A Stabilized Headless Measles Virus Attachment Protein Stalk Efficiently Triggers Membrane Fusion

Abstract

Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved.

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

“…Multiple studies have shown that retargeted MeV H or NDV HN heads binding noncognate receptors are able to trigger F (45)(46)(47)(48)(49) and chimeric attachment proteins bearing globular heads or stalks from different viruses in various combinations are functional when paired with the cognate F protein corresponding to the stalk domain portion of the chimeric attachment protein (31,42,(49)(50)(51)(52)(53). Recent data have also shown that "headless" PIV5 HN, MeV H, and NiV G proteins, lacking the globular head domains in their entirety, are sufficient to trigger their cognate F proteins (42,54,55). Thus, counterintuitively, it appears that across the paramyxovirus family in general, receptor specificity or even receptor binding by the HN, H, or G heads is not a major requirement for F triggering.…”

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors

“…To determine infection kinetics, 293T cells transfected with the plasmids encoding the DSP 1-7 or DSP [8][9][10][11] (37), respectively, were mixed at equal ratios, preloaded with EnduRen life cell substrate as described (60), and spin-inoculated with recRSV A2-L19F (1,000 × g; 30 min; 4°C; MOI = 6 pfu per cell) in the presence of GPAR-3710 or DMSO. Activity of reconstituted luciferase was recorded at the specified time points.…”

Probing the Functions of the Paramyxovirus Glycoproteins F and HN with a Panel of Synthetic Antibodies

“…This movement exposes critical residues in the stalk domain that interact with and trigger F to initiate membrane fusion (41,62,63). This model is based on the following observations: (i) mutations in HN that affect only fusion activity map to the stalk region of the attachment protein of several paramyxoviruses (12,18,42,43,64,65); (ii) headless PIV5 and NDV HN, MeV H, and NiV G are sufficient to trigger fusion (41,63,66,67); (iii) the head domains of HN have been observed by EM to be in various orientations relative to the stalk for NDV (12) and PIV5 (41); (iv) a crystal structure of the tetrameric NDV HN ectodomain reveals the heads in the down position, forming an interface with the stalk that overlaps with the critical fusion (69) was used to morph high-resolution structures of HN, F, and the humanized anti-HER2 Fab, 4D5 (which the sAb phage library was based on), to 15-Å surface representations. Protein Data Bank (PDB) entries 3T1E and 3TSI were overlaid and used for the HN four-heads-down model.…”