Alternative conformations of a major antigenic site on RSV F

Jones, Harrison G.; Battles, Michael B.; Lin, Chun-Chi; Bianchi, Siro; Corti, Davide; McLellan, Jason S.

Recent vaccine strategies targeting viruses with class I fusion proteins have focused on stabilizing the prefusion conformation of the fusion protein for use as an immunogen. A common approach to achieve prefusion stabilization has been through introduction of one or more proline residues within the loop of a helix-loop-helix motif that refolds into a continuous alpha helix in the postfusion conformation. This strategy has been used successfully to stabilize several class I viral fusion glycoproteins including RSV F, MPV F, influenza HA, HIV Env, and coronavirus Spike [41, [57] [58] [59] [60] . Proline-based stabilization utilizes the restricted phi-psi angles of proline residues and disruption of the alpha-helix hydrogen bond network to inhibit the hinge motion required of the helix-loop-helix motif and the subsequent coil-to-helix structural transition required for refolding into the postfusion conformation. However, for wild-type prefusion RSV F there is a conserved proline residue (Pro205) within the middle of the α4-helix, N-terminal to the α4-α5 loop that may act as a hinge during refolding [41] . Crystal structures of prefusion RSV F demonstrate a variety of alternate conformations adopted by antigenic site Ø, all of which diverge near the conserved Pro205. This indicates that Pro205 may disfavor a rigid alpha-helical structure, which in turn facilitates conformational flexibility of site Ø and the tendency of prefusion RSV F to trigger. We note that Pro205 is absolutely conserved in all human and bovine RSV strains, and a proline at an identical position is also commonly found in F proteins from viruses within the Pneumoviridae and Paramyxoviridae families, with the exception of human metapneumoviruses (Fig 5D) . The functional importance of this well-conserved proline residue will need to be evaluated in future studies investigating the triggering mechanism and refolding of pneumovirus and paramyxovirus F proteins.