Program Director/Principal Investigator (Last, First, Middle): Weliky, David Paul Project Summary/Abstract The long-term goal of the proposed research is detailed understanding of the basis for viral fusion protein- induced membrane fusion. 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 hemagglutinin HA2 and gp41 fusion proteins of the influenza virus and human immunodeficiency virus, respectively. These proteins are chosen because these viruses cause major human diseases and because the proteins serve as prototypes for other class I viral fusion proteins. Although the protein sequences are non-homologous, both proteins are single-pass transmembrane proteins of the viral membrane with ~180-residue ectodomain regions which lie outside the virus. There is particular emphasis on the ~25-residue N-terminal “fusion peptide” (FP) domain which plays a key role in fusion catalysis and is thought to insert into the host cell membrane early in the fusion process. There are four Aims which include structural distributions in membrane of the FP's of HA2(Aim 1), and gp41(Aim 2), and the membrane locations of the FP's of HA2(Aim 3) and gp41(Aim 4). Each Aim is divided into (a) FP-only and (b) FP+soluble ectodomain+transmembrane domain sections. The FP's of both proteins can adopt either α helical or antiparallel β sheet structure, and both structures catalyze membrane fusion. The project focuses on the α HA2 and β gp41 FP structures. Structure/function correlations are further developed with corollary measurements on HA2 G1E and gp41 V2E mutants which both result in greatly impaired fusion and viral infection. The main technique to elucidate FP structure and membrane contacts is solid-state nuclear magnetic resonance(SSNMR) with particular emphasis on rotational-echo double resonance(REDOR) which can elucidate internuclear proximities and distances ≤10Å. REDOR will be used to determine interhelical HA2 FP structures and antiparallel registries of β gp41 FP structures. Fractional populations will be determined for the different structures as well as how the structures and populations change with fusion-impairing mutations. There will be REDOR measurements of FP/membrane contacts as well as complementary SSNMR measurements of FP/water contacts using paramagnetic relaxation and 1H spin diffusion. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page