The goal of the proposed studies is to determine how retroviruses select and package their diploid RNA genomes. During the current funding period, we obtained strong in vivo and in vitro evidence that the Moloney Murine Leukemia Virus (MLV) uses an RNA structural switch mechanism, in which high affinity nucleocapsid (NC - the protein domain responsible for genome selection) binding sites are sequestered by base pairing in the monomeric RNA and become exposed to allow NC binding upon dimerization. Having nearly completed studies of this model retrovirus, we now intend to focus almost exclusively on the Human Immunodeficiency Virus (HIV-1). Methods developed over the past 3 1/2 years now enabled us to obtain NMR spectra of outstanding quality for the intact HIV-1 5'-UTR in its monomeric (356 nucleotides) and dimeric (712 nt) states. Based on these and other unpublished findings, we now believe that genome dimerization and packaging are mediated by a novel allosteric "nucleotide displacement" RNA switch mechanism, in which residues near the gag start codon induce a global rearrangement that simultaneously exposes a dimer-promoting stem loop (DIS) and high affinity NC binding sites that were sequestered in the momomeric conformer. Preliminary in vivo packaging experiments support this mechanism. 5'-UTR mutants that exclusively adopt either the monomer or dimer conformation have been prepared, and NMR spectra of outstanding quality have been collected for these constructs. We are thus poised to determine the high-resolution 3D structure of the HIV-1 5'-UTR in conformations relevant to the mechanism of diploid genome selection. NMR studies of such large RNAs are technically challenging - the average size of all NMR-derived RNA structures in RNA Structure Database is only 25 nucleotides - but the potential payoff is substantial, and could ultimately lead not only to a more detailed understanding of how HIV replicates, but also to the development of new approaches for the treatment of AIDS, cancers, and other virally-induced human diseases.

Public Health Relevance

The aim of this project is to develop understanding of the mechanism that HIV-1 and other retroviruses use to select and package their RNA genomes. These studies should lead not only to a better understanding of how retroviruses replicate, but also to the development of new approaches for the treatment of AIDS, Cancer, and other virally-induced human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042561-25
Application #
8465235
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
1989-07-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
25
Fiscal Year
2013
Total Cost
$576,864
Indirect Cost
$94,750
Name
University of Maryland Balt CO Campus
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
061364808
City
Baltimore
State
MD
Country
United States
Zip Code
21250
Keane, Sarah C; Van, Verna; Frank, Heather M et al. (2016) NMR detection of intermolecular interaction sites in the dimeric 5'-leader of the HIV-1 genome. Proc Natl Acad Sci U S A 113:13033-13038
Keane, Sarah C; Heng, Xiao; Lu, Kun et al. (2015) RNA structure. Structure of the HIV-1 RNA packaging signal. Science 348:917-21
Brown, Joshua D; Summers, Michael F; Johnson, Bruce A (2015) Prediction of hydrogen and carbon chemical shifts from RNA using database mining and support vector regression. J Biomol NMR 63:39-52
Tran, Thao; Liu, Yuanyuan; Marchant, Jan et al. (2015) Conserved determinants of lentiviral genome dimerization. Retrovirology 12:83
Johnson, Silas F; Collins, John T; D'Souza, Victoria M et al. (2014) Determinants of Moloney murine leukemia virus Gag-Pol and genomic RNA proportions. J Virol 88:7267-75
Barton, Shawn; Heng, Xiao; Johnson, Bruce A et al. (2013) Database proton NMR chemical shifts for RNA signal assignment and validation. J Biomol NMR 55:33-46
Yildiz, F Zehra; Babalola, Kathlene; Summers, Michael F (2013) Identification of a high affinity nucleocapsid protein binding element from the bovine leukemia virus genome. Virus Res 171:278-86
Johnson, Silas F; Garcia, Eric L; Summers, Michael F et al. (2012) Moloney murine leukemia virus genomic RNA packaged in the absence of a full complement of wild type nucleocapsid protein. Virology 430:100-9
Heng, Xiao; Kharytonchyk, Siarhei; Garcia, Eric L et al. (2012) Identification of a minimal region of the HIV-1 5'-leader required for RNA dimerization, NC binding, and packaging. J Mol Biol 417:224-39
Summers, Michael F (2011) Training the next generation of protein scientists. Protein Sci 20:1796-801

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