| Hendra virus, a member of the Paramyxoviridae family, first emerged in Brisbane, Australia in 1994 resulting in the deaths of fourteen horses and two humans. Like other paramyxoviruses, Hendra virus possesses an RNA genome of negative polarity, and has two major glycoproteins: the fusion (F) protein, shown to be responsible for the promotion of membrane fusion and the G protein, which functions in the attachment to ephrin-B2. The Hendra virus fusion (F) protein is produced as a precursor protein, F0, which is proteolytically processed by cathepsin L to a disulfide linked heterodimer, F1 + F2. The Hendra F1 protein contains a fusion peptide, two heptad repeats, and a transmembrane domain (TMD).; The Hendra virus fusion (HeV F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine to alanine mutations indicated that two sites in the F2 subunit (N67 and N99) and two sites in the F1 subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active, as shown by syncytia and reporter gene assays. As N464 lies within heptad repeat B, these results contrast with those seen with several paramyxovirus F proteins.; Helical wheel construction revealed four serine residues and a glycine (5487, S490, S493, S501, and G508), are potentially located on the same face of the HeV F TMD. Analysis of the HeV F TMD single serine to alanine mutations indicated three of these serines (490, 493, and 501) possess roles in membrane fusion. Though S493A and S501A disrupted activity, the uncleaved form of S490A was retained at the cell surface and may be necessary for proper endocytosis or recycling of HeV F. The serine to alanine mutations (except S487A) as well as G508I and G508L (though not G508A) resulted in loss of syncytia and fusion activities, due to reduced cell surface expression of the cleaved form. We hypothesize that glycine and polar residues possess potential roles in HeV F protein mediated membrane fusion. |
