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Journal of Virology, October 2000, p. 8867-8875, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Eva Lee and Mario Lobigs*

Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Australian Capital Territory, Australia

Received 3 May 2000/Accepted 10 July 2000

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp390, which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp390 on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly390, Ala390, and His390) showed 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 glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

* Corresponding author. Mailing address: Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. Phone: 61-2-62494048. Fax: 61-2-62492595. E-mail: Mario.Lobigs{at}anu.edu.au.

Journal of Virology, October 2000, p. 8867-8875, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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