Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Abstract

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membraneadjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3-to 5-fold. These results were corroborated in a reversepseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper-or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ϳ20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P ‫؍‬ 0.007, r 2 ‫؍‬ 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation

“…Henipavirus, like many paramyxoviruses, requires the presence of two surface glycoproteins for virus-cell and cell-cell fusion (8,9,36): the fusion protein, F, which mediates the membrane fusion event, and the attachment protein, G, which binds cellular receptors ephrin B2 (6,22) and ephrin B3 (23) and which is required for F-mediated membrane fusion. Interactions between the fusion and attachment proteins of a number of paramyxoviruses have been observed previously (30,33,35), and interactions between the henipavirus F and G proteins have been demonstrated by coimmunoprecipitation (1)(2)(3)(4)(5)18). However, important questions remain concerning the timing of these interactions and the mechanism by which the attachment protein regulates F-mediated fusion.…”

“…An alternative model for paramyxovirus fusion has suggested that attachment proteinfusion protein interactions occur only after the attachment protein binds the receptor. However, analysis of henipavirus G and F mutants suggests that F-G avidity inversely correlates with fusion (2,3,5), and Hendra virus G protein mutants deficient in receptor binding also lose the ability to coimmu- FIG. 3.…”

The Transmembrane Domain Sequence Affects the Structure and Function of the Newcastle Disease Virus Fusion Protein

“…The cytoplasmic domain has been implicated in the fusion activity of some paramyxovirus F proteins, since mutations in the CT domain or extensions of this domain can affect fusion activity (1,5,52,63,66). The role of the TM domain in the structure and function of paramyxovirus F proteins has not, however, been extensively investigated.…”

The Paramyxovirus Fusion Protein C-Terminal Region: Mutagenesis Indicates an Indivisible Protein Unit

“…The cytoplasmic tails of viral fusion proteins have also been implicated in fusion activity in several studies, and many mutations have been identified that reduce fusion activity (1,16,38,44). For PIV5 F, deletion of the cytoplasmic tail impairs fusion pore enlargement (16), and increasing the length of the cytoplasmic tail such that it has the propensity to form a three-helix bundle abolishes fusion (48).…”