Several other studies have addressed the role of E H244 in the flavivirus lifecycle. Experiments in TBEV evaluated particle production and membrane fusion activity using a VLP system [56] . Mutation of H248 (TBE numbering) to A or I blocks VLP secretion, in agreement with our results. However, an H248N mutant efficiently produces VLP, and these particles show WT levels of fusion activity. WNV E H246A or Q mutations inhibit release of infectious reporter virus particles from cells, as do a number of other substitutions at this position [57] . Replacement of H246 with aromatic residues such as phenylalanine allows both particle release and infectivity. An H244A mutation in DENV2 NGC inhibits infectious virus production [43] . E H244 and its interacting partners D63 and D65 on pr are conserved within the flaviviruses, and thus these data from several flaviviruses plus our DENV results support an important role for the E 244 position. However, a histidine residue at this position does not seem to be strictly required for particle production, suggesting that substitutions such as 244F and 244N can support the interaction of E with pr.