Date of Award

4-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Medical Science)

Department

Medical Microbiology and Immunology

First Advisor

Prof. Tahir A. Rizvi

Second Advisor

Dr. Ahmed Al-Marzouqi

Third Advisor

Dr. Gulfaraz Khan

Abstract

The “diploid” genome of retroviruses consists of two strands of RNA that are non-covalently linked as a “dimer” at their 5’ ends. The ubiquitous presence of a dimeric genome among retroviruses suggests that dimerization plays a crucial role in gRNA packaging during viral life-cycle. For almost all retroviruses, determinants of gRNA dimerization and packaging, which are for the most part physically and genetically indistinguishable, reside at the 5’ end of the gRNAs and have been shown to assume higher order structures. Employing a combination of genetic and structural prediction approaches, we have earlier shown that Mason-Pfizer monkey virus (MPMV) and mouse mammary tumor virus (MMTV) packaging determinants comprise sequences at the 5’ end of the genome, starting from R and extending into the beginning of Gag. Sequences encompassing these regions were predicted to fold into stable RNA secondary structures comprising several structural motifs. In an attempt to establish structure-function relationship of the higher order features of MPMV and MMTV packaging signal RNAs, we first validated their predicted structures employing a novel chemo-enzymatic probing strategy, selective 2’hydroxyl acylation primer extension (SHAPE). The SHAPE-analyzed structures of MPMV and MMTV packaging signal RNAs validated the major structural motifs, including U5/Gag long range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive GC-rich palindromic (pal) helix loop. Minimum free-energy structure predictions, phylogenetic, and in silico analyses of different MPMV and MMTV strains further suggested the existence of these major structural motifs. viii To test the importance of the pal sequences in MPMV and MMTV gRNA dimerization and packaging, we introduced a series of mutations in the pal helix loops. Tests of these mutations employing in vitro and in vivo complementary approaches, phylogenetic, and structure prediction analyses revealed pal helix loops (5’ CGGCCG 3’ in MPMV and 5’ CGGCCG 3’ in MMTV ) containing a canonical “GC” dyad functioning as dimerization initiation sites (DISs) controlling MMTV and MPMV gRNA dimerization and packaging. Furthermore, in MMTV, a second pal within the primer binding site (PBS) was also observed that was found to be involved in gRNA dimerization. Concomitant mutational analysis of pal II and PBS pal suggests that both pals are required for efficient RNA dimerization, packaging and propagation of MMTV gRNA.

Acknowledgments

First and foremost, I want to thank Allah (the most Merciful, the All-knowing and the most Beneficent) whose constant guidance and support has helped me throughout my life. I am grateful to Prof. Rizvi for being an extraordinary supervisor who showed me the road and helped me to get started on the path to the PhD degree. His enthusiasm, encouragement and faith in me throughout have been extremely helpful. He was always available for my questions and he was positive and was generous with his time and vast knowledge. I found a very nice and unforgettable association with Dr. Roland Marquet’s mentorship during our collaboration for my PhD thesis work. I appreciate his and his family’s kindness towards me during my stay in France. I am grateful to all my committee members (Dr. Farah Mustafa, Dr. Ahmed Al-Marzouqi and Dr. Gulfaraz Khan), who were very helpful to manage time for me from their busy schedules to arrange committee meetings every semester. My sincere appreciation and regards go to Dr. Farah Mustafa who is like a role model. I appreciate her patience that has allowed me to extract her from her busy schedule at any time for discussions. I am extremely grateful to my current and past lab mates (Arshad, Ayesha, Elsa, Gayathri, Jaicy, Lizna, Pretty, Reka, Sahla, Soumeya, Zahabar) in UAE without whose support I would not have been able to graduate. I am thankful to the people in Dr. Roland Marquet’s lab (Delphine, J.C. Paillart, Julien, Patrick, Redmond, Santiago, Valerie) for their friendliness and for making my work in France quite enjoyable. xii My deep appreciation goes to my friends, Varun, Rawan, Nabila, Jasbir, Subash, Waqar and Karima, who supported me during some of my toughest times. In the end, my special thanks go to my family: my father, Mohd. Saleh Ahmed, who unfortunately did not live long enough to see the fruits of his hard work towards his children’s education, my strong mother, Shamim Akter who supported me in all my decisions, my brother, brother-in-law and sisters who were always available for moral support and to provide all concerned facilities to fulfill my needs by clearing away all the difficulties of my life. And also to my little sister, Amy, who helped me with my acknowledgement, with mostly minor editing (some of which I neglected to pay any attention to) and who also added this comment.

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