This project, focused on characterization of the three-dimensional structures of RNA molecules in biologically relevant environments, links broadly important problems in biomedical science. First, the human immunodeficiency virus (HIV) currently infects 35 million individuals globally with roughly 3 million new infections per year. Studies of the protein components of the virus have led to the development of multiple effective drugs, but additional effective antiretroviral agents are needed. In principle, the RNA genome itself is a compelling drug target. The conserved, functional motifs in the HIV-1 genomic RNA that we have identified - and elements that we will characterize as part of this proposal - have significant promise as novel targets and pathways for antiretroviral drug development. Second, the extraordinary density of information encoded in the higher-order structure of the HIV-1 RNA genome represents a key component of the genetic code, one that we understand poorly at present. This research program will enhance our understanding of the principles that govern RNA-mediated gene regulation. It will also advance our understanding of the organization and compaction of RNA virus genomes and will illuminate principles that apply to densely organized RNAs including lncRNAs, nuclear bodies, and other regulatory elements. Third, the SHAPE probing strategy, pioneered by our lab, yields quantitative but highly specific nucleotide-resolution information, and, therefore, we are now focusing on developing multiple, alternative, complementary strategies that specifically detect true through-space and higher-order interactions in RNA. Finally, throughout this project, we will create integrated strategies that meld multiple, complementary, experimentally concise, and physically accurate structure probing technologies with user-friendly data processing to make possible facile and comprehensive functional analyses of large RNAs in relevant cellular contexts by non-expert laboratories. Our collaborative team, consisting of HIV virologists, RNA structural biologists, and experts in bioinformatics, will tackle the following Aims: (1) Analyze the structure of complete HIV-1 genomes inside authentic virions using the genome-scale SHAPE-MaP strategy; (2) identify through-space interactions across the entire HIV-1 genome by large-scale, single-molecule, correlated chemical probing experiments; (3) comprehensively define the intra- and inter-molecular interactions that mediate the RNA interactome of packaged HIV-1 genomic RNAs; and (4) create robust platform-independent software for fully automated analysis of chemical probing experiments that specifically measure through-space interactions in RNA.

Public Health Relevance

This work will use multiple distinctive and innovative, high-throughput chemical probing technologies, invented in the project laboratory, to determine structures for complete HIV RNA genomes and to understand the viral genetic code. The structure probing technologies and user-friendly software developed to analyze these experiments will be broadly useful for many other infectious disease and biomedical problems. Over the long term, nucleotide-resolution information about HIV genome structure will reveal multiple novel frameworks for design of new anti-retroviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI068462-14
Application #
9685840
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Refsland, Eric William
Project Start
2006-02-01
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
14
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Busan, Steven; Weeks, Kevin M (2018) Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24:143-148
Liu, Yang; Chen, Jianbo; Nikolaitchik, Olga A et al. (2018) The roles of five conserved lentiviral RNA structures in HIV-1 replication. Virology 514:1-8
Busan, Steven; Weeks, Kevin M (2017) Visualization of RNA structure models within the Integrative Genomics Viewer. RNA 23:1012-1018
Weidmann, Chase A; Mustoe, Anthony M; Weeks, Kevin M (2016) Direct Duplex Detection: An Emerging Tool in the RNA Structure Analysis Toolbox. Trends Biochem Sci 41:734-736
Smola, Matthew J; Rice, Greggory M; Busan, Steven et al. (2015) Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10:1643-69
Weeks, Kevin M (2015) RNA clubs. RNA 21:760-1
Lavender, Christopher A; Lorenz, Ronny; Zhang, Ge et al. (2015) Model-Free RNA Sequence and Structure Alignment Informed by SHAPE Probing Reveals a Conserved Alternate Secondary Structure for 16S rRNA. PLoS Comput Biol 11:e1004126
Lavender, Christopher A; Gorelick, Robert J; Weeks, Kevin M (2015) Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes. PLoS Comput Biol 11:e1004230
Weeks, Kevin M (2015) Review toward all RNA structures, concisely. Biopolymers 103:438-48
Rice, Greggory M; Leonard, Christopher W; Weeks, Kevin M (2014) RNA secondary structure modeling at consistent high accuracy using differential SHAPE. RNA 20:846-54

Showing the most recent 10 out of 37 publications