Probing the Spatial Organization of Measles Virus Fusion Complexes

Abstract

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein heterooligomers 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 ␣-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.Paramyxoviruses infect cells through fusion of the viral envelope with target cell membranes. For all members of the Paramyxovirinae subfamily, this involves the concerted action of two envelope glycoproteins, the fusion (F) and attachment (H, HN, or G, depending on the Paramyxovirinae genus) proteins. Both proteins feature short lumenal tails, a single transmembrane domain, and large ectodomains. The F protein, in type I orientation, forms homotrimers, while homodimers or homotetramers have been suggested as functional units for attachment proteins of different Paramyxovirinae subfamily members (7,14,28,41,49,50,66). For entry, upon receptor binding, the attachment protein is considered to initiate a series of conformational rearrangements in the metastable prefusion F protein (15, 77), which ultimately brings together transmembrane domains and fusion peptides and, thus, donor and target membranes (3,32,45,53,80).Multiple studies have demonstrated that specific interactions between compatible F and attachment proteins of paramyxovirinae are imperative for the formation of functional fusion complexes (6,29,36,42,43,56,75). However, the molecular nature of these interactions and the spatial organization of functional glycoprotein hetero-oligomers remain largely unknown. Individual ectodomain and partial ectodomain crystal stru...

Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements

“…Nipah's G protein assembles as a homo-tetramer and its ectodomain contains both the ephrin binding and F activation sites (4,5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The F activation site is located in the N-terminal portion of the ectodomain (Fig.…”

“…The F activation site is located in the N-terminal portion of the ectodomain (Fig. 1), which is generally referred to as the stalk domain, or the F activation domain (FAD) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The receptor binding sites are located in the C-terminal portion of the ectodomain, and are >2 nm away from the F activation site.…”

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

“…Cell-to-cell fusion was assessed using an established luciferase reporter assay as previously described (60). In fusion experiments containing TCEP, the TCEP was added at the time of target cell overlay.…”

The Near-to-Native-State Architecture of Measles Virus Assembly Sites and Isolated Measles Virus Particles

“…Images were recorded at a magnification of 20,000×, corresponding to a pixel size of 2.94 Å on a DE-20 direct electron detector camera at a rate of 12 frames per second. Data was processed as previously described [3] and tomograms were reconstructed using the IMOD software package [7].Thin areas along the periphery of Measles virus-infected cells were surveyed to locate sites of virus assembly and to identify released virus particles. Regions of interest were identified and cryo-ET data was acquired.…”

“…Images were recorded at a magnification of 20,000×, corresponding to a pixel size of 2.94 Å on a DE-20 direct electron detector camera at a rate of 12 frames per second. Data was processed as previously described [3] and tomograms were reconstructed using the IMOD software package [7].…”