The structures of flavivirus E proteins and domain III subunits that have been determined to date show an overall similarity and conservation of the protein fold. However, more detailed studies of the physical and functional properties of these proteins, including the results presented here, are identifying key differences in the sequences and structural characteristics of these viral proteins that will more clearly define the specific molecular determinants of their antigenicity and functional properties.
Previous studies have described engineered mutations at residues in E domain III of some flaviviruses that had significant effects on virus multiplication or mouse virulence phenotypes (Hurrelbrink and McMinn, 2001
; Lee and Lobigs, 2000
; Mandl et al., 2000
). However, with the exception of the cysteines that form the conserved disulphide bridge, this is the first example of a single domain III residue - Y329 - that appears to be strictly required for viability of a flavivirus. In addition to the critical requirement for Y329 for proper folding of domain III, the conserved residues G331 and D333 appeared to play important roles in the infectivity of WNV, as mutations of either residue were associated with significant attenuation of WNV multiplication in Vero cells and of mouse neuroinvasivness. Mutations of G331 and D333 were also associated with reductions in detectable binding of neutralizing anti-WNV MAbs in Western blots () and reductions in neutralization () to levels that were comparable to those mutations previously described at residues 330 and 332 that facilitated complete or partial neutralization escape from one or more of those antibodies. These observations support our hypothesis that all BC loop residues make some contribution to antigenicity but some are subject to structural and/or functional constraints that limit divergence.
The BC loop sequence of WNV and most other mosquito-borne flaviviruses matches a “reverse Δ4 tyrosine corner” feature (sequence YXGXD/G) previously identified in a small number of viral capsid proteins, including the non-enveloped picornavirus poliovirus VP1 and rhinovirus VP1 (Hemmingsen et al., 1994
). Tyrosine corners with varying sequences have been identified in a wide range of beta-barrel/sandwich protein structures and physicochemical studies of protein folding have suggested a specific requirement for tyrosine corner hydrogen bonding to stabilize the folding of the Ig-like domains in which those motifs occur (Hamill et al., 2000
; Hemmingsen et al., 1994
; Nicaise et al., 2003
). The inability to recover infectious virus and the improper folding of recombinant WNV domain III proteins encoding mutations at Y329 in this study suggest that this tyrosine and the BC loop may play a similar structural role to that of the tyrosine corner motifs in other Ig-like domains.
Unlike WNV and other JE and DEN complex mosquito-borne flaviviruses, viruses in the YF group have a shorter predicted BC loop sequence and do not encode a tyrosine ()(Grard et al., 2009
). A recent structural analysis of YFV domain III has confirmed predicted differences in the lengths of the surface loops compared to other mosquito-borne flaviviruses and demonstrated that two proline residues at the N and C termini of the YFV BC loop may stabilize the surface loop structure rather than the tyrosine corner-like sequence found in other mosquito-borne flaviviruses (Volk et al., 2009
). However, other YF group viruses (e.g. Edge Hill virus, Wesselsbron virus in ) (Grard et al., 2009
) do not encode either the N-terminal BC loop proline found in YFV strains or the tyrosine found in other mosquito-borne flaviviruses. Similarly, evolution of mammalian tick-borne viruses (Gaunt et al., 2001
; Grard et al., 2007
) appears to have resulted in shortening of the BC loop and replacement of the tyrosine by phenylalanine (). This suggests that, despite the apparent similarities in domain III structures for different flaviviruses, the specific intramolecular interactions that define these structures are significantly different and would, at least in part, explain why attempts to transplant a DENV group reactive neutralizing epitope to other mosquito-borne flavivirus E-III molecules were much less successful when using YFV E-III as the backbone compared to either WNV or JEV (Lisova et al., 2007
). These differences are also supported by biophysical and biochemical comparisons of domain III from some tick- and mosquito-borne flaviviruses that have shown distinct differences in the stability of those proteins, despite the overall similarities in their secondary structure and hydrodynamic properties (Yu et al., 2004
In summary, the amino acid residues in the WNV BC loop clearly play significant roles in E protein domain III folding, virus infectivity, virulence and antigenicity. These findings also suggest that there are varying constraints on the plasticity of residues encoded in the surface loops of WNV domain III, which may have important implications for the design and evaluation of antibodies and other therapeutic compounds targeted to this domain (i.e. optimizing interactions with functionally important residues may limit the potential for emergence of resistant variants). The absence of an equivalent “tyrosine corner”-like motif in YF group viruses and many tick-borne flaviviruses suggests that flaviviruses have evolved with different intramolecular interactions supporting the folding and stability of domain III which are presumably associated with the functional and antigenic differences that exist between them. The results presented here provide a basis for future studies to define the specific relationships between amino acid sequence, structure, function and antigenicity of the putative receptor binding domains encoded by different flaviviruses that may also aid the rationale design of novel molecules based on domain III for use as improved vaccine immunogens and serodiagnostic antigens.