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Journal of Virology, October 2009, p. 10480-10493, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.01195-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Probing the Spatial Organization of Measles Virus Fusion Complexes{triangledown}

Tanja Paal,1,# Melinda A. Brindley,1,# Courtney St. Clair,1,# Andrew Prussia,2,# Dominika Gaus,1 Stefanie A. Krumm,1 James P. Snyder,2 and Richard K. Plemper1,3,4*

Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia 30322,1 Department of Chemistry, Emory University, Atlanta, Georgia 30322,2 Department of Microbiology & Immunology, Emory University School of Medicine,3 Children's Healthcare of Atlanta, Atlanta, Georgia 303224

Received 10 June 2009/ Accepted 24 July 2009

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid {alpha}-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Pediatrics, 520 Children's Center, 2015 Uppergate Drive, Emory University School of Medicine, Atlanta, GA 30322. Phone: (404) 727-1605. Fax: (404) 727-9223. E-mail: rplempe{at}emory.edu

{triangledown} Published ahead of print on 5 August 2009.

# These authors contributed equally to the study.

Journal of Virology, October 2009, p. 10480-10493, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.01195-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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