Role of Rna Genome Structure and Paraspeckle Proteins in Hepatitis Delta Virus Replication

ROLE OF RNA GENOME STRUCTURE AND PARASPECKLE PROTEINS IN HEPATITIS DELTA VIRUS REPLICATION Yasnee Beeharry Thesis submitted to the Faculty of Graduate and Postdoctoral Studies In partial fulfillment of the requirements For the PhD degree in Biochemistry Thesis Supervisor: Dr Martin Pelchat Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Yasnee Beeharry, Ottawa, Canada, 2016 ABSTRACT The Hepatitis Delta Virus (HDV) is an RNA pathogen that uses the host DNA-dependent RNA polymerase II (RNAP II) to replicate. Previous studies identified the right terminal domain of genomic polarity (R199G) of HDV RNA as an RNAP II promoter, but the features required for HDV RNA to be used as an RNA promoter were unknown. In order to identify the structural features of an HDV RNA promoter, I analyzed 473,139 sequences representing 2,351 new R199G variants generated by high-throughput sequencing of a viral population replicating in 293 cells. To complement this analysis, I also analyzed the same region from HDV sequences isolated from various hosts. Base pair covariation analysis indicates a strong selection for the rod-like conformation. Several selected RNA motifs were identified, including a GC-rich stem, a CUC/GAG motif and a uridine at the initiation site of transcription. In addition, a polarization of purine/pyrimidine content was identified, which might represent a motif favourable for the binding of the host Polypyrimidine tract-binding protein-associated-splicing-factor (PSF), p54 and Paraspeckle Protein 1 (PSP1). Previously, it was shown that R199G binds both RNAP II and PSF, that PSF increased the HDV levels during in vitro transcription and that p54 binds R199G. In the present study, I showed that PSP1 also associates with HDV RNA and I investigated whether these proteins are required for HDV replication. My results show that knockdown of PSF, p54 and PSP1 resulted in a decrease of HDV accumulation. These proteins are highly concentrated in paraspeckles, which are nuclear structures involved in storage of transcripts generated by RNAP II. I found that upon viral replication in 293 cells, PSP1 appeared as bigger foci present outside of the nucleus, while PSF and p54 foci remained in the nucleus. NEAT1 is a long non-coding RNA essential for the formation of paraspeckles. Upon HDV replication, I found an increase of the intensity and size of NEAT1 foci that correlates with an increase of NEAT1 transcripts. ii Altogether, these data suggest that HDV replication results in an alteration of the paraspeckles structures, providing foundation for further investigation of the paraspeckles role in HDV cycle. Overall, the present study addresses the importance of the HDV RNA structure and of the host paraspeckle proteins for HDV replication. iii ACKNOWLEDGEMENTS I would like to thank Dr Martin Pelchat for his mentorship, his enthusiasm for science (from tiny RNAs to the solar system), for the many learning opportunities I had during my PhD and for the time he invested in teaching bio-informatics. I am sincerely thankful to my thesis advisory committee members, Dr Alain Stinzi, Dr Jean- François Couture and Dr Derrick Gibbings, as well as Dr Earl Brown for their guidance and their input. I am grateful to Dr Jocelyn Côté’s laboratory and CBIA Core for their advice and help for immunofluorescence and microscopy techniques. I am grateful to previous and current members of the laboratory for their support and good humour. Thank you Lynda Rocheleau for your kindness and for your help with the bio-informatics. To DJ and Gabrielle, thank you for always being willing to help and your sense of humour. I am indebted to you Dorota, for your invaluable support during the long days of experimental optimization, for the scientific advice, the daily help and for being such a great friend. To the hard-working and passionate people I met in this research community, I will not enumerate you all because the list would be too long, but I address you a deep thank you for the insightful scientific discussions, help and for the good laughs. I have to mention the very exceptional people that are my friends Mohammed, Helina, Céline and Nicole, that were always by my side through my long PhD journey, with the magic of your conversations, laughs and your support, you filled my graduate student life with joy. To my dearest friends Dorota, Alexis, Helina and Julien, I am grateful to you for the proofreading of my thesis. To my precious family and friends, here and overseas, I have always been blessed with your unconditional love, support and understanding when I was always busy with work all these years. Even if I spent most of my hours and days in another world, working in the laboratory or on my computer, thanks for the talks that kept me always connected to the world outside of the lab. A Rubin, Reedima et mes beaux-parents, merci de toujours veiller sur moi, l’amour et les perpétuels encouragements. Merci maman Joanne pour tous les bons petits plats et de m’avoir conduit pendant cette dernière année où j’étais blessée au genou. A toi mon amour, merci pour ta patience et ton inébranlable positivité. Un mari comme toi est une telle force motrice pour affronter les obstacles en tout genre et toujours persévérer pour de bonnes causes. “Ce n’est qu’au prix d’une ardente patience que nous pourrons conquérir la cité splendide qui donnera la lumière, la justice et la dignité à tous les hommes. Ainsi la poésie n’aura pas chanté en vain.” (Rimbaud) Merci de m’avoir (au sens propre et figuré) soutenue et de toujours me rappeler que tout est une question d’équilibre. “De temps en temps il est bon d’arrêter notre quête du Bonheur et d’être tout simplement heureux.”(Apollinaire) iv A vous mes très chers parents, je vous aime. Merci de m’avoir donné l’opportunité de poursuivre ces longues études et de m’avoir motivée sans relâche. Maman, tu es mon exemple de force morale, merci pour avoir partagé toutes les peines et joies durant mon doctorat et d’avoir toujours été de bon conseil. A mon formidable papa qui a toujours été là pour moi, j’admire ton dur labeur et tes grands accomplissements, tu es ma source d’inspiration. v Table of Contents ABSTRACT .......................................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................................. iv Table of Contents ............................................................................................................................ vi List of Abbreviations ..................................................................................................................... ix List of Figures ................................................................................................................................ xiii List of Tables ................................................................................................................................. xiv Chapter1: INTRODUCTION ........................................................................................................... 1 1.1 Epidemiology and pathogenesis ................................................................................................... 2 1.2 Classification, phylogeny and evolution .................................................................................... 3 1.3 Virion structure and viral antigen modifications ................................................................... 4 1.4 Symmetrical rolling circle replication ........................................................................................ 8 1.5 Models used to study HDV replication ..................................................................................... 11 1.5.1 Study of HDV replication in cellular culture ................................................................................. 11 1.5.2 Pathogenesis in cellular culture......................................................................................................... 12 1.6 Evolution rate of HDV ..................................................................................................................... 12 1.7 Involvement of the RNA Polymerase II in replication and transcription .................... 14 1.7.1 RNAPII pre-initiation complex and DNA promoter features in the human cell ............ 14 1.7.2 RNAP II used by HDV.............................................................................................................................. 16 1.7.3 RNAP II RNA promoter features ........................................................................................................ 17 1.8 Localization of the HDAg-S and the HDV genomes in the host cell and interaction with the nuclear structures ................................................................................................................. 18 1.8.1 Localization of HDAg .............................................................................................................................. 18 1.8.2 Study of the link between the HDV foci and HDV transcription ........................................... 22 1.8.3 Localization of genomic and antigenomic HDV RNAs .............................................................. 23 1.9. Host proteins linked to HDV cycle............................................................................................. 25 1.9.1 Host proteins

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