title: 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 membrane-adjacent 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 reverse-pseudotyped 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 approximately 20% 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.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, 609 Charles E. Young Drive East, Los Angeles, CA 90095, USA.
authors.forename: Hector C
authors.initials: HC
authors.lastname: Aguilar
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Matreyek
authors.forename: Daniel Y
authors.initials: DY
authors.lastname: Choi
authors.forename: Claire Marie
authors.initials: CM
authors.lastname: Filone
authors.forename: Sophia
authors.initials: S
authors.lastname: Young
authors.forename: Benhur
authors.initials: B
authors.lastname: Lee
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D047468:Immunoprecipitation; D008561:Membrane Fusion / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D045405:Nipah Virus / Q000235:genetics*; D011487:Protein Conformation; D015398:Signal Transduction / Q000235:genetics*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D014760:Viral Fusion Proteins