Esi-Fticrms Characterization of the HIV-1 Packaging Signal as a Viable Anti-Viral Target - Development of High-Throughput Methods for Drug Screening and Target Identification. (Paperback)

A primary cause of failure of the highly active antiretroviral therapy (HAART) used to control the infection of human immunodeficiency virus type 1 (HIV-1) is the rapid emergence of strains that are resistant to one or more of the active agents used in typical multidrug regimens. Mutations of genes coding for inhibitor targets, such as protease, reverse transcriptase, and gp41, result in a reduced response to treatment from patients infected by resistant strains. The magnitude reached by the AIDS pandemic and the daunting problem of drug resistance elevate the need for new therapeutic agents with alternative mechanisms of action. In the life cycle of HIV-1, the processes of genome recognition, dimerization, and packaging provide very attractive opportunities for the development of novel anti-retroviral strategies. These essential functions are mediated by the interactions between the nucleocapsid (NC) domain of the Gag polyprotein and a highly conserved region of genomic RNA, known as the packaging signal, or Psi-RNA. Located in the 5'-untranslated leader (5'-UTR), the ∼120 nucleotides comprising Psi-RNA have been shown to fold into relatively stable secondary structures identified as stem-loop 1 through 4 (SL1-4), which serve as possible binding sites for NC during viral replication. The characteristics and structural determinants of these protein-RNA interactions have been extensively investigated to elucidate the role played by the different elements and enable the identification of viable targets. Different strategies have been proposed to disrupt the mechanism of genome recognition, dimerization, and packaging by targeting key structures involved in these processes. We have employed an approach based on electrospray ionization (ESI) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) to investigate ligand-RNA interactions and evaluate the stability of NC-hairpin complexes in the presence of ligands. Furthermore, we have explored the possibility of employing tandem mass spectrometry to map the actual binding sites of small molecule ligands on individual stemloop domains of Psi-RNA. This approach was also extended to determine the effects of these ligands on the chaperone activity presented by NC. Finally, we have taken advantage of the high mass limits and resolution of ESI-FTICRMS to study the binding interactions of a peptide library with the complete Psi-RNA. These biophysical studies have provided an ideal platform to screen possible therapeutics, as well as identifying new viral targets, which could lead to the development of antiretroviral agents that are capable of interfering with these key steps in the viral life cycle.