Our goal is to determine the structural basis and mechanism of retroviral genome selection and packaging. During the current funding period we (1) showed that genome packaging by the Moloney Murine Leukemia Virus (MoMuLV) is mediated by a dimerization-dependent structural switch, (2) determined the 3D structure of a MoMuLV core encapsidation element (132 nucleotide dimer) using a novel NMR/cryo-electron tomography (cryo-ET) approach, (3) developed a 2H- and 13C-edited NMR method that enabled direct NMR detection of structural elements within the intact HIV-1 5'-leader (712 nucleotide dimer);(4) demonstrated that HIV-1 genome dimerization and packaging are regulated by a novel allosteric """"""""nucleotide displacement"""""""" RNA switch mechanism, in which base pairing of residues near the gag start codon with those of an upstream element induces a global RNA rearrangement that simultaneously exposes a dimer-promoting stem loop and high affinity nucleocapsid (NC) binding sites;and (5) identified a minimal HIV-1 core encapsidation signal that exhibits NMR, dimerization, and NC binding properties of the intact leader and can efficiently direct the packaging of heterologous cytoplasmic RNAs into assembling particles. In addition, we have obtained unpublished NMR evidence that the HIV-1 packaging signal adopts a novel 3D structure that is unlike any of the >25 structures predicted previously on the basis of chemical reactivity probing and modeling. We are now on the verge of completing the 3D structure of the HIV-1 core encapsidation signal which, when finished, will provide insights into the RNA structures and protein-RNA interactions that direct HIV-1 genome selection. We have also developed NMR tools that enable identification of intermolecular contacts in the native, dimeric HIV- 1 5'-leader, and we are poised to determine the 3D structure of the full-length HIV-1 5'-leader in its monomeric and dimeric states. Preliminary ITC and biophysical studies indicate that an HIV-1 Gag fragment comprising the CA-through-NC domains (GagCANC) binds the minimal packaging signal RNA with high affinity and 12:2 Gag:RNA stoichiometry. By using our hybrid NMR/cryo-ET approach, it should now be possible to determine the structure of the Gag:RNA complex that is trafficked to the plasma membrane and nucleates virus assembly. NMR studies of the monomeric HIV-1 5'-leader and a spliced HIV-1 mRNA will provide insights into the interactions and mechanisms that block packaging of these RNAs, and comparisons with HIV-2 and MoMuLV will help establish if RNA-centered control mechanisms are evolutionarily conserved. NMR studies of large RNAs are technically challenging - the average size of NMR-derived RNA structures in the RNA Structure Database is only 27 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 human immunodeficiency virus (HIV) selectively packages two copies of its full-length RNA genome as the virus assembles in infected cells -- a requirement for viral infectivity. Understanding the molecular structures and mechanisms responsible for genome packaging will 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. New NMR and hybrid NMR/cryo-EM methodologies developed in the course of these studies should be broadly applicable to the rapidly growing field of RNA biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM042561-26
Application #
8789513
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sakalian, Michael
Project Start
1989-07-01
Project End
2018-08-31
Budget Start
2014-09-15
Budget End
2015-08-31
Support Year
26
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21250
Gaines, Christy R; Tkacik, Emre; Rivera-Oven, Amalia et al. (2018) HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting. J Mol Biol 430:2113-2127
Marchant, Jan; Bax, Ad; Summers, Michael F (2018) Accurate Measurement of Residual Dipolar Couplings in Large RNAs by Variable Flip Angle NMR. J Am Chem Soc 140:6978-6983
Kharytonchyk, Siarhei; Brown, Joshua D; Stilger, Krista et al. (2018) Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function. J Mol Biol 430:2066-2079
Zhang, Kaiming; Keane, Sarah C; Su, Zhaoming et al. (2018) Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach. Structure 26:490-498.e3
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; Summers, Michael F (2016) NMR Studies of the Structure and Function of the HIV-1 5'-Leader. Viruses 8:
Kharytonchyk, Siarhei; Monti, Sarah; Smaldino, Philip J et al. (2016) Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome. Proc Natl Acad Sci U S A 113:13378-13383
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

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