Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Integrative Microbiology and Biochemistry

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

J. Stephen Lodmell

Committee Co-chair

Jean-Marc Lanchy

Commitee Members

Scott Samuels, Mark Grimes, J. B. Alexander Ross

Keywords

Dimerization, HIV-2, in vitro, in vivo, Packaging, SELEX

Abstract

The investigation of sequences and structures in the 5' untranslated leader region (5'UTR) of HIV genomic RNA is essential for understanding viral replication because the 5'UTR regulates several essential functions by the alternate presentation and sequestration of signals through conformational changes. Our main focus in this study was to understand those sequences and structures that are involved in dimerization and packaging of HIV-2 RNA. Progressing from previous findings, we studied in detail a 10-nucleotide palindrome sequence (pal; 5'-GGAGUGCUCC-3') of the 5'UTR, located within the major packaging signal, upstream of the dimerization signal (SL1: stem loop-1). Pal has been shown to interact intramolecularly with SL1 to inhibit SL1-mediated HIV-2 leader RNA dimerization in vitro. We carried out three lines of experiments in an effort to understand the roles of pal in viral RNA dimerization and packaging.

The first study was achieved through randomization of pal and the subsequent selection from a population of random-pal RNAs those with enhanced or diminished dimerization properties in vitro. We showed that the 3'-pal motif (3'-pal; GCUCC-3') is involved in intramolecular interactions with a sequence downstream of SL1 that regulates SL1-mediated HIV-2 leader RNA dimerization. The second study was designed to investigate the role of 5'-pal motif (5'-GGAGU) in RNA packaging in vivo. Our findings indicated that the 5'-pal is essential for viral replication and genomic RNA packaging. Based on these findings, we proposed that 5'-pal is a binding element for the packaging proteins. Therefore, a third study was designed in which HIV-2 packaging proteins (Gag and NC) were expressed, purified, and assayed for binding with wild-type and mutant 5'-pal RNAs in vitro. These results suggested that the 5'-pal is a binding element for Gag protein. In summary, we have showed that pal is an important regulator of dimerization and packaging processes of HIV-2 RNA. We also demonstrated that pal is composed of two motifs with distinct functions. Overall, our study significantly contributes to the understanding of HIV-2 RNA dimerization and packaging, which may ultimately lead to the identification of novel antiretroviral targets.

Download

Share

COinS
 

© Copyright 2008 Tayyba Tabassum Baig