Respiratory syncytial virus (RSV) is a major global paediatric viral pathogen, causing severe lower respiratory tract diseases such as pneumonia and bronchiolitis1. The virion surface glycoprotein F is an essential player in virus entry into cells, being responsible for mediating virion-cell membrane fusion. RSV F expressed at the infected cell surface also results in the formation of multinucleate syncytia. The F protein exists in a metastable pre-fusion form, that transitions into the more energy-favourable, post-fusion form following receptor engagement. This post-fusion form is stabilized by the formation of a six-helix bundle made of two helical heptad repeats (HR) in each of the three monomers, referred to as HRA and HRB2 . An additional heptad repeat, heptad repeat C (HRC), was investigated in this project since it is predicted to undergo significant re-arrangement between both protein structures and also lies in close proximity to HRA in the pre-fusion form. An alanine scan was performed on the HRC domain using site-directed mutagenesis and the effect on fusion phenotype of recombinantly expressed F was assessed through syncytia formation in cell culture. Utilizing the recently-published pre-fusion structure3, a striking pattern was observed in that mutants that enhanced fusion aligned on one helical face, while mutants that abrogated fusion lay on another. It is clear that HRC plays a pivotal role in the fusion process and it is even postulated that HRC may engage the cellular receptor nucleolin, causing a small conformational change in HRC that is the trigger for fusion. The clustering of these residues and proposed mechanism of action provide opportunities that will direct further research and insights that may prove beneficial to anti-viral drug design.