Date of Award

5-2015

Document Type

Thesis

Degree Name

Master of Science in Medical Sciences (MSMS)

Department

Medical Microbiology and Immunology

First Advisor

Tahir A . Rizvi

Second Advisor

Dr. Ahmed AI-Marzouqi

Third Advisor

Dr. Farah M ustafa

Abstract

Employing a combination of genetic and biochemical approaches, we have recently shown that the 5’ end of MPMV genome (from the first nucleotide in R up to 120 nt of Gag) contains a number of sequence and structural motifs important for MPMV gRNA packaging and dimerization. A distinguishing feature of the higher order structure of MPMV packaging signal RNA is two long-range interactions (LRI) between U5 and Gag complementary sequences, LRI-I and LRI-II, which are phylogenetically conserved among different MPMV strains. These LRIs have been suggested to play a role in stabilizing the RNA secondary structure of the 5’ UTR sequences that are important for MPMV RNA packaging. The overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from distal parts of the 5’ UTR and Gag, excluding Gag sequences involved in forming U5-Gag LRIs.

To provide functional evidence for the biological significance of U5-Gag LRIs and the three stem loops to the MPMV life cycle, a series of mutations were introduced in these structural motifs and their effects on MPMV RNA packaging and propagation tested in a genetic trans-complementation assay. Test of LRIs mutants revealed that disrupting the complementary base pairing of the LRI structural motifs affected both gRNA packaging and propagation, confirming their functional role during MPMV life cycle. Our results further revealed that the two LRIs function at different levels. Specifically, LRI-I functions at the secondary structural level, where LRI-II functions at both the primary sequence as well as in its native structural context levels. Finally, Mutational analysis of the sequences involved in forming SL3, Gag SL1, and Gag SL2 revealed that they do not play crucial role at individual levels during MPMV gRNA packaging and propagation. These findings suggest that U5-Gag LRIs have a more important architectural role in stabilizing the structure of the 5’ UTR sequences, while the three stem loops (SL3, Gag SL1, and Gag SL2) may have a more secondary role in stabilizing the overall RNA secondary structure, providing a better understanding of the molecular interactions that take place during MPMV gRNA packaging.

Keywords: Retroviruses; Mason-Pfizer monkey virus; RNA secondary structure; RNA packaging; long range interactions (LRI).

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