The technological novelty is three-fold, i) Establishing a correlative multi-mode imaging system using live cell single particle imaging, super-resolution single molecule imaging, and cryoET imaging advances our previously established correlative live-cell and cryoET methodology to determine HIV-i protein interactions with host cell factors. At one end of the imaging spectrum, the dynamic behavior of a specific target in the context of a living biological system can be characterized with compromised resolution, while at the other end, high resolution structural snapshots of regions of interest can be obtained. Super-resolution molecular imaging bridges these methods and allows identification and localization of specific target proteins associated with cryoET structures. 2) Creating cell lamella in vitreously frozen cells, similar to yo-ultramicrotomy, but without sectioning artifacts, is a novel approach to generate suitably thin vitreous biological specimens and enhances the applicability of cryoET for high resolution 3D structural analysis of native cells and tissues. 3) Using a short fusion peptide with high affinity to a gold surface constitutes a novel approach to overcome some of the difficulties in developing a GFP-equivalent taggable EM label. Ultimately the innovation of the technology program relies on the integration of several approaches developed by us (correlative light microscopy/cryoEM imaging) and by others (super resolution imaging). Our proposed studies build on our prior work and develop new capabilities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
5P50GM082251-07
Application #
8546406
Study Section
Special Emphasis Panel (ZRG1-AARR-K)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
7
Fiscal Year
2013
Total Cost
$269,010
Indirect Cost
$91,446
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Gupta, Rupal; Lu, Manman; Hou, Guangjin et al. (2016) Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies. J Phys Chem B 120:329-39
Van Oss, S Branden; Shirra, Margaret K; Bataille, Alain R et al. (2016) The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6. Mol Cell 64:815-825
Perilla, Juan R; Gronenborn, Angela M (2016) Molecular Architecture of the Retroviral Capsid. Trends Biochem Sci 41:410-20
Sharaf, Naima G; Ishima, Rieko; Gronenborn, Angela M (2016) Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study. Biochemistry 55:3864-73
Byeon, In-Ja L; Byeon, Chang-Hyeock; Wu, Tiyun et al. (2016) Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity. Biochemistry 55:2944-59
Saito, Akatsuki; Ferhadian, Damien; Sowd, Gregory A et al. (2016) Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74. J Virol 90:5808-23
Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L et al. (2016) In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway. Nat Commun 7:13689
Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik et al. (2016) The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes. J Biol Chem 291:11809-19
Ramalho, Ruben; Rankovic, Sanela; Zhou, Jing et al. (2016) Analysis of the mechanical properties of wild type and hyperstable mutants of the HIV-1 capsid. Retrovirology 13:17
Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana et al. (2016) Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion. J Virol Methods 233:62-71

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