Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV(3929)), including the hinge region (E273–277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV(3929) binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV(3929) exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV(3929) as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV(3929) in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273–277 and E154–156 of ALFV(3929) were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV(3929), with the E327/E273–277 combination most dominant. These data demonstrate that attenuation of ALFV(3929) is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.