New imaging strategy to address challenges in the field. The lethal and transmissible nature of the paramyxoviruses Hendra virus and Nipah virus (HeV, NiV) makes these pathogens of serious concern. These are also ideal models for developing an innovative new imaging platform at the interface between bioengineering and virology. Despite the relative abundance of crystal structure data for the paramyxovirus fusion machinery, the dynamic processes - starting with the binding of virus to the host cell and ending with infection -- are poorly understood. The interplay between the envelope viral glycoproteins that leads to activation of the fusion process has eluded rigorous analysis. Imaging the viral fusion nano-machinery will deepen our understanding of viral membrane fusion and will allow us to identify strategies for interfering with the process and arresting the infection cycle. We propose to capture, immobilize, and visualize activated intermediate states of the fusion process, exposing the conserved domains that are essential for viral entry. While the activated conformation is normally present only at the surface of live cells, we propose to use engineered liposomes to capture and immobilize transitional states. We will use receptor molecules, presented in a biomimetic fashion on the surface of the liposomes, to """"""""trigger"""""""" the fusion protein and activate the conformational change in the viral fusion machinery. We will then use fusion inhibitory peptides to arrest the fusion machinery in its activated state, and thus immobilize the captured intermediates with the liposomes. These complexes will then be analyzed by cryo-electron tomography.
Aim 1. Generate NiV fusion machinery intermediate states 1.1 Trapping intermediate states of the NiV fusion machinery. We will capture the NiV fusion machinery at intermediate stages of the fusion reaction, by first activating the fusion process using cellular receptor molecules presented on liposomes, and then trapping fusion intermediates with fusion-inhibitory peptides that block the conformational refolding of the fusion protein that is required for completing membrane fusion. 1.2 Functional and morphological studies of different states of the fusion process. Fluorescence spectroscopy and negative-stain transmission electron microscopy will be used to determine the stability of the captured complexes from sub aim 1.1.
Aim 2. Determine how the receptor binding and fusion proteins interact during binding and fusion activation Electron cryo-tomography (ECT) imaging of NiV fusion machinery. Using ECT we will visualize the process and requirements for the NiV receptor binding protein (G) to trigger fusion (F) protein-mediated fusion and subsequent steps in F structural rearrangement.
The goal of this project is to develop a novel imaging strategy based on the use of artificial liposomes to attract and lock intermediate states of viral fusion complexes. The proposed research seeks to gain a detailed understanding of the function of the viral fusion nano-machinery that paramyxoviruses use to invade host cells. Such an understanding is necessary in order to identify how best to interfere with the cycle of viral infection.
|Palgen, Jean-Louis; Jurgens, Eric M; Moscona, Anne et al. (2015) Unity in diversity: shared mechanism of entry among paramyxoviruses. Prog Mol Biol Transl Sci 129:1-32|
|Talekar, Aparna; Moscona, Anne; Porotto, Matteo (2013) Measles virus fusion machinery activated by sialic acid binding globular domain. J Virol 87:13619-27|
|Talekar, Aparna; DeVito, Ilaria; Salah, Zuhair et al. (2013) Identification of a region in the stalk domain of the nipah virus receptor binding protein that is critical for fusion activation. J Virol 87:10980-96|