The long-term goal of the proposed research is to understand the basis for viral fusion protein-induced membrane fusion with atomic-resolution detail. Fusion of viral and target cell membranes is a key step in infection for many viruses important in disease and a detailed understanding of fusion mechanisms will aid development of anti-viral therapeutics whose mode of action is fusion inhibition. Fusion proteins are also a target of viral vaccine development. The proposed research focuses on the gp41 and the hemagglutinin HA2 fusion proteins of the respective human immunodeficiency virus and influenza virus. These proteins are chosen because the viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the N-terminal "fusion peptide" (FP) domains of the fusion proteins, which represent ~20-residue apolar regions at the N-termini of the proteins. Numerous biochemical and biophysical studies have shown that the FP plays a key role in fusion and that chemically synthesized peptides with the FP sequence can serve as useful model systems to understand some aspects of viral/target cell fusion. The long-term objectives will be achieved with three specific aims.
Aim 1 focuses on production of multi-mg quantities of large constructs of gp41 and HA2 proteins that contain FPs, including full-length HA2. In addition, gp41 constructs will be made which contain the C-terminal membrane-proximal external region (MPER) and viral transmembrane (TM) domains. MPER and TM are important in fusion and MPER is also an HIV vaccine target. Residue-specific conformation of the membrane-associated constructs will be determined with solid- state nuclear magnetic resonance (SSNMR) measurements of specific carbonyl (13CO) chemical shifts. There will be particular focus on: (1) testing whether structural models of FP and MPER from short peptide studies are valid for these regions in the large constructs;(2) correlating FP and MPER structure to the observed fusogenicity of the construct;and (3) examining whether the gp41 FP is close to either the MPER or TM. Occlusion of MPER by FP could reduce MPER efficacy as an immunogen.
Aim 2 focuses on determining the membrane locations of FP, MPER, and TM regions in these large gp41 and HA2 constructs using SSNMR measurements of distances between protein 13CO and lipid or cholesterol 31P, 19F, or 2H. These experiments will test the general structure-function model that deep but not transmembrane FP membrane insertion correlates with greater membrane perturbation and more rapid fusion catalysis.
Aim 3 focuses on determining the distribution of antiparallel registries of b sheet FP in several gp41 constructs with very different FP membrane locations and fusogenicities. The free energies of FP membrane insertion will be estimated for the observed registries and correlated with membrane locations and fusogenicities. The overall goal is to connect high-resolution FP structure, membrane insertion depth, and fusogenicity.

Public Health Relevance

The proposed research will provide insight into entry of human immunodeficiency virus and influenza virus into host cells and how this entry can be stopped. In addition, the research will provide information about how a HIV vaccine should be designed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047153-12
Application #
8209021
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Salzwedel, Karl D
Project Start
2000-09-30
Project End
2016-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
12
Fiscal Year
2012
Total Cost
$328,416
Indirect Cost
$103,416
Name
Michigan State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Ratnayake, Punsisi U; Sackett, Kelly; Nethercott, Matthew J et al. (2015) pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant ? sheet fusion peptide conformation. Biochim Biophys Acta 1848:289-98
Banerjee, Koyeli; Weliky, David P (2014) Folded monomers and hexamers of the ectodomain of the HIV gp41 membrane fusion protein: potential roles in fusion and synergy between the fusion peptide, hairpin, and membrane-proximal external region. Biochemistry 53:7184-98
Sackett, Kelly; Nethercott, Matthew J; Zheng, Zhaoxiong et al. (2014) Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel * sheet fusion peptide structure in the final six-helix bundle state. J Mol Biol 426:1077-94
Xie, Li; Ghosh, Ujjayini; Schmick, Scott D et al. (2013) Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and ¹³C-²H rotational-echo double-resonance solid-state NMR. J Biomol NMR 55:11-7
Gabrys, Charles M; Qiang, Wei; Sun, Yan et al. (2013) Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the * helical and * sheet peptide structures. J Phys Chem A 117:9848-59
Vogel, Erica P; Weliky, David P (2013) Quantitation of recombinant protein in whole cells and cell extracts via solid-state NMR spectroscopy. Biochemistry 52:4285-7
Ghosh, Ujjayini; Xie, Li; Weliky, David P (2013) Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone (13)CO- (15)N rotational-echo double-resonance solid-state NMR. J Biomol NMR 55:139-46
Sackett, Kelly; TerBush, Allan; Weliky, David P (2011) HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion. Eur Biophys J 40:489-502
Sackett, Kelly; Nethercott, Matthew J; Epand, Raquel F et al. (2010) Comparative analysis of membrane-associated fusion peptide secondary structure and lipid mixing function of HIV gp41 constructs that model the early pre-hairpin intermediate and final hairpin conformations. J Mol Biol 397:301-15
Qiang, Wei; Weliky, David P (2009) HIV fusion peptide and its cross-linked oligomers: efficient syntheses, significance of the trimer in fusion activity, correlation of beta strand conformation with membrane cholesterol, and proximity to lipid headgroups. Biochemistry 48:289-301

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