Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H▿

• Type of Material: Article
• Authors: Melinda A. Brindley, Emory University; Richard Karl Plemper, Emory University
• Language: English
• Date: 2010-12
• Publisher: American Society for Microbiology (ASM)
• Publication Version: Final Published Version
• Copyright Statement: © 2010, American Society for Microbiology
• Final Published Version (URL): http://jvi.asm.org/content/84/23/12174
• Title of Journal or Parent Work: Journal of Virology
• Volume: 84
• Issue: 23
• Start Page: 12174
• End Page: 12184
• Grant/Funding Information: This work was supported, in part, by U.S. Public Health Service grants AI071002 and AI083402 (to R.K.P.) from the NIH/NIAID and a seed grant from the Childrens Healthcare of Atlanta Vaccines & Immunology Center (to R.K.P.).; M.A.B. was funded by Molecular Mechanism of Microbial Pathogenesis training grant T32 AI007470 from the NIH.
• Research Categories: Biology, Virology; Health Sciences, Immunology; Biology, Microbiology

Abstract

Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex.