Protein-mediated membrane fusion is essential for maintaining eukaryotic cell organization and propagation of major human viruses. Many clinically relevant members of the paramyxovirus family rely on the concerted action of two envelope glycoproteins, the attachment and fusion protein, to fuse their envelope with the target cell plasma membrane for cell entry. However, despite their clinical importance, fundamental mechanistic principles that govern the organization and function of native paramyxovirus fusion complexes are not understood. Towards the overarching goal of elucidating these principles, this project will pursue the problem in three basic questions: What is the spatial organization of the envelope glycoprotein hetero-oligomer complexes in the native, metastable conformation displayed on the surface of infectious particles? How does receptor binding affect attachment protein organization? What is the molecular mechanism that links receptor binding with fusion protein refolding into the thermodynamically stable postfusion conformation? With crystal structures of isolated ectodomains of different paramyxovirus glycoproteins at hand, the proposed studies will focus on measles virus envelope glycoproteins to address these questions in a comprehensive, interdisciplinary approach that interfaces innovative imaging, biochemical, functional, and computational experimental strategies. Cryo-electron tomography combined with envelope glycoprotein engineering will elucidate the overall spatial organization of hydrated fusion complexes displayed on the surface of viral particles, alone and after treatment with soluble receptor (aim 1). Native gel electrophoresis, H oligomer stabilizing and destabilizing modifications, and H bimolecular complementation will extract functional information by assessing the effect of receptor binding on attachment protein organization and characterizing the molecular nature of the signal that initiates fusion protein refolding (aim 2). Molecular modeling-guided mutagenesis, biochemical contact domain mapping, peptide binding and mass spectrometry will cross-examine, expand and functionally characterize candidate intermolecular contacts found in pilot studies, resulting in the identification of discrete microdomains in either glycoprotein that line the hetero-oligomer interface and control the structural integrity of a native paramyxovirus fusion complex (aim 3).

Public Health Relevance

Elucidating how viruses infect human cells at a molecular level is crucial not only for better understanding the basic cell entry mechanisms that viruses have developed, but also for the design of new drugs that can block viral infection. Paramyxoviruses are a group of viruses that cause major human diseases, including measles, mumps, and flu-like illnesses, and several important animal diseases. This project will determine the arrangement of proteins on the paramyxovirus surface that are responsible for virus entry into human cells, and define precisely the mechanism of how these proteins work together to mediate infection. Although much-investigated, these questions have yet to be answered. Insight gained from this study will improve public health by enabling the future development of innovative antiviral drugs that efficiently block essential steps of the entry process of this important group of viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083402-04
Application #
8488398
Study Section
Virology - A Study Section (VIRA)
Program Officer
Cassetti, Cristina
Project Start
2011-07-01
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$330,460
Indirect Cost
$95,460
Name
Georgia State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
837322494
City
Atlanta
State
GA
Country
United States
Zip Code
30302
Cox, Robert; Plemper, Richard K (2016) Structure-guided design of small-molecule therapeutics against RSV disease. Expert Opin Drug Discov :1-14
Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa et al. (2015) Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head. J Virol 89:1445-51
Weisshaar, Marco; Cox, Robert; Plemper, Richard K (2015) Blocking Respiratory Syncytial Virus Entry: A Story with Twists. DNA Cell Biol 34:505-10
Brindley, Melinda A; Chaudhury, Sukanya; Plemper, Richard K (2015) Measles virus glycoprotein complexes preassemble intracellularly and relax during transport to the cell surface in preparation for fusion. J Virol 89:1230-41
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Avila, Mislay; Alves, Lisa; Khosravi, Mojtaba et al. (2014) Molecular determinants defining the triggering range of prefusion F complexes of canine distemper virus. J Virol 88:2951-66
Kiss, Gabriella; Chen, Xuemin; Brindley, Melinda A et al. (2014) Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications. Microsc Microanal 20:164-74
Brindley, Melinda A; Plattet, Philippe; Plemper, Richard Karl (2014) Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain. Proc Natl Acad Sci U S A 111:E3795-804

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