Enveloped viruses enter cells using their fusion proteins, which merge the virus envelope and the cell membrane. Determining the atomic-resolution structures and structural evolution of viral fusion proteins along the fusion pathway is important for understanding how these proteins cause membrane curvature and dehydration and for developing antiviral drugs to inhibit virus entry. Although crystal structures of the water-soluble ectodomains of several viral fusion proteins have been determined, much less is known about the hydrophobic N-terminal fusion peptide (FP) and the C-terminal transmembrane domain (TMD). In the last funding period, we showed that the FP and TMD of the parainfluenza virus 5 (PIV5) fusion protein, F, adopt membrane-dependent conformations. In phosphatidylethanolamine (PE)-rich membranes, the ?-strand conformation dominates and induces negative Gaussian curvature, which is characteristic of hemifusion intermediates, to the membrane. This suggests that the ?-strand structure is important for the prefusion to hemifusion transition. Building on these results, we will now determine the oligomeric structure of the FP and TMD of PIV5 and investigate the structure of the TMD-including region of the HIV fusion protein, gp41. These experiments will address whether the FP and TMD are trimeric when they are apart from each other, as in the early fusion stages, and whether they form a trimer of hairpins when they are brought into close proximity, as in the post-fusion state.
In Aim 1, we will measure the oligomeric structure and intermolecular distances of the PIV5 FP and TMD peptides to probe the early- fusion structures. 19F and 13C CODEX experiments will be performed to measure intermolecular distances for both the ?-helical and ?-sheet conformations of the peptides. These experiments will be done as a function of membrane composition, and lipid mixtures that mimic the cell membrane and virus envelope will be used.
In Aim 2, we will investigate late-fusion structures of PIV5 by studying a chimera that links the FP and TMD. Vesicle fusion assays show that the chimera is fusogenic, with lipid mixing activities that are approximately the sum of the activities of the FP and TMD peptides. We will develop improved 13C and 1H spin diffusion techniques to measure inter-domain FP-TMD distances as well as intermolecular homo- oligomeric distances. These distance constraints will test whether a six-helix bundle is formed by the FP and TMD inside the membrane.
In Aim 3, we will extend our structural studies to HIV gp41, focusing on the antibody-targeted MPER region and the membrane-bound TMD. We will investigate the conformation and curvature-inducing ability of this domain in lipid membranes, conduct 19F-19F and 13C-19F distance experiments to measure the relative orientation of the MPER and TMD, their depths of insertion in the membrane, and the oligomeric structure of this protein. These experiments are expected to provide fundamental new insights into the protein structural transitions that underlie virus-cell fusion.

Public Health Relevance

Enveloped viruses enter cells using their fusion proteins, which merge the virus lipid envelope and the cell membrane. The design of antiviral drugs and vaccines that inhibit viral entry requires knowledge of the high-resolution structures of these fusion proteins bound to biologically relevant lipid membranes. We will use solid-state NMR spectroscopy to determine the structures of two crucial membrane-bound domains in the fusion proteins of two viruses: the parainfluenza virus, which is responsible for infant respiratory diseases such as pneumonia and bronchitis, and the human immunodeficiency virus, which infects ~37 million people worldwide.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Wu, Mary Ann
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Kwon, Byungsu; Lee, Myungwoon; Waring, Alan J et al. (2018) Oligomeric Structure and Three-Dimensional Fold of the HIV gp41 Membrane-Proximal External Region and Transmembrane Domain in Phospholipid Bilayers. J Am Chem Soc 140:8246-8259
Roos, Matthias; Wang, Tuo; Shcherbakov, Alexander A et al. (2018) Fast Magic-Angle-Spinning 19F Spin Exchange NMR for Determining Nanometer 19F-19F Distances in Proteins and Pharmaceutical Compounds. J Phys Chem B 122:2900-2911
Gelenter, Martin D; Hong, Mei (2018) Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination. J Phys Chem B 122:8367-8379
Lee, Myungwoon; Yao, Hongwei; Kwon, Byungsu et al. (2018) Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity. J Mol Biol 430:695-709
Shcherbakov, Alexander A; Hong, Mei (2018) Rapid measurement of long-range distances in proteins by multidimensional 13C-19F REDOR NMR under fast magic-angle spinning. J Biomol NMR 71:31-43
Mandala, Venkata S; Williams, Jonathan K; Hong, Mei (2018) Structure and Dynamics of Membrane Proteins from Solid-State NMR. Annu Rev Biophys 47:201-222
Liao, Shu Y; Lee, Myungwoon; Hong, Mei (2018) Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR. J Struct Biol :
Lee, Myungwoon; Wang, Tuo; Makhlynets, Olga V et al. (2017) Zinc-binding structure of a catalytic amyloid from solid-state NMR. Proc Natl Acad Sci U S A 114:6191-6196
Wang, Tuo; Jo, Hyunil; DeGrado, William F et al. (2017) Water Distribution, Dynamics, and Interactions with Alzheimer's ?-Amyloid Fibrils Investigated by Solid-State NMR. J Am Chem Soc 139:6242-6252
Fritzsching, Keith J; Hong, Mei; Schmidt-Rohr, Klaus (2016) Conformationally selective multidimensional chemical shift ranges in proteins from a PACSY database purged using intrinsic quality criteria. J Biomol NMR 64:115-30

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