Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry

The flavivirus receptor-binding domain has been putatively assigned to a hy drophilic region (FG loop) in the envelope (E) protein. In some flaviviruse s this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD), One of us has shown earlier that host cell adaptation of Murray Valley encephaliti s virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-le ngth, infectious cDNA clone of MVE was constructed and employed to systemat ically investigate the impact of single amino acid changes at Asp(390) on c ell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showe d pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells, The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on g lycosaminoglycans (determined by inhibition of virus infectivity by heparin ) for attachment of the three mutants to different mammalian cells, and (ii i) the loss of virulence in mice. These results confirm a functional role o f the FG loop in the flavivirus E protein in virus entry and suggest that e ncephalitic flaviviruses can enter cells via attachment to glycosaminoglyca ns. However, it appears that additional cell surface molecules are also use d as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.