………………………………………………………………...64 Figure 2.9: Level of viral genome and protein in replicon-containing cells compared to rgRSV-infected cells…………………………………………………...67 viii Chapter 3: Figure 3.1: Production of virions by replicon-containing HeLa cells……………..82 Figure 3.2: Gene Trees of expressed genes from replicon-containing A546 cells and RSV infected A549 cells at 24 hr p.i. compared to non-infected A549 control cells……………………………………………………………………………………...88 Figure 3.3: Modular Map of gene expression in replicon-containing A546 cells and RSV infected A549 cells…………………………………………………………91 Figure 3.4: Expression of pro-apoptotic genes in RSV-infected (24 h p.i.) and replicon containing A549 cells………………………………………………………..94 Figure 3.5: Expression of anti-apoptotic genes in RSV-infected (24 h p.i.) and replicon containing A549 cells………………………………………………………..94 Figure 3.6: List of genes highly overexpressed in the replicon-containing A549 cells but not RSV-infected A549 cells (24 h p.i.)…………………………………...96 Figure 3.7: List of genes highly overexpressed in RSV-infected A549 cells (24 h p.i.) but not replicon-containing A549 cells…………………………………………97 Figure 3.8: Replicon Network 1………………………………………………………99 Figure 3.9: RSV Network 19………………………………………………………...100 Figure 3.10: Side-by-side comparison of Replicon Network 1 with RSV Network 19………………………………………………………………………………………101 Figure 3.11: Replicon Network 2……………………………………………………102 Figure 3.12: RSV Network 1………………………………………………………...103 Figure 3.13: RSV Network 2………………………………………………………...104 Figure 3.14: Plasmid containing the RSV replicon cDNA with the Renilla green fluorescent protein (RrFP) gene and the humanized Renilla luciferase (hRLuc) gene……………………………………………………………………………………107 Figure 3.15: Luciferase expression driven by the ts or wt luciferase GS in RSV replicons incubated at 33°C or 38°C and shifted to 33°C………………………..110 ix List of Tables Chapter 2: Table 2.1: Interferon released by replicon-containing A549 cells, determined by bioassay………………………………………………………………………………...70 Chapter 3: Table 3.1: RSV replicon and virion cDNA constructs with a constant number of genes and with the viral glycoprotein genes in their natural positions………….80 x Abbreviations A Adenine AFP Alpha-fetal protein AU Arbitrary Units A549 Type II alveolar epithelial lung carcinoma cell line BHK Baby hamster kidney immortalized cell line BSD Blasticidin S deaminase cDNA Complementary DNA CMV Cytomegalovirus cpRSV Cold-passage RSV CsCl Cesium Chloride Cys Cysteine DAOY IFN-sensitive medulloblastoma cell line DMEM Dulbecco’s modified Eagle medium DNA Deoxyribonucleic acid DRbz Hepatitis D virus ribozyme F Fusion protein F0 Precursor form of the fusion protein F1 Larger subunit of the fusion protein formed after furin cleavage xi F2 Smaller subunit of the fusion protein formed after furin cleavage fbs Fetal bovine serum FI-RSV Formalin-inactivated RSV G Glycoprotein (attachment) G Guanine GAGs Glycosaminoglycans GAPDH Glyceraldehyde-3-phosphate dehydrogenase GE Gene end GFP Green fluorescent