Possible neutralization mechanisms by HMAb 1F4.

1. HMAb 1F4 may block DC-SIGN from binding to the glycosylation site on N67. One DC-SIGN molecule binds across two N67 glycans on the virus surface. When Fab 1F4 is allowed to bind to the virus surface, it engages the N67 glycan on molecule B. This would cause steric hindrance thereby preventing the virus from binding to DC-SIGN on the surface of dendritic cells (Pokidysheva et al, 2006). Superposition of the cryo-EM complex structures of DENV1-Fab 1F4 and the DENV2-carbohydrate recognition domain (CRD) of DC-SIGN (PDB code 2B6B) showed that the Fab and the CRD molecules clashed. This indicates that simultaneous binding of these molecules on the virus surface is not possible. E protein molecules A, B and C are colored in light gray and molecules A’, B’ and C’ in gray. DC-SIGN is shown as a transparent orange surface. The glycosylation sites on N67 and N153 are marked as red stars and blue squares, respectively. The clashes between the Fab and DC-SIGN molecules are indicated by arrows.
2. Fab 1F4 may interfere with the E protein dimer to trimer structural changes during fusion of the virus to the endosomal membrane. (i) The DI-DII hinge of the whole virus E protein has a different angle compared to the crystal structure of the trimeric post- fusion rE protein. This suggests that the binding of HMAb 1F4 to virus may lock the E protein hinge thus preventing the kl loop from undergoing structural changes to the post-fusion structure. The E protein on the virus and the post-fusion trimeric state are colored in blue and pink, respectively. (ii) Superposition of the cryo-EM Fab 1F4-DENV1 E protein structure onto the post-fusion trimeric E protein crystal structure. The Fab 1F4 molecule clashed with DIII of a neighboring E protein in the post-fusion trimeric structure (indicated by arrow). Two molecules of E protein are shown as surfaces (colored in light gray and dark gray) and one molecule is shown as ribbons. Fab 1F4 is drawn as a transparent surface.