Identification of Novel Compounds That Inhibit HIV-1 Gene Expression by Targeting Viral RNA Processing by Ahalya Balachandran A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Ahalya Balachandran 2015 Identification of Novel Compounds That Inhibit HIV-1 Gene Expression by Targeting Viral RNA Processing Ahalya Balachandran Master of Science Department of Molecular Genetics University of Toronto 2015 Abstract Novel strategies targeting different stages of the HIV lifecycle are vital for continued success in combating viral infection. Since HIV gene expression is dependent upon controlled splicing of the viral transcript, small molecule modulators of RNA processing hold tremendous promise as novel drugs. To this end, we screened splicing modulators for their effect on HIV-1 gene expression. We identified four compounds, 191, 791, 833 and 892, that strongly suppressed accumulation of HIV-1 incompletely spliced RNA and expression of viral structural/regulatory proteins. Furthermore, compound treatment had limited effects on alternative splicing of host RNA splicing events. Subsequent studies confirmed anti-HIV activity of two compounds in the context of peripheral blood mononuclear cells. The distinct effects of these compounds from previously characterized HIV-1 RNA processing inhibitors validate targeting this stage of the virus lifecycle. Elucidating the mechanism by which these compounds alter HIV-1 gene expression holds key insights for novel therapeutic strategies. ii Acknowledgments I would like to thank my supervisor, Dr. Alan Cochrane, for the opportunity to work on this project in his laboratory over the past few years. I would also like to thank my committee members, Dr. Lori Frappier, Dr. Craig Smibert, and Dr. Peter Roy, for their continued guidance and support. It has been a pleasure working with all members of the Cochrane Lab over the past few years. I’d like to thank all the students and post docs for their help and support along the way. Special thanks go to Raymond Wong for taking me under his wing when I was an undergraduate student and for sharing his knowledge and experience about the drug screening projects in our lab. I’d also like to thank Dr. Alex Chen for training me in preparation for working with replicative HIV in the BSL3 facility and the Scott Gray-Owen Lab for source of PBMCs. Last but not least, I’d like to thank our collaborators Dr. Peter Stoilov at West Virginia and Dr. Sandy Pan from the Blencowe Lab for examining the effect of the compounds on cellular alternative splicing. The work presented here would not be possible without funding provided by CIHR grants, as well as the Ontario Graduate Scholarship Award. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices ......................................................................................................................... xi Abbreviations ................................................................................................................................ xii 1 Introduction .................................................................................................................................1 1.1 mRNA processing ................................................................................................................1 1.1.1 mRNA capping ........................................................................................................1 1.1.2 Constitutive splicing and the spliceosome ...............................................................2 1.1.3 Alternative splicing ..................................................................................................2 1.1.4 Polyadenylation........................................................................................................4 1.1.5 RNA export ..............................................................................................................4 1.1.6 Translational initiation .............................................................................................6 1.1.7 Interdependence of events in mRNA processing .....................................................6 1.2 Regulation of mRNA splicing .............................................................................................7 1.2.1 Role of cis elements in splicing ...............................................................................7 1.2.2 Role of trans factors in splicing .............................................................................10 1.2.2.1 SR-protein family of splicing factors ......................................................10 1.2.2.2 Heterogeneous nuclear ribonucleoproteins (hnRNPs) ............................10 1.2.3 Regulation of splicing factors ................................................................................11 1.2.4 Splicing factors and signaling pathways ................................................................12 1.3 Perturbation of alternative splicing in disease ...................................................................14 1.4 HIV-1 utilizes host alternative splicing machinery for viral gene expression ...................15 iv 1.4.1 Overview of the HIV-1 lifecycle ...........................................................................15 1.4.2 Current treatment strategies for HIV-1 ..................................................................16 1.4.3 Limitations of current HIV-1 therapies ..................................................................18 1.4.4 HIV-1 RNA processing..........................................................................................19 1.4.5 Regulation of HIV-1 RNA splicing .......................................................................19 1.4.6 HIV-1 gene expression and Rev-dependent export ...............................................24 1.5 Modulation of RNA splicing as a therapeutic strategy ......................................................27 1.5.1 Modulation of AS using small molecules ..............................................................27 1.5.1.1 Spliceosome inhibitors ............................................................................29 1.5.1.2 Histone deacetylase (HDAC) inhibitors ..................................................29 1.5.1.3 Topoisomerase (Topo I) inhibitors ..........................................................30 1.5.1.4 Kinase and phosphatase inhibitors ..........................................................30 1.6 Effect of splicing modulators on HIV-1 gene expression ..................................................31 1.7 Research objective and rationale .......................................................................................33 2 Materials and Methods ..............................................................................................................34 2.1 HIV-1 provirus doxycycline-inducible cell lines ...............................................................34 2.2 Assess activity of compounds on HIV-1 gene expression .................................................34 2.2.1 Preparation of compounds .....................................................................................34 2.2.2 Compound treatment assay ....................................................................................34 2.3 HIV-1 p24 antigen ELISA .................................................................................................36 2.4 XTT cytotoxicity assay ......................................................................................................36 2.5 Analysis of HIV-1 protein expression ...............................................................................37 2.6 Analysis of HIV-1 RNA expression and localization ........................................................38 2.6.1 RNA extraction and reverse transcription ..............................................................38 2.6.2 Quantification of HIV-1 mRNA expression by qPCR ..........................................38 2.6.3 Analysis of splice site selection within the HIV-1 MS RNA ................................39 v 2.6.4 Analysis of HIV-1 US RNA subcellular localization ............................................40 2.7 Monitoring protein synthesis by SUnSET .........................................................................42 2.8 Viral protein degradation assay .........................................................................................44 2.9 Proteasomal degradation protection assay .........................................................................44 2.10 Analysis of cellular alternative splicing events by RT-PCR .............................................45 2.11 Analysis of cellular alternative splicing by RNA sequencing ...........................................45 2.11.1 Sample preparation for RNA sequencing (RNAseq) .............................................45