Two mutations in the HR2 region of Newcastle disease virus fusion protein with a cleavage motif "RRQRRL" are critical for fusogenic activity

Background: Newcastle disease virus (NDV) causes severe diseases in avian species. Its fusion protein cleavage site (Fcs) is a major contributor to virulence and membrane fusion. Previous studies showed that a change from phenylalanine (F) to lysine (L) at position 117 of the virulent strain fusion protein, which has the polybasic amino acid Fcs motif " (112)RRQKR down arrow F-117", blocked syncytium formation. However, we observed that F proteins of the virulent strain F48E9 and avirulent strain LaSota substituted with an identical cleavage motif, "(112)RRQRR down arrow L-117", induced extensive and slight syncytium formation, respectively. Accordingly, we hypothesized that the difference in syncytium formation is caused by other regions of the fusion protein. Results: The exchanged regions between the fusion proteins of two strains, F48E9 and LaSota, showed that the region from amino acid 118- 499 plays an important role in modulation of fusogenic activity in transfected cells. Further dissection of this region indicated that replacement of two amino acids (N479D, R486S) in heptad repeat 2 (HR2) of the avirulent fusion protein by the virulent counterpart resulted in fusion promotion. Moreover, the role of these two amino acids in fusion is dependent on the unique Fcs sequence "RRQRR down arrow L". Conclusions: Our results demonstrated that two amino acids (D479, S486) of the virulent strain F protein with this unique Fcs were critical for promoting fusogenic activity, and residue F or L at position 117 did not affect syncytium formation. These findings provide novel insights into fusogenic triggering by the fusion protein and may be useful for designing antiviral peptides.