Date of Award

4-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Medical Science)

Department

Medical Education

First Advisor

Tahir A. Rizvi

Second Advisor

Dr. Ahmed Al-Marzouqi

Third Advisor

Dr. Gulfaraz Khan

Abstract

The “diploid” genome of retroviruses consist 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, determinant 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.

To test the importance of the pal sequence 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.

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