Structural and dynamic investigation of HIV-1 capsid assemblies and capsid maturation by magic angle spinning NMR spectroscopy
Date
2018
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Human immunodeficiency virus (HIV) is the causative agent for the acquired immune deficiency syndrome (AIDS), a global pandemic. The curative treatments or vaccines for HIV are still lacking to date. The HIV-1 capsid (CA) protein plays essential roles in the HIV-1 life cycle, involving in two critical assembly events, formation of the immature viral particle as a component of Gag polyprotein and reassembly into a mature capsid core after Gag cleavage. During CA maturation, the Gag polyprotein cleaves into its constituent domains. The final step in the Gag processing cascade is the cleavage of spacer peptide 1 (SP1) from the C-terminal domain of CA. Following maturation, approximately 1000-1500 copies of CA protein arrange into a conical core to protect the viral genome in a mature virion. Initial research has identified that maturation inhibitors (MI) prevent the cleavage of CA from SP1, and formation of infectious virions. Thus, the HIV-1 CA maturation is an attractive target for therapeutic intervention. However, atomic-level understanding of CA maturation and maturation inhibition is lacking. ☐ In this dissertation, I employed magic angle spinning (MAS) solid-state NMR spectroscopy to investigate the structural rearrangements and dynamic changes accompanying capsid maturation. By examining the final-step maturation intermediate, CA-SP1, I demonstrate the presence of as well as quantified dynamic helix-coil equilibrium in the CA-SP1 assemblies, and discovered that it is inhibited by the T8I mutation in the SP1 domain that phenocopies the MI-bound state. I have combined MAS NMR and molecular dynamics (MD) simulations to obtain unprecedented atomic-level quantitative insights into the assemblies of maturation intermediates, unavailable from other techniques. Overall, the results indicate that modulation of protein dynamics appears to be a determining factor in capsid maturation and maturation inhibition. This study is described in Chapter 4. ☐ To further understand the role of dynamics in the capsid assembly, I have also applied MAS NMR to probe atomic-resolution structures of the CA capsid protein assemblies of different morphologies and of model pentameric and hexameric CA building blocks. This study is described in Chapter 3. ☐ In Chapter 5, I have explored 19F as an NMR probe for applications to biological assemblies. A robust protocol for 19F MAS NMR spectroscopy has been developed based characterization of structure and dynamics in fluorinated solids by examining fluorosubstituted tryptophans. I have applied this approach to the HIV-1 capsid protein assemblies. The results exhibit the fast and ultrafast MAS frequencies are beneficial to resolution enhancement and recording interfluorine distances. ☐ I have incorporated 5-F-Trp into CA protein, and resonances were assigned by mutagenesis. The 19F chemical shifts for the five tryptophans are distinct, reflecting differences in local environment. Spin diffusion and radio frequency driven recoupling (RFDR) experiments were performed at fast MAS frequencies of 35-60 kHz and permitted establishing the 19F-19F correlations, yielding interatomic distances as long as 23 Å. Fast MAS frequencies of 35-60 kHz are essential and yield narrow lines. I demonstrate the potential of 19F NMR for structural analysis in large protein assemblies by MAS NMR.