The specific aims of this study are the elucidation of the determinants of the structural polymorphism manifested by the 5'-untranslated region (5'-UTR) of Human Immunodeficiency Virus type 1 (HIV-1), the dentification of the binding sites of the nucleocapsid (NC) domain of Gag on the different forms assumed by the 5'-UTR fold, and the evaluation of deoxyribozymes as functional probes and possible interferents of 5'- UTR dynamics. 5'-UTR is involved in key steps of viral replication, including reverse transcription, splicing, translation, genome recognition, dimerization, and packaging. Facilitated by the chaperone activity of NC, 5'-UTR can fold into polymorphic forms defined by the different pairing arrangement of complementary sequences ocated in distal positions of the HIV-1 leader. The equilibrium between alternative conformations has been interpreted as a possible riboswitch mechanism for regulating the 5'-UTR functions. Understanding the riboswitch determinants and the role played by NC in switch actuation would enable the development of new antiviral strategies aimed at disrupting the 5'-UTR processes. We propose to investigate the 5'-UTR polymorphism in vitro using an approach based on bifunctional crosslinking, chemical footprinting, and high-resolution mass spectrometry (MS3D). Not limited by considerations of size and crystallization behavior, this approach will provide valuable information on the spatial organization and long range interactions between secondary structures formed by the different conformers of leader RNA. The remodeling of local hairpins and loops with formation of new base pairs and tertiary interactions will be investigated in the context of full-length 5'-UTR mutants that fold in the specific conformations. The effects of NC on the stability of the new structures will be investigated through binding and crosslinking experiments. These experiments will also enable the possible identification of new NC binding sites formed by distal regions of RNA, which are brought into close proximity by the global fold of 5'- UTR. The effects of deoxyribozyme interference on the observed RNA-RNA and protein-RNA interactions will be determined to elucidate the mechanism of riboswitch actuation and support the design of new 5'-UTR inhibitors. The long term goal is to understand the molecular basis for the 5'-UTR processes and the mechanism regulating its different roles during viral replication. New strategies that interfere with 5'-UTR function and regulation would provide a much needed complement to the current treatments based on protease and reverse-transcriptase inhibitors, which are particularly affected by the emergence of drug-resistant strains.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM064328-10
Application #
8135155
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Edmonds, Charles G
Project Start
2001-08-01
Project End
2010-09-29
Budget Start
2010-06-01
Budget End
2010-09-29
Support Year
10
Fiscal Year
2009
Total Cost
$166,423
Indirect Cost
Name
State University of New York at Albany
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
152652822
City
Albany
State
NY
Country
United States
Zip Code
12222
Lippens, Jennifer L; Mangrum, J B; McIntyre, William et al. (2016) A simple heated-capillary modification improves the analysis of non-covalent complexes by Z-spray electrospray ionization. Rapid Commun Mass Spectrom 30:773-83
Rose, Rebecca E; Pazos 2nd, Manuel A; Curcio, M Joan et al. (2016) Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response. Mol Cell Proteomics 15:932-44
Lippens, Jennifer L; Ranganathan, Srivathsan V; D'Esposito, Rebecca J et al. (2016) Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry. Analyst 141:4084-99
Spell, Sarah R; Mangrum, John B; Peterson, Erica J et al. (2016) Au(iii) compounds as HIV nucleocapsid protein (NCp7)-nucleic acid antagonists. Chem Commun (Camb) 53:91-94
Rose, Rebecca E; Quinn, Ryan; Sayre, Jackie L et al. (2015) Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics. RNA 21:1361-74
Tosoni, Elena; Frasson, Ilaria; Scalabrin, Matteo et al. (2015) Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription. Nucleic Acids Res 43:8884-97
Welch, Joshua D; Slevin, Michael K; Tatomer, Deirdre C et al. (2015) EnD-Seq and AppEnD: sequencing 3' ends to identify nontemplated tails and degradation intermediates. RNA 21:1375-89
Scalabrin, Matteo; Siu, Yik; Asare-Okai, Papa Nii et al. (2014) Structure-specific ribonucleases for MS-based elucidation of higher-order RNA structure. J Am Soc Mass Spectrom 25:1136-45
Doria, Filippo; Nadai, Matteo; Folini, Marco et al. (2013) Targeting loop adenines in G-quadruplex by a selective oxirane. Chemistry 19:78-81
Quinn, Ryan; Basanta-Sanchez, Maria; Rose, Rebecca E et al. (2013) Direct infusion analysis of nucleotide mixtures of very similar or identical elemental composition. J Mass Spectrom 48:703-12

Showing the most recent 10 out of 19 publications